Brainstorm

2025.01.06

We have formally established the 2025 JLU-NBBMS team.

2025.01.14

First Brainstorming Session

This marks our inaugural brainstorming session, requiring team members to share their conceptual ideas.

Min Yingxuan proposed employing the MBR method to isolate carbon sources from oily industrial wastewater, then utilising synthetic biology to enable chassis organisms to utilise these carbon sources, ultimately synthesising ginsenosides.

You Mingyu proposed utilising engineered Bacillus subtilis to degrade harmful substances such as phenolic acids in soil, thereby improving ginseng growth conditions and enhancing saponin yield. He further suggested immobilising the engineered strain using biochar to ensure experimental safety.

Zhang Jiayi and Li Ruoxi proposed constructing anti-diabetic ginsenoside nanocarriers based on DNA origami. They utilised Y-shaped scaffold DNA tiling technology to prepare rigid DNA tetrahedral ROTs. A multifunctional nanoparticle system featuring folic acid and tripeptide dual-modified PLGA was developed to progressively overcome continuous absorption barriers.

Ou Liyang proposed constructing a multi-enzyme assembly system within Saccharomyces cerevisiae to synthesise ginsenosides, alongside designing sensors and adhesion proteins to facilitate yeast colonisation at intestinal tumour sites.

Chang Xuanhe proposed preparing ginsenosides as swellable nanoparticles to load trace amounts of ginsenoside Rg3, with lipid carrier NLC enhancing ginsenoside targeting and delivery efficiency.

Sun Jiayin proposed constructing a mesenchymal stem cell-ginseng exosome fusion vesicle as a carrier, containing specific molecular tools. The carrier could be engineered to target diseased cells, thereby treating psoriasis.

Song Jie proposed utilising compartmentalised expression technology to engineer ginsenoside production, followed by the self-assembly of traditional Chinese medicine components into natural small-molecule gels exhibiting pharmacological effects such as anti-ageing properties.

2025.02.08

Second Brainstorming Session

This marks our second brainstorming session. Following the initial session, we conducted preliminary feasibility analyses of our ideas, exchanged questions and feedback, and selected the most viable projects for group work. This is our first presentation since forming the groups.

Group One: Synthesis of Ginsenosides from Edible Oil Industry Wastewater Building upon the initial presentation, the team gained deeper insights into the Membrane Bioreactor (MBR) process. They proposed a stepwise synthesis pathway from edible oils to ginsenosides, identified key genes and synthesis strategies through literature review, and outlined a design concept for a gene oscillator. However, the approach was questioned due to the large number of genes involved, which would prevent sustained product output post-oscillation, necessitating revision. The team proposed organelle compartmentalisation for production, though this requires integration with the synthesis process.

The second group focused on improving soil for ginseng continuous cropping using biochar-immobilised Bacillus subtilis. Following concerns raised during the initial presentation about the safety of the engineered strain, the team optimised the genetic circuit based on the principle of population effects. They designed a suicide switch to enhance project safety and refined the biochar preparation and modification protocols.

The third group developed a ginsenoside DNA origami sponge for moisture retention and anti-photoaging. Building upon their initial presentation, they designed a three-dimensional spatial structure for the origami sponge, enabling oxidative stress gating during ginsenoside experiments.

The fourth group focused on Saccharomyces cerevisiae expressing ginsenosides, colonised at colon cancer sites to provide adjuvant therapy. Building upon their initial presentation, they refined the customisation and therapeutic systems, constructing a relatively complete sensor.

Project promotion

2025.02.10

First Principal Investigator (PI) Presentation: This marked our inaugural formal engagement with the PI. During this session, each group presented their design proposals, with the PI providing specific feedback to refine project selection.

(1) The first group focused on synthesising ginsenosides from industrial waste oils. Members presented in four sections, providing an in-depth explanation of the MBR process. They outlined a stepwise synthesis pathway from oils to ginsenosides (from oils to MVA, and from MVA to ginsenosides), identified key genes and synthetic strategies, proposed a design concept for a genetic oscillator, and divided the synthesis into two systems (production and control systems). The Principal Investigator (PI) expressed initial approval, noting the project's strong embodiment of synthetic biology principles alongside environmental sustainability. The system design was deemed relatively robust with high experimental feasibility. The PI offered suggestions for refinement, highlighting that the extensive target gene selection and numerous synthesis modules risked exceeding yeast's plasmid capacity limits. The final recommendation was to prioritise key elements during design.

(2) The second group proposed using biochar-immobilised Bacillus subtilis to remediate ginseng-contaminated soils. Team members suggested employing synthetic biology to degrade phenolic acids, thereby reducing phytohormone toxicity, and utilising biochar to immobilise Bacillus subtilis for synergistic soil remediation. The PI noted this project well aligns with iGEM's founding principles but highlighted the difficulty in modifying biochar and the relatively low experimental feasibility.

(3) The third group proposed a ginsenoside DNA origami suction cup for moisture retention and anti-photoaging. Team members suggested employing DNA origami principles to enable DNA self-assembly into suction cups, thereby addressing skin photoaging caused by UV radiation. The PI acknowledged the project's strong innovation but highlighted challenges in achieving the research objectives and forming the multi-enzyme complex.

(4) The fourth group proposed colonising colon cancer sites with Saccharomyces cerevisiae expressing ginsenosides for adjuvant therapy. Team members suggested constructing therapeutic and colonisation systems. The PI noted that building multi-enzyme complexes requires not only multiple genes but also connecting necessary components, involving numerous plasmids with potentially limited efficacy.

Summary: Based on project feasibility, synthetic biology principles, and the original intent of environmental sustainability and efficiency, the Principal Investigator ultimately selected the project 'Synthesis of Ginsenosides from Waste Oils and Fats'. This was deemed highly feasible, consistent with synthetic biology characteristics, and capable of achieving favourable outcomes leveraging the laboratory platform.

2025.03.09

This marks the inaugural group discussion to determine the project's thematic focus.

Improvements over previous designs include:

(1) Selection of a specific ginsenoside type—Oleanolic acid-type ginsenoside Ro. This compound exhibits well-defined pharmacological effects yet remains underutilised in biosynthetic approaches. Given its anti-ageing and antioxidant properties, subsequent applications may extend to cosmetic production.

(2) Establishing Saccharomyces cerevisiae as the biological chassis, owing to its native MVA pathway in the cytoplasm and essential saponin synthesis systems including cytochrome monooxygenases and glycosyltransferases.

(3) Determining the method for generating acetyl-CoA from fatty acids—by targeting key MVA pathway enzymes to the peroxisomes.

(4) Establishing the specific Ro synthesis strategy—including glycosyltransferase and key genes for saponin aglycone synthesis.

Proposing multiple optimisation approaches—reducing bypass metabolism, optimising key enzymes, enhancing promoter efficiency, and improving fermentation conditions.

2025.03.15

We visited Professor Gao Renjun at the School of Life Sciences to interview him regarding enzyme modification. We provided Professor Gao with a detailed explanation of the project and raised questions concerning the enzyme modification engineering.

The improvements made were:

(1) For the glycerol to 2,3-OSQ metabolism pathway, it is preferable to utilise the organism's own metabolic pathway. This approach reduces the number of plasmids required and prevents metabolic disruption in the yeast.

(2) Project focus should prioritise the subsequent conversion of OA to Ro; thus, the ergosterol pathway need not be inhibited to avoid compromising yeast viability.

(3) A website for glycosyltransferases was recommended. Screening enzymes from databases via molecular dynamics simulations is advised to reduce time expenditure, or AI methods could accelerate progress.

(4) Given the numerous glycosylation steps in ginsenoside synthesis requiring substantial UDPG consumption, enhancing endogenous UDPG pathways is advised.

Summary: Professor Gao emphasised holistic project management, advocating targeted focus on the latter stages. Regarding enzyme engineering, he proposed two more feasible approaches: AI integration and database-driven screening.

2025.3.19

Second PI Report

This time, incorporating expert feedback and group discussion outcomes, we present specific optimisation proposals, including:

(1) Reducing the additional glycerol metabolic pathway by utilising acetyl-CoA generated through its natural metabolic pathway.

Redesigning the Ro pathway to generate glycosylation pathways, allocating distinct metabolic fluxes to R1 and 4a. Through in vitro optimisation of the glycosyltransferase, we have engineered differing metabolic flux levels between the two pathways. This ensures that the enzyme UGT73F3 exhibits significantly greater affinity for 4a than for CE.

2025.04.03

Visited the School of Pharmacy to interview Professor Zhang Xinmin, an expert in synthetic biology. We provided Professor Zhang with a detailed explanation of the project and conducted a preliminary assessment of its experimental feasibility.

Improvements include:

(1) When loading two to three genes onto a single plasmid vector, employing a promoter-terminator fusion approach would be more conducive to protein expression. Additionally, note that different selection conditions should be chosen for different plasmids; consideration should be given to using nutritional selection conditions to reduce experimental costs.

(2) The final ginsenoside Ro product should be validated using high-performance liquid chromatography (HPLC). As unexpected outcomes may arise during experimentation, separate experimental groups should be established with distinct experimental conditions.

(3) This project embodies ecological conservation and waste recycling principles. Glycerol is inexpensive, being the primary by-product of biodiesel production, and numerous studies currently explore its use as a feedstock for value-added products. The approach of introducing key enzymes into peroxisomes for fatty acid metabolism via the mevalonate pathway demonstrates innovation. However, prior formulation of the culture medium composition is essential.

2025.04.10

This was a group discussion during which we assigned roles to members of the experimental group and, incorporating Professor Zhang's insights, highlighted common pitfalls in plasmid design. We determined that the plasmids we require must synthesise the following functions:

(1) Utilising fatty acids to generate acetyl-CoA for MVA synthesis

(2) Producing the key enzyme necessary for oleanolic acid (OA) generation

(3) The key enzyme required for converting oleanolic acid (OA) into the rare ginsenoside Ro

(4) The key enzyme required for the UDPG pathway

2025.04.10-04.25

Team members design plasmid maps, determine vector types, screen for labels, and select promoter-terminator combinations and tag types.

2025.04.29

A further visit to Professor Zhang Xinmin's laboratory was made to finalise specific experimental details, during which the application of carbon sources was discussed in depth. Given that fatty acids are categorised as either saturated or unsaturated, with unsaturated fatty acids existing in liquid form and saturated fatty acids in solid form, the formulation of the culture medium must consider how yeast can utilise fatty acids. This may involve various approaches, such as the addition of emulsifiers.

2025.05.01-06.01

Group members shall procure materials relevant to the experiment as required by the experimental needs and under the guidance of the Principal Investigator, and shall finalise the experimental protocols and schedule.

2025.08.20

Visited Professor Zhang Zuoming, an expert in synthetic biology at the School of Life Sciences, for an interview.

Upon obtaining the HPLC yield results for the rare ginsenoside Ro, we observed that alongside the target product peak, the HPLC results also revealed a deglycosylation product of Ro—a disaccharide ginsenoside. Based on the Ro synthesis process, we hypothesised this was caused by accumulation of intermediate products. We consulted Professor Zhang regarding this issue, proposing the following suggestions:

(1) Determine whether the issue lies with plasmid expression or a defect in the metabolic pathway encoded by the key gene?

(2) Validate successful plasmid transfection and expression via PCR and Western Blot analysis.

(3) Assess enzyme activity through in vitro enzymatic assays. Professor Zhang suggested that the heterologous gene, derived from the plant Panax notoginseng, may undergo excessive glycosylation upon transfection into yeast, leading to enzyme inactivation.

(4) Conduct in vitro experiments to assess conversion efficiency across both pathways, and implement a 'source-cutting and flow-interception approach through dry experiments.

Convert glycerol and fatty acids into MVA

Cultivation and Cryopreservation of Yeast

Experimental time: 2025.06.25

Experimental personnel: Zhao Xiaoqing, Sun Jiayin

Objectives of the Experiment

1. Cultivate and freeze-store Saccharomyces cerevisiae to prepare for subsequent plasmid transfection in the production of competent cells.

2. Prepare the plasmid as a solution for subsequent transfection.

Experimental Equipment and Reagents

Equipment

Shaker, refrigerator, laminar flow hood, spectrophotometer

Reagents

YPD medium, plasmid powder, sterile water, glycerol

Reagent Preparation

Experimental Procedure

Cultivation of Saccharomyces cerevisiae

(1) Irradiate the ultra-clean workbench with ultraviolet light for 30 minutes beforehand. Prepare YPD medium and microwave-treat it in advance for later use;

(2) Take a 50 mL centrifuge tube and pour 5 mL of YPD liquid medium into it;

(3) Pick a single colony of Saccharomyces cerevisiae W303 from the culture plate and inoculate into the tube. Incubate on a shaking incubator (30°C, 200 rpm);

(4) After approximately 7 hours, measure the OD600. If OD600 = 0.3, add 2 mL of the culture to 100 mL of YPD medium.

Prepare the plasmid

(1) Retrieve the plasmid powder tube and centrifuge (4μg);

(2) Add 30μL sterile water to the tube;

(3) Transfer 10μL to an EP tube, then add 32μL sterile water for later use;

(4) Store the remaining 20μL plasmid at -20°C.

Freeze-preserve experimental strains

Take an EP tube, add 750μL bacterial suspension and 750μL glycerol (50%) in a 1:1 ratio, mix, and store at -80°C. Replenish the remaining bacterial suspension in the centrifuge tube with appropriate medium.

Experimental Results

Analysis and Discussion

Yeast exhibited robust growth and is suitable for preparing competent cells for subsequent experiments.

Precautions

(1) Maintain aseptic technique when isolating single-colony yeast to prevent contamination by extraneous microorganisms.

(2) When preparing YPD medium, avoid excessive temperatures during autoclaving. Typically, autoclave at 115°C for 15 minutes; higher temperatures may cause glucose to darken.

(3) Ensure culture duration does not exceed recommended limits. If optical density (OD) exceeds 1.2, the strain is unsuitable for subsequent procedures.

Preparation of Sc-Trp Medium and Competent Cells

Experiment Date: 2025.06.29

Experiment operator: Zhao Xiaoqing, Sun Jiayin

Objectives

1. Prepare Sc-Trp medium and plate it to facilitate subsequent cultivation of Saccharomyces cerevisiae following plasmid transfection.

2. Prepare competent cells for plasmid transfection.

Experimental Equipment and Reagents

Equipment

Laminar flow hood, spectrophotometer, autoclave, shaking incubator

Reagents

SC-Trp, arginine, adenine, glucose, YNB, ammonium sulphate, agar, sterile water, LiAC/TE buffer

Reagent Preparation

Experimental Procedure

Preparation of SC-Trp Medium

(1) Take 0.4 g SC-Trp, 0.04 g arginine, 0.2 g adenine, and 4 g glucose; add to 60 mL water and place in an empty bottle;

(2) Take 4 g agar and add to 120 mL water; place in another empty bottle and add a rotor;

(3) Place both bottles alongside an empty 100 mL bottle into an autoclave for sterilisation (115°C, 15 minutes);

(4) Weigh 1.6 g of YNB yeast and 5 g of ammonium sulphate into a beaker. Add 100 mL of sterilised water and stir with a stirrer rod until completely dissolved;

(5) Sterilise the solution by filtration through a 0.22μm filter, then transfer to a 100mL sterile autoclave bottle for later use;

(6) Take 20mL of the sterilised YNB solution and add to the sterilised agar bottles. While hot, pour the SC-Trp solution into the agar bottles containing YNB and mix thoroughly using a magnetic stirrer;

(7) Swiftly pour the mixture into sterile Petri dishes to form plates. Allow to stand at room temperature for 4 hours before refrigerating for later use.

Preparation of Competent Cells

(1) Place sterile water in a 4°C refrigerator for later use. Dilute LiAC/TE to 0.1 mol/L for later use;

(2) Measure the OD600 of the Saccharomyces cerevisiae culture from the previous day. Proceed to the next step if OD600 = 0.8;

(3) Take 50 mL of the culture, centrifuge (3500 rpm, 2 min), and discard the supernatant;

(4) Resuspend in 50 mL of cold sterile water, centrifuge (3500 rpm, 2 min), and discard the supernatant;

(5) Resuspend in 1 mL 0.1 mol/L LiAC/TE buffer, centrifuge briefly (4000 rpm, 30 s), and discard supernatant;

(6) Resuspend in 400 μL 0.1 mol/L LiAC/TE buffer, mix thoroughly, and refrigerate overnight on ice.

Experimental Results

Precautions

(1) After filtration and sterilisation, the YNB solution must be rapidly poured into agar while hot to prevent uneven distribution causing abnormal solidification of the medium.

(2) Once prepared, the medium must be promptly poured into plates to prevent premature solidification due to prolonged exposure.

(3) Monitor the OD value of Saccharomyces cerevisiae to prevent excessive incubation time, which may render the sample unsuitable for subsequent experiments.

Plasmid ① transfection; Saccharomyces cerevisiae culture post-transfection

Experiment date: 2025.06.30

Experiment operator: Wang Quanhao

Experimental Objectives

(1) Utilise prepared competent cells for plasmid transfection;

(2) Transfect Saccharomyces cerevisiae competent cells with Plasmid ① to utilise fatty acids;

(3) Prepare YPD medium and Trp-deficient solid medium for future use;

Experimental Equipment and Reagents

Equipment

Laminar flow hood, shaker, 30°C incubator, water bath, rocking incubator, oven, electronic balance, magnetic stirrer, autoclave

Reagents

PEG, LiAc (1 mol/L), salmon sperm, plasmid, yeast competent cells, YPD medium, PEG, DMSO Glucose, peptone, arginine, adenine, SC-Trp;

Reagent Preparation

3.1 YPD medium (500 mL)

3.2 Trp-deficient solid medium (180 mL)

3.3 Preparation of YNB Medium

Experimental Procedure

Plasmid Transfection

1.1 Boil salmon sperm in water for 5 minutes, then promptly place into an ice-water mixture (on ice).

1.2 Add approximately 50 μL of the yeast competent cells prepared the previous day to each Eppendorf tube. Centrifuge (4000 rpm, 30 seconds) and discard the supernatant.

1.3 Add sequentially to the EP tube: 42 μL plasmid, 10 μL salmon sperm (10 mg/mL), 240 μL PEG, and 36 μL LiAc (1 mol/L). Mix thoroughly by repeated pipetting with a 1 mL pipette, then incubate at 30°C for 30 minutes.

1.4 Add 36 μL DMSO to each EP tube. Mix thoroughly by pipetting and heat-shock at 42°C for 20 minutes.

Post-transfection yeast culture

2.1 Remove EP tubes from water bath. Centrifuge (3000 rpm, 20 seconds) and discard supernatant. Resuspend in 1 mL YPD medium and incubate on shaking incubator for 3 hours.

2.2 Centrifuge (3000 rpm, 20 s), discard supernatant, resuspend in 100 μL water. Spread culture onto plates (Sc-Trp) using glass beads and incubate at 30°C for approximately 3 days.

Preparation of Trp-deficient solid medium

3.1 Prepare Trp-deficient solution as per Section 3.2 under 'II', then prepare YNB solution as per Section 3.3.

3.2 Place glass bottles containing the solutions into an autoclave for sterilisation (115°C, 15 min).

3.3 Sterilise the YNB solution by filtration through a 0.22 μm filter, then transfer to a 100 mL sterile autoclave bottle for storage.

3.4 Take 20 mL of the sterilised YNB solution and add to the Trp double-deficient solution, mixing thoroughly.

3.5 Pour the medium into sterile Petri dishes (approximately 15-20 mL per dish). Allow to solidify at room temperature before inverting and storing at 4°C (prevent condensation accumulation).

Preparation of YPD Medium

4.1 Prepare YPD medium as per Section 3.1 of Part II. Follow the remaining steps as per Section 4 of Part III. Sterilise by UV irradiation as a liquid medium.

Precautions

1. Salmon sperm must be pre-boiled for 5 minutes in boiling water, then rapidly transferred to an ice-water mixture to preserve exogenous plasmids.

2. All reagents required for plasmid transfection must be thoroughly mixed to maximise cell membrane permeability and enhance transfection efficiency.

3. Incubation times in the cell culture incubator and heat shock in the water bath must be strictly controlled to prevent reduced transfection efficiency due to prolonged exposure.

Growth curve measurement of single yeast clones containing plasmid ① in different glycerol-fatty acid media
Cycle 1

Experiment date: 2025.07.02

Experiment operator: Sun Jiayin, Min Yingxuan, Wang Jingning

Objective

Yeast strains transformed with plasmid ① and the wild-type w303 strain were used as controls, while various glycerol-fatty acid media were prepared to screen for the yeast strain exhibiting the highest selective utilization of a specific fatty acid.

Equipment and Reagents

Equipment

Laminar flow hood, autoclave, shaker, microplate reader, 96-well plates

Reagents

Sc-Trp、Arg、Adenine sulfate、ddH2O、Glycerol、Lauric acid、Oleic acid、Palmitic acid

Reagent Preparation

(1) Place the following ingredients into a clean bottle;

(2) Add X g of solid stearic acid and Y g of lauric acid separately into two 1.5 mL centrifuge tubes. Add 150 μL of anhydrous ethanol to each centrifuge tube and dissolve in a 60 °C water bath. Immediately transfer to sterile caps of clean 50 mL centrifuge tubes. Subsequently, dry the dissolved solids in a fume hood until no residual liquid remains on the surfaces of the centrifuge tubes and all anhydrous ethanol has completely evaporated.

(3) Transfer dried palmitic acid and lauric acid separately into EP tubes, add 1.8 mL of water and 0.18 mL of Tween-80, then dissolve completely by heating in a water bath with continuous stirring.

(4) Take another EP tube, add Z mL of oleic acid, M mL of glycerol, and 0.18 mL of Tween-80, then heat in a water bath and mix thoroughly.

(5) Preheat the solution obtained in step (1) while stirring continuously. Add palmitic acid, lauric acid, oleic acid, and glycerol sequentially into the container prepared in step (1), mix thoroughly, and sterilize by autoclaving at 115°C for 15 minutes.

(6) Finally, add 20 mL of YNB solution to the container to complete the preparation. Transfer the dried palmitic acid into EP tubes, adding 1.8 mL of water and 0.18 mL of Tween-80, respectively. Heat in a water bath and stir thoroughly.

Experimental Procedures

(1) The biosafety cabinet was irradiated with ultraviolet light 30 minutes prior to use;

(2) Take a 50 mL centrifuge tube and add 15 mL of glycerol-fatty acid liquid medium into the tube;

(3) Pick a single yeast clone from the Saccharomyces cerevisiae plate transformed with Plasmid ① and add it into the centrifuge tube, then incubate on a shaker at 30℃ and 200 rpm;

(4) Every 2 hours, take an aliquot of the liquid culture of the single yeast clone transformed with Plasmid ① and observe yeast growth by checking for turbidity in the liquid medium.

(5) Using a 96-well plate, every 2 hours, take 100 µL of yeast suspension from each medium and add it into the plate, with three replicates for each group; measure OD600 using a microplate reader and calculate the average.

Experimental Results

Analysis and Discussion of Results

Overall, yeast transformed with Plasmid ① exhibited significantly greater growth than the wild-type yeast W303, indicating that the transformed yeast can effectively utilize the glycerol-fatty acid medium for growth, confirming the successful introduction of Plasmid ①.

The OD values measured by the microplate reader for yeast transformed with Plasmid ① during the growth plateau phase across fatty acid media followed the order: glycerol:palmitic acid = 1:1 > glycerol:oleic acid = 1:1 > glycerol:lauric acid = 1:1, demonstrating that yeast utilize palmitic acid and oleic acid significantly more effectively than lauric acid. This may be attributed to the fact that yeast cells transformed with Plasmid ① possess an inherent mechanism for the efficient metabolism of long-chain fatty acids. Palmitic acid (16 carbons) and oleic acid (18 carbons) are the predominant fatty acid components in the phospholipids of yeast cell membranes, with their synthesis and degradation pathways being well established. In contrast, lauric acid, a 12-carbon medium-chain fatty acid, is not a principal component of the yeast's endogenous metabolism. Its shorter carbon chain may cause the lauroyl-CoA produced upon activation to exhibit low affinity toward yeast acyl-CoA metabolic enzymes, such as those involved in β-oxidation, thus impeding its efficient incorporation into central energy metabolism or biosynthetic pathways and resulting in decreased utilization efficiency.

In subsequent experiments, palmitic acid and oleic acid will be selected as the main fatty acid components. A glycerol-to-fatty acid ratio of 1:1 will be maintained for liquid culture of the strain carrying plasmid ①, in order to observe plasmid ①'s preference for different types of fatty acid mixtures.

Notes

(1) Set up the wild-type w303 strain without plasmid ① as a control group, to better assess whether plasmid ① is successfully expressed.

(2) Measure the growth OD value approximately every 2 hours to allow timely monitoring of yeast growth rate and extent.

Growth curve determination of plasmid ① yeast monoclonal cultures in glycerol-fatty acid media at varying ratios
Cycle 2

Experiment date: 2025.07.05

Experiment operator: Jiayin Sun, Yingxuan Min, Jingning Wang

Experimental objective

Building upon Cycle 1, palmitic acid and oleic acid were selected as the principal fatty acid components, maintaining a glycerol:fatty acid ratio of 1:1 for liquid culture of plasmid ①-transformed strains to assess plasmid ① preference for different fatty acid mixtures.

Experimental Equipment and Reagents

Equipment

Biosafety cabinet, autoclave, shaker, microplate reader, 96-well plate

Reagents

Sc-Trp, Arg, Adenine sulfate, ddH 2 O, Glycerol, Lauric acid, Oleic acid, Palmitic acid, Tween 80, Anhydrous ethanol

Reagent Preparation

(1) Add the following components into empty bottles;

(2) Add X g of solid stearic acid and Y g of lauric acid separately into two 1.5 mL centrifuge tubes. Add 150 μL of anhydrous ethanol to each centrifuge tube and dissolve in a 60°C water bath. Immediately transfer to the sterile cap of a clean 50 mL centrifuge tube. Dry the dissolved solid material in a fume hood until no liquid residue remains on the centrifuge tube surface and all anhydrous ethanol has completely evaporated.

(3) Transfer the dried palmitic acid and lauric acid separately into EP tubes, add 1.8 mL of water and 0.18 mL of Tween-80, then heat in a water bath with thorough stirring to dissolve.

(4) In another EP tube, add Z mL of oleic acid, M mL of glycerol, and 0.18 mL of Tween-80, then place it in a water bath for heating and thorough mixing.

(5) Preheat the solution obtained in step (1) with continuous stirring.

(6) Add palmitic acid, lauric acid, oleic acid, and glycerol sequentially to the container prepared in step (1). After thorough mixing, sterilize by autoclaving at 115°C for 15 minutes.

(7) Finally, add 20 mL of YNB solution to the container to complete the preparation. Transfer the dried palmitic acid to EP tubes and add 1.8 mL of water and 0.18 mL of Tween-80, respectively. Heat in a water bath and stir thoroughly.

Experimental procedure

(1) Irradiate the biosafety cabinet with UV light for 30 minutes prior to use;

(2) Transfer 15 mL of glycerol-fatty acid liquid culture medium into a 50 mL centrifuge tube;

(3) Inoculate single yeast clones from Saccharomyces cerevisiae plates transformed with plasmid ① into the centrifuge tube and incubate on a shaker at 30℃ and 200 rpm;

(4) Every 2 hours, transfer liquid cultures of monoclonal yeast carrying plasmid ① and observe the yeast growth by monitoring the turbidity of the liquid medium.

(5) Using one 96-well plate, collect 100 µL of culture from each medium every 2 hours and add it into the 96-well plate. Each group includes three biological replicates as controls. Measure OD600 values using a microplate reader and calculate the average.

Experimental Results

Results Analysis and Discussion

(1) Optical density (OD) values measured by microplate reader for yeast containing plasmid ① during the mid-logarithmic growth phase in fatty acid media were as follows: Glycerol: palmitic acid: oleic acid = 2:1:1 > Glycerol: palmitic acid: oleic acid: lauric acid = 3:1:1:1 > Glycerol: lauric acid: palmitic acid = 2:1:1 ≈ Glycerol: lauric acid: oleic acid = 2:1:1, indicating yeast selective preference for palmitic acid and oleic acid. Palmitic acid and oleic acid are the two most common fatty acids found in domestic wastewater; this engineered yeast strain may serve as a biosafety yeast capable of overcoming the high-fat environment of wastewater and facilitating the bioconversion of lipids in wastewater.

(2) In subsequent experiments, PCR and western blot methods will be used to further verify the normal expression of the protease encoded by plasmid ①.

Notes

(1) Always maintain the glycerol-to-fatty acid ratio at 1:1 to ensure the total lipid content remains the same, thereby enabling a better comparison of yeast preference for different carbon sources.

(2) Measure the growth OD value approximately every 2 hours to allow timely monitoring of yeast growth rate and extent.

Yeast culture following plasmid ① transfection; PCR; agarose gel electrophoresis

Experiment date: 2025.07.09

Experiment operator: Wang Quanhao, Min Yingxuan

Experimental objective

(1) Re-streak single clones for liquid yeast culture to prepare for genomic DNA extraction and PCR analysis.

(2) Extract two-thirds of the plasmid for use in PCR and plasmid transfection.

(3) Verify the integration of overexpressed enzyme genes Erg10, Erg13, and HMG1 into Saccharomyces cerevisiae and evaluate plasmid expression levels in engineered strains.

Experimental Equipment and Reagents

Equipment

Laminar flow cabinet, microplate reader, analytical balance, centrifuge, fume hood, shaker, ultrasonic homogenizer, PCR machine, shaker, electrophoresis tank, microwave oven, gel imaging system;

Reagents

LiAC/TE solution, sterile water; Buffer P1、Buffer P2、Buffer P3、Buffer DW1、Wash Solution、ElutionBuffer; Glass beads, chloroform, methanol; 2× TransFast Taq PCR SuperMix (+ dye), DNA template, specific primers, 1× TAE buffer, agarose, nucleic acid stain EB, DNA marker;

Reagent Preparation

Experimental Procedures

1. Yeast Culture

1.1 Irradiate the ultraclean workbench with UV light for 30 minutes prior to use.

1.2 Take a 50 mL centrifuge tube and add 15 mL of single dropout liquid medium.

1.3 Inoculate a single colony of Saccharomyces cerevisiae containing plasmid ① from the culture plate into the centrifuge tube, then incubate in a shaker at 30℃ and 200 rpm.

1.4 Every 2 hours, transfer liquid cultures of monoclonal yeast carrying plasmid ① and observe the yeast growth by monitoring the turbidity of the liquid medium.

1.5 Using one 96-well plate, collect 100 µL of culture from each medium every 2 hours and add it into the 96-well plate. Each group includes three biological replicates as controls. Measure OD600 values using a microplate reader and calculate the average.

2. Plasmid Extraction

2.1 Confirm whether RNase A has been added to Buffer P1.

2.2 Confirm whether anhydrous ethanol has been added to the Wash Solution.

2.3 Inspect Buffers P2 and P3 for the presence of precipitates.

2.4 Collect 1.5–5 mL of overnight yeast culture; centrifuge at 8,000×g for 2 minutes to pellet the cells, then discard the supernatant.

2.5 Add 250 μL of Buffer P1 to the pellet and thoroughly resuspend the cells.

2.6 Add 250 μL of Buffer P2, immediately invert the centrifuge tube gently 5–10 times to mix, then incubate at room temperature for 2–4 minutes.

2.7 Add 350 μL of Buffer P3, immediately invert the centrifuge tube gently 5–10 times to mix.

2.8 Centrifuge at 12,000×g for 10 minutes. Transfer the supernatant to the adsorption column and incubate for 3–4 minutes. Centrifuge at 8,000×g for 30 seconds, then discard the liquid from the collection tube.

2.9 Add 500 μL of Buffer DW1, centrifuge at 9,000×g for 30 seconds, then discard the liquid from the collection tube.

2.10 Add 500 μL of Wash Solution, centrifuge at 9,000×g for 30 seconds, then discard the liquid from the collection tube. Repeat step 2.10 once.

2.11 Centrifuge the empty adsorption column at 9,000×g for 1 minute.

2.12 Place the adsorption column into a clean 1.5 mL centrifuge tube, add 50–100 μL of Elution Buffer onto the center of the adsorption membrane, incubate at room temperature for 1 minute, then centrifuge for 1 minute. Collect and preserve the DNA solution in the tube.

3. Genomic DNA Extraction from Saccharomyces cerevisiae

3.1 Aliquot 200 μL of yeast culture, centrifuge at 4000 rpm for 30 s, and discard the supernatant;

3.2 Resuspend the cell pellet in 100 μL of LiOAc-1% SDS solution, then incubate at 70°C for 5 minutes;

3.3 Add 300 μL of anhydrous ethanol, mix thoroughly by repeated pipetting, centrifuge at 15000 rpm for 3 minutes, and discard the supernatant;

3.4 Wash the precipitate with 70% ethanol solution, centrifuge, and discard the supernatant;

3.5 Dissolve the precipitate in 100 μL of water and store at -20 ℃.

PCR and Agarose Gel Electrophoresis

4.1 Prepare the PCR reaction system according to Table 1;

4.2 Set the PCR reaction program according to Table 2 and perform PCR;

4.3 Prepare 2% agarose gel according to Table 3, dissolve by microwave heating;

4.4 Add 9 μL of EB to the agarose gel, mix well, pour into the casting tray with combs in place, lay flat in the refrigerator, and allow the gel to solidify;

4.5 Place the gel in the electrophoresis tank, add an appropriate amount of 1×TAE electrophoresis buffer. After loading samples, run electrophoresis at a constant voltage of 140 V for 25 min;

4.6 After electrophoresis, observe and photograph under UV light.

Experimental Results

Results Analysis and Discussion

All three target genes were detected with clear bands in the positive control group PC (plasmid ①), indicating that the three target genes were successfully cloned into the plasmid vector.

The three target genes all showed positive bands in the target strain, suggesting that the genes were successfully introduced into Saccharomyces cerevisiae.

The expression levels of the three target genes in the target yeast culture were significantly higher than those in the negative control group, indicating successful overexpression of the target genes in Saccharomyces cerevisiae.

Protein Extraction from Strains Transformed with Plasmid ①

Experiment Date: 2025.07.11

Experiment operator: Jingning Wang, Quanhao Wang, Yingxuan Min, Jiayin Sun

Experimental objective

(1) Protein extraction from samples to prepare for western blot analysis verifying proper plasmid expression

Experimental Equipment and Reagents

Equipment

Biosafety cabinet, centrifuge, water bath, gel casting plates, electrophoresis tank, vertical electrophoresis apparatus, transfer apparatus, destaining shaker, chemiluminescence imaging system;

Reagents

NaOH, β-glycerophosphate sodium salt, sodium orthovanadate, NaF, EDTA, PMSF Reagent Preparation

Experimental Procedure

Sample Preparation

1.1 Prepare yeast lysis buffer as detailed in the table below: using NaOH as the base, add the specified reagents per 1 mL of NaOH; protease and phosphatase inhibitors may be added as required by the experiment (optional);

1.2 Take 200 μL of yeast culture, centrifuge (4000 rpm, 30 s) and discard the supernatant;

1.3 Add lysis buffer to the collected cell pellet and lyse on ice for 10 minutes;

1.4 Centrifuge (5000 rpm, 1 min) and discard the supernatant;

Add 80 μL of 1× loading buffer to the 1.5 mL precipitate and mix thoroughly; incubate in a metal bath at 100°C for 5 minutes, then store at -20°C for subsequent use

Experimental Results

Western Blot Detection of Protein Expression Success

Experiment Date: 2025.07.13

Experiment operator: Wang Jingning, Wang Quanhao, Min Yingxuan, Sun Jiayin

Experiment Objective

(1) To verify the successful introduction of ERG10, ERG13, and HMG1 enzyme genes into Saccharomyces cerevisiae

Experimental Equipment and Reagents

Equipment

Laminar flow clean bench, centrifuge, water bath, gel casting plates, electrophoresis tank, vertical electrophoresis system, transfer electrophoresis system, decolorization shaker, chemiluminescence imaging system

Reagents

Tris, glycine, SDS, methanol, polyacrylamide, APS, TEMED, 1.5M Tris-HCl (pH 8.8), 1.0 M Tris-HCl (pH 6.8), KCl, NaCl, Tween-20, ddH₂O

Reagents

Experimental Procedure

Protein Loading and Gel Electrophoresis

1.1 Prepare 5% stacking gel and 12% resolving gel according to Tables 4 and 5. The resolving gel is incubated at room temperature for 20-30 min, and the stacking gel is incubated at room temperature for approximately 5-10 min. Insert the gels into the electrophoresis apparatus and fill the tank with electrophoresis buffer. During sample loading, add 10μL of protein sample into each well, and add 4 μL of marker into one well. Handle samples gently to avoid bubble formation.

1.2 Electrophoresis: Electrophoresis conditions: stacking gel at 80 V for 30 min ; Lower separation gel 120 V, 60-80 min;

1.3 Transfer: Upon completion of electrophoresis, remove the gel plates, carefully detach the gel, and cut the PVDF membrane and filter paper according to the gel dimensions; Prior to transfer, the PVDF membrane must be activated in methanol for approximately 20-30s. Conduct the transfer in an ice-water mixture at a constant current of 200 mA for 2h;

1.4 Milk Blocking and Hybridization: Remove the PVDF membrane and wash with 1×TBST on a horizontal shaker 3 times, 10min each. Block with 5% skim milk at room temperature on a horizontal shaker for 2h.

1.5 After blocking for 2h, wash with TBST three times, 5min each, then incubate with the appropriately diluted primary antibody (dilution ratio according to the specific antibody datasheet, diluted in TBST). After adding the primary antibody, place at 4℃ overnight. The next day, wash three times with TBST on a shaker, 10min each, then incubate with the secondary antibody for 1h. Finally, wash again with TBST three times, 10min each.

1.6 ECL Detection: According to Table 7 and the Thermo Fisher ECL instructions, mix solution A and solution B at a 1:1 volume ratio. After mixing, incubate in the dark at room temperature for 1-2min. Capture images using a gel imaging system and analyze the band gray values with its image processing software, followed by statistical analysis.

Experimental Results

Sapogenin - Construction of oleanolic acid

Cultivation and Cryopreservation of Saccharomyces cerevisiae

Experiment Date: 2025.07.21

Experiment operator: Zhao Xiaoqing, Sun Jiayin

Objectives

1. To cultivate and cryopreserve Saccharomyces cerevisiae for subsequent preparation of competent cells for plasmid transfection.

2. To formulate the plasmid into a solution for convenient transfection.

Experimental Equipment and Reagents

Equipment

Shaker, refrigerator, laminar flow hood, spectrophotometer

Reagents

YPD medium, plasmid powder, sterile water, glycerol

Reagent Preparation

Experimental Procedure

Cultivation of Saccharomyces cerevisiae

(1) Irradiate the ultra-clean workbench with ultraviolet light for 30 minutes beforehand. Prepare YPD medium and microwave-treat it in advance for later use;

(2) Take a 50 mL centrifuge tube and pour 5 mL of YPD liquid medium into it;

(3) Pick a single colony of Saccharomyces cerevisiae W303 from the culture plate and inoculate into the tube. Incubate on a shaking incubator (30°C, 200 rpm);

(4) After approximately 7hours, measure the OD600. If OD600 = 0.3, add 2 mL of the culture to 100 mL of YPD medium.

Prepare the plasmid

(1) Retrieve the plasmid powder tube and centrifuge (4μg);

(2) Add 30μL sterile water to the tube;

(3) Transfer 10μL to an EP tube, then add 32μL sterile water for later use;

(4) Store the remaining 20μL plasmid at -20°C.

Freeze-preserve experimental strains

Take an EP tube, add 750μL yeastl suspension and 750μL glycerol (50%) in a 1:1 ratio, mix, and store at -80°C. Replenish the remaining yeastl suspension in the centrifuge tube with appropriate medium.

Experimental Results

Analysis and Discussion

Yeast exhibited robust growth and is suitable for preparing competent cells for subsequent experiments.

Precautions

(1) Maintain aseptic technique when isolating single-colony yeast to prevent contamination by extraneous microorganisms.

(2) When preparing YPD medium, avoid excessive temperatures during autoclaving. Typically, autoclave at 115°C for 15 minutes; higher temperatures may cause glucose to darken.

(3) Ensure culture duration does not exceed recommended limits. If optical density (OD) exceeds 1.2, the strain is unsuitable for subsequent procedures.

Preparation of Sc-Trp Medium and Competent Cells

Experiment Date: 2025.07.23

Experiment operator: Zhao Xiaoqing, Sun Jiayin

Objectives

1. Prepare Sc-Trp medium and plate it to facilitate subsequent cultivation of Saccharomyces cerevisiae following plasmid transfection.

2. Prepare competent cells for plasmid transfection.

Experimental Equipment and Reagents

Equipment

Laminar flow hood, spectrophotometer, autoclave, shaking incubator

Reagents

SC-Trp, arginine, adenine, glucose, YNB, ammonium sulphate, agar, sterile water, LiAC/TE buffer

Reagent Preparation

Experimental Procedure

Preparation of SC-Trp Medium

(1) Take 0.4 g SC-Trp, 0.04 g arginine, 0.2 g adenine, and 4 g glucose; add to 60 mL water and place in an empty bottle;

(2) Take 4 g agar and add to 120 mL water; place in another empty bottle and add a rotor;

(3) Place both bottles alongside an empty 100 mL bottle into an autoclave for sterilisation (115°C, 15 minutes);

(4) Weigh 1.6 g of YNB yeast and 5 g of ammonium sulphate into a beaker. Add 100 mL of sterilised water and stir with a stirrer rod until completely dissolved;

(5) Sterilise the solution by filtration through a 0.22μm filter, then transfer to a 100mL sterile autoclave bottle for later use;

(6) Take 20mL of the sterilised YNB solution and add to the sterilised agar bottles. While hot, pour the SC-Trp solution into the agar bottles containing YNB and mix thoroughly using a magnetic stirrer;

(7) Swiftly pour the mixture into sterile Petri dishes to form plates. Allow to stand at room temperature for 4 hours before refrigerating for later use.

Preparation of Competent Cells

(1) Place sterile water in a 4°C refrigerator for later use. Dilute LiAC/TE to 0.1 mol/L for later use;

(2) Measure the OD600 of the Saccharomyces cerevisiae culture from the previous day. Proceed to the next step if OD600 = 0.8;

(3) Take 50 mL of the culture, centrifuge (3500 rpm, 2 min), and discard the supernatant;

(4) Resuspend in 50 mL of cold sterile water, centrifuge (3500 rpm, 2 min), and discard the supernatant;

(5) Resuspend in 1 mL 0.1 mol/L LiAC/TE buffer, centrifuge briefly (4000 rpm, 30 s), and discard supernatant;

(6) Resuspend in 400 μL 0.1 mol/L LiAC/TE buffer, mix thoroughly, and refrigerate overnight on ice.

Precautions

(1) After filtration and sterilisation, the YNB solution must be rapidly poured into agar while hot to prevent uneven distribution causing abnormal solidification of the medium.

(2) Once prepared, the medium must be promptly poured into plates to prevent premature solidification due to prolonged exposure.

(3) Monitor the OD value of Saccharomyces cerevisiae to prevent excessive incubation time, which may render the sample unsuitable for subsequent experiments.

Transfection of Saccharomyces cerevisiae Plasmid ②

Experiment Date: 2025.07.25

Experiment operator: Zhao Xiaoqing, Sun Jiayin

Objectives

To transfect Saccharomyces cerevisiae competent cells with a plasmid for synthesising the intermediate compound oleanolic acid (OA).

Experimental Equipment and Reagents

Equipment

Laminar flow hood, centrifuge, 30°C incubator, water bath, shaking incubator, drying oven.

Reagents

PEG, LiAc (1 mol/L), salmon sperm, plasmid, yeast competent cells, YPD medium

Reagent Preparation

Experimental Procedure

Plasmid Transfection

(1) Add approximately 50 μL of the yeast competent cells prepared the previous day to each Eppendorf tube. Centrifuge (4000 rpm, 30 seconds) and discard the supernatant.

(2) Add sequentially to the EP tube: 42 μL plasmid, 10 μL salmon sperm extract (10 mg/mL), 240 μL PEG, and 36 μL LiAc (1 mol/L). Mix thoroughly by pipetting up and down with a 1 mL pipette, then incubate at 30°C for 30 minutes;

(3) Add 36 μL DMSO to each EP tube. Mix thoroughly by pipetting and heat-shock at 42°C for 20 minutes.

Post-transfection yeast culture

(1) Remove EP tubes from water bath. Centrifuge (3000 rpm, 20 seconds) and discard supernatant. Resuspend in 1 mL YPD medium and incubate on shaking incubator for 3 hours.

(2) Centrifuge (3000 rpm, 20 s), discard supernatant, resuspend in 100 μL water, spread culture onto plates (Sc-Trp) using glass beads, and incubate at 30°C for approximately 3 days.

Precautions

(1) Salmon sperm must be pre-treated by boiling for 5 minutes, then rapidly immersed in an ice-water bath to preserve exogenous plasmids.

(2) All reagents required for plasmid transfection must be thoroughly mixed to maximise cell membrane permeability and enhance transfection efficiency.

(3) Incubation times in the incubator and heat shock duration in the water bath must be strictly controlled to prevent reduced transfection efficiency due to prolonged exposure.

Yeast Monoclonal Culture Following Plasmid Transfection

Experiment Date: 2025.07.27

Experiment operator: Zhao Xiaoqing, Sun Jiayin

Objectives

(1) To transfer successful plasmid-transfected yeast clones, selected under nutritional conditions, from plates to YPD medium and incubate on a shaking incubator;

(2) To prepare YPD and LB media for subsequent use.

Experimental Equipment and Reagents

Equipment

Laminar flow hood, autoclave, shaking incubator, centrifuge.

Reagents

Peptone, yeast extract, sodium chloride, glucose, sterile water.

Reagent Preparation

Experimental Procedure

Monoclonal Culture of Transfected Yeast

(1) Remove the transfection-treated yeast culture plate from the incubator and observe yeast growth;

(2) Prepare six 50mL centrifuge tubes, each containing 10mL YPD medium;

(3) Pick individual colonies from the transfection plate and transfer them to the tubes. Incubate on a shaking incubator for 24 hours.

Medium Preparation

Prepare LB and YPD liquid media according to recipes. Sterilise in an autoclave (115°C, 15 min).

Experimental Results

As illustrated, plasmid-transfected Saccharomyces cerevisiae exhibited robust growth.

Results Analysis and Discussion

As shown, high yeast growth density complicated single-colony isolation.

Solution: Reduce the amount of yeast competent cells in the transfection system or shorten the incubation time.

Precautions

(1) When transferring from culture plates to centrifuge tubes, single colonies must be picked to obtain strains with stable genetic backgrounds.

(2) When adding medium to centrifuge tubes, to avoid contamination from residual medium, transfer a portion to a 50 mL centrifuge tube for later use.

Growth Curve Measurement of Yeast Single Clones Following Plasmid Transfection

Experiment Date: 2025.07.28

Experiment operator: Wang Jingning, Wang Quanhao

Objective

Every 12 hours, yeastl suspensions from two single clone colonies of single plasmid yeast 3, which exhibited higher OA production after one day of culture, were transferred into a 96-well plate containing Trp-deficient liquid medium. Optical density (OD600) measurements were taken to indirectly assess strain growth. Monitoring was continued for 96 hours, culminating in the plotting of the growth curve of the single clone colonies.

Experimental Equipment and Reagents

Equipment

Class II biosafety cabinet, autoclave, shaker, microplate reader, 96-well plate

Experimental Procedure

1、OA High-Yield Yeast Growth Curve Measurement

(1) At 10:00 AM and 10:00 PM, respectively, remove two 3 mL centrifuge tubes containing single clone yeast harboring a single plasmid from the shaker after one day of liquid culture. Observe yeast growth by checking for turbidity in the liquid medium.

(2) Take one 96-well plate and add 100 µL of yeast culture to each well. Establish the Trp-deficient medium as the control group. Both types of yeast cultures, as well as the single-deficient medium, are set up with three replicates each. The loading order is shown in the following figure. Measure OD₆₀₀ using a microplate reader and calculate the average values.

Experimental results

Results analysis and discussion

(1) Compared to the Trp-deficient medium group, the centrifuge tubes were visibly turbid after shaker incubation, and the OD values measured by the microplate reader were higher than those of the control group, indicating substantial growth of strain ③;

(2) Comparison of OD values for strains incubated at 10:00 AM and 10:00 PM showed no significant differences. The strains exhibited slow growth in Trp-deficient medium; subsequent data collection may occur every 24 hours;

(3) At the early cultivation stage, the average OD value of strain 3② was higher than that of strain 3①; continuous observation will determine whether strain 3② is the strain with better growth.

(4) Overall, no significant growth was observed in the strains, possibly due to the omission of essential nutrients during culture medium preparation, which requires timely correction.

Precautions

(1) Establish a control group using Trp-deficient medium to better reflect the growth status of the yeast culture.

(2) Measure the growth OD value approximately every 12 hours to enable timely monitoring of the yeast growth rate and extent.

Measurement of yeast single-clone growth curves following Plasmid ② transfection

Experiment date: 2025.08.01

Experimental personnel: Wang Jingning, Wang Quanhao

Objective

Every 2 hours, 100 μL of culture was taken from three single-clone colonies transfected with Plasmid ②, cultivated in Trp-deficient liquid medium, transferred into a 96-well plate, and optical density (OD600) was measured to indirectly assess strain growth. Continuous monitoring was conducted over 96 hours, and the growth curve of each single-clone colony was subsequently plotted.

Experimental Equipment and Reagents

Equipment

Biosafety cabinet, autoclave, shaker, microplate reader, 96-well plate

Experimental Procedure

1. Irradiate the biosafety cabinet with ultraviolet light for 30 minutes in advance.

2. Add 15 mL of glycerol fatty acid liquid medium to a 50 mL centrifuge tube.

3. Pick a single yeast clone from the culture plate transfected with Plasmid ② and add it to the centrifuge tube, then incubate on a shaker at 30°C and 200 rpm.

4. Observe yeast growth by assessing the turbidity of the liquid medium.

5. Prepare one 96-well plate and add 100 μL of yeastl suspension to each well. Establish a Trp-deficient media control group, as well as three replicates each for three single-clone yeastl suspensions and single-deficient media controls. Measure the OD₆₀₀ values using a microplate reader and calculate the averages.

Experimental Results

Results Analysis and Discussion

(1) The Trp-deficient media group exhibited visibly turbid centrifuge tubes following shaking incubation. The OD600 values measured by the microplate reader for the three single-clone colonies were elevated compared to the control group, indicating substantial growth of yeast transfected with Plasmid ②;

(2) Continuous monitoring of yeast growth showed that yeast reached the stationary phase at approximately 28 h.

(3) Compared to the previous growth curve, significant strain growth was observed after re-preparing the liquid media, confirming that the prior media preparation lacked essential nutrients.

Precautions

(1) Include a Trp-deficient media control group to better assess the extent of yeast growth.

(2) Measure OD600 values approximately every 2 hours to enable timely monitoring of yeast growth rate and extent.

Glycoflavin streaking; PCR and agarose gel electrophoresis Verify the successful transfection of plasmid ② enzyme gene

Experiment time: 2025.08.03

Experiment operator: Sun Jiayin, Wang Quanhao, Min Yingxuan

Experimental Objective

(1) Monoclonal cells were prepared by glycohydrolase streaking.

(2) To determine whether the genes of MtCPR, CYP716A12 and β-AS were transferred into yeast, and to detect the expression of plasmids in yeast 1, 2 and 3.

Experimental equipment and reagents

Equipment

Clean workbench, centrifuge, micropipette and gun head, PCR instrument, electrophoresis instrument, gel imaging system

reagent

Plasmid 2, glycerol-free freeze-dried culture medium, Amoxicillin LB solid agar plates, 2× TransFast Taq PCR SuperMix (with dye), DNA templates, specific primers, TAE buffer, agarose, EB DNA stain, DNA markers

Reagent preparation

3.1 PCR reaction system

3.2 PCR reaction procedure

3.3 1% agarose gel

Experimental Procedure

Glycojugate streaking

(1) The ultra-clean workbench was sterilized with ultraviolet light 30 minutes in advance, and the experimental table was wiped with 75% alcohol;

(2) Mark the name of the strain, date, operator and other information on the bottom of the tablet with a marker pen;

(3) The bamboo stick is burned in the outer flame of the alcohol lamp, cooled, and then dipped directly into a small amount of yeastl liquid (visible drops can be seen);

(4) The bamboo stick dipped in the yeastl solution is gently drawn in a zigzag pattern on one side of the plate (about 1/4 area, marked as A area). The line should be dense but not overlapping. The bamboo stick should be drawn at an angle of about 30°-45°. After drawing the line, discard the bamboo stick;

(5) The second marking step (Zone B) involves passing cooled bamboo sticks 1-2 times from the end of Zone A, extending to Zone B (approximately half of the area) to create sparse zigzag patterns. The third marking step (Zones C/D) repeats the process, tracing from the end of Zone B to Zone C (the remaining area) with even finer lines, ultimately forming a gradient dilution where Zone A contains concentrated yeast and Zone D has diluted yeastl content.

Yeast genome extraction

(1) Take 200μL yeast culture solution, centrifuge (4000rpm, 30s) and discard the supernatant;

(2) Resuspend the cell precipitate in 100μL LiOAc-1%SDS solution, and incubate in 70℃ oven for 5min;

(3) Add 300μL anhydrous ethanol, repeatedly blow and mix, centrifuge (15000rpm, 3min) and discard the supernatant;

(4) Wash the precipitate with 70% ethanol solution, and discard the supernatant after centrifugation;

(5) The precipitate was dissolved in 100μL water and stored in a-20℃ refrigerator.

PCR

(1) The PCR reaction system was prepared according to 3.1 in "I";

(2) Set the PCR reaction program according to 3.2 in "I" and carry out PCR;

Agarose gel electrophoresis

(1) Prepare 2% agarose gel by microwave heating according to 3.3 in "I";

(2) Add 9 μ L EB and shake well, then pour it into the mixing tank where the comb has been placed. Place it flat in the refrigerator and wait for the gel to solidify;

(3) Put the gel into the electrophoresis tank and pour in an appropriate amount of 1× TAE electrophoresis buffer. After sampling, electrophoresis for 25min under constant voltage of 140V;

(4) After electrophoresis, observe and take photos under ultraviolet lamp;

Experimental Results

Results analysis and discussion

1. The three target genes were not detected in the negative control group NC, indicating that the primer amplification was specific;

2. The three target genes were detected in the positive control group AC (plasmid 2) with clear bands and normal molecular weight, indicating that the three target genes were successfully cloned into the plasmid vector;

3. MtCPR

The MtCPR gene exhibited distinct bands in yeastl strains 2 and 3 during this experiment, consistent with the results from Experiment 7.22, indicating successful gene transfection in these two strains. Although the banding was not prominent in strain 1, the 7.22 experiment revealed clear bands for this strain, confirming successful gene transfection. The experimental results may be attributed to either an increase in amplified fragment length or insufficient sample loading during electrophoresis.

4. CYP716A12

The CYP716A12 gene was detected as weakly positive in bands 1 and 2 of yeastl cultures, consistent with the results from Experiment 7.22, indicating successful transfection of the gene. A distinct band at 100bp was observed, likely caused by primer dimer formation. The absence of bands in yeastl culture 3 and the faint banding in Experiment 7.22 suggest low transfection efficiency in culture 3, which may be attributed to amplified fragments being too long for effective PCR amplification.

5. β-AS

In this experiment, the β-AS gene showed no detectable bands in yeastl strains 1, 2, and 3, all presenting negative results. The 7.22 experimental result indicated weak positive for strain 1, while both strains 2 and 3 remained negative. This suggests successful but low-efficiency transfection of the gene in strain 1, with the negative outcomes likely attributed to reduced PCR efficiency due to extended amplification fragments. Both strains 2 and 3 demonstrated negative results in subsequent experiments, indicating unsuccessful transfection of the gene in these two yeastl colonies.

matters need attention

1. The tail of the bamboo stick is picked out with tweezers and then roasted.

2. Start the line from the end of the last line. The lines should be dense but not overlapping.

3. The PCR instrument should stop working first, then open the lid, and finally shut down.

4. Set the electrophoresis instrument to constant voltage.

Plasmid extraction; Plasmid transfection; Preparation of YPD and LB media; PCR and agarose gel electrophoresis

Experiment time: 2025.08.05

Experiment operator: Sun Jiayin, Wang Quanhao, Min Yingxuan

Objective

(1) Plasmids were extracted from overnight cultured Ganoderma lucidum to prepare for subsequent plasmid transfection and PCR;

(2) Saccharomyces cerevisiae was transfected with plasmid 3 to synthesize the final product ginsenoside Ro;

(3) Prepare YPD medium and Trp-Leu double deficiency solid medium for standby;

(4) To determine whether the genes of three enzymes, MtCPR, CYP716A12 and β-AS were transferred into Saccharomyces cerevisiae and to detect the expression of plasmids in yeast 1, 2 and 3

Experimental equipment and reagents

Equipment

laminar flow hood, shaker, 30℃ incubator, water bath, shaker, oven, electronic balance, magnetic stirrer, autoclave, PCR instrument, electrophoresis system, gel imaging system, adsorption column

reagent

Buffer P1、Buffer P2、Buffer P3、Buffer DW1、Wash Solution、ElutionBuffer; PE, LiAc (1mol/L), salmon sperm, plasmid, yeast receptive state, YPD medium; Glucose, peptone, arginine, adenine, SC-Trp-Leu; 2×TransFast Taq PCR SuperMix (+dye), DNA template, specific primers, 1×TAE buffer, agarose, DNA dye EB, DNA Marker;

Reagent configuration

3.1 YPD medium (50 0mL)

3.2 Trp-Leu double deficient liquid medium (180 mL)

3.3 YNB solution

3.4 PCR reaction system

3.5 PCR reaction procedure

3.6 1% agarose gel

Experimental Procedure

Viral transfection

1.1 Salmon sperm was boiled in boiling water for 5min and then rapidly placed in an ice water mixture (on ice)

1.2 Add about 50μL of yeast receptive state prepared the previous day to each EP tube, and centrifuge (4000rpm, 30s) and discard the supernatant;

1.3 Add 42μL plasmid, 10μL croaker sperm (10 mg/mL), 240μL PEG, and 36μL LiAc (1 mol/L) sequentially into the EP tube. After repeatedly pipetting to mix thoroughly with a 1mL pipette, incubate the mixture in a 30℃ incubator for 30 minutes.;

1.4 Add 36μL DMSO to each EP tube, mix by repeated blowing and then put it into a 42℃ water bath for 20min.

Yeast culture after transfection

2.1 The EP tube was taken out of the water bath, and the supernatant was discarded after centrifugation (3000rpm, 20s). The suspension was resuspended in 1 mL LYPD medium and cultured for 3h in a shaker.

2.2 After centrifugation (3000rpm, 20s), the supernatant was discarded, and the yeastl suspension was resuspended with 100μL water. The yeastl suspension was spread on the plate (Sc-Trp) with glass beads, and cultured in a 30℃ incubator for about 3 days.

Protein extraction

3.1 Check whether RNase A has been added to buffer P1.

3.2 Check whether anhydrous ethanol has been added to the Wash Solution.

3.3 Check for precipitation of buffer P2 and P3.

3.4 Take 1.5-5mL of the yeastl liquid that has been cultured overnight, and collect the yeastl body by centrifugation at 8,000×g for 2 minutes, and discard all the culture medium.

3.5 Add 250 μL BufferP1 to the precipitate and resuspend the yeast thoroughly.

3.6 Add 250 μL Buffer P2 and gently invert the centrifuge tube 5-10 times immediately to mix. Let it stand at room temperature for 2-4 minutes.

3.7 Add 350μL Buffer P3 and gently invert the centrifuge tube 5-10 times immediately to mix.

3.8 Centrifuge at 12,000×g for 10 minutes. Transfer the supernatant to the adsorption column and let it stand for 3-4 minutes. Centrifuge at 8,000×g for 30 seconds, then discard the liquid from the collection tube.

3.9 Add 500μ L Buffer DW1 and centrifuge at 9000×g for 30 seconds. Discard the liquid in the collection tube.

3.10 Add 500 μ L Wash Solution and centrifuge at 9000×g for 30 seconds. Pour off the liquid in the collection tube.

3.11 Repeat 3.10 once.

3.12 The air adsorption column was centrifuged at 9000×g for 1 min.

3.13 The adsorption column was placed in a clean 1.5mL centrifuge tube, and 50-100 μL ElutionBuffer was added to the center of the adsorption membrane. After 1 min at room temperature, the centrifuge was spun for 1 min. The DNA solution was stored in the tube.

Prepare YPD medium

4.1 Follow 3.1 of Part II.Prepare the solution;

4.2 Place the glass bottle with the solution into the autoclave and sterilize (115℃,15min);

Preparation of Trp-Leu double deficient liquid medium

5.1 Follow 3 of section 22. Prepare a double deficiency solution of Trp-Leu according to 3.3. Prepare YNB solution;

5.2 Place the glass bottle with the solution into the autoclave and sterilize (115℃,15min);

5.3 After filtering and sterilizing the YNB solution through a 0.22μm filter, it was transferred to a 100mL empty high-pressure sterilized bottle for standby;

5.4 Take 20 m L of filtered YNB solution and add it to the Trp-Leu double deficiency solution and mix thoroughly;

PCR and agarose gel electrophoresis

6.1 According to 3.4. Configuration of PCR reaction system;

6.2 According to 3.5. Set the PCR reaction program and carry out PCR;

6.3 According to 3.6. Prepare 1% agarose gel by microwave heating and dissolution;

6.4 Add 9 μ L EB and shake well, then pour it into the mixing tank where the comb has been placed. Place it flat in the refrigerator and wait for the gel to solidify;

6.5 Put the gel into the electrophoresis tank and pour in an appropriate amount of 1× TAE electrophoresis buffer. After sampling, electrophoresis for 15min under a constant voltage of 1 50 V;

6.6 After electrophoresis, observe and take photos under ultraviolet lamp;

Experimental Result

Results analysis and discussion

1. The three target genes were clearly band in yeast 1, yeast 2 and yeast 3, and all were strongly positive, indicating that the three target genes were successfully transferred into yeast

2. The band of the NC negative control was caused by insufficient solidification time of the agarose gel, resulting in soft gel, proper adhesion of the sample well, and sample leakage during sampling (the negative control samples used in the first two experiments did not show the band, and the negative control samples used in this time were the same as those in the first two experiments);

3. A distinct band is present at 100bp, which is caused by the formation of dimer of primer itself;

4. The electrophoresis time was short, so the band differentiation was slightly not obvious.

matters need attention

1. Salmon sperm need to be boiled in boiling water for 5min in advance, and then quickly put into ice water mixture to play the role of protecting exogenous plasmids

2. All kinds of reagents required for particle transfection should be mixed evenly to maximize cell membrane permeability and improve transfection efficiency.

3. The incubation time of the titer transfection incubator and the heat shock time of the water bath must be strictly controlled to prevent the decrease of transfection efficiency after too long time.

4. In electrophoresis, if the length of DNA is short, the voltage can be adjusted to control the electrophoresis speed.

Yeast culture after plasmid ②transfection; PCR; agarose gel electrophoresis

Experiment time: 2025.08.09

Experiment operator: Wang Quanhao, Min Yingxuan

experimental objective

(1) Yeast genome extraction and PCR experiment preparation;

(2) 2/3 of the extracted plasmid was used for PCR and plasmid transfection;

(3) To determine whether the genes of three enzymes expressed MtCPR, CYP716A12 and β-AS were introduced into Saccharomyces cerevisiae, and to detect the expression of plasmids in the engineered yeast;

Experimental Equipment and Reagents

Equipment

Clean workbench, enzyme label instrument, electronic balance, centrifuge, fume hood, oscillator, ultrasonic crusher, PCR instrument, oscillator, electrophoresis tank, microwave oven, gel imaging system;

Reagent

LiAC/TE solution, sterile water; Buffer P1、Buffer P2、Buffer P3、Buffer DW1、Wash Solution、ElutionBuffer; Glass beads, chloroform, methanol; 2×TransFast Taq PCR SuperMix (+dye), DNA template, specific primers, 1×TAE buffer, agarose, nucleic acid dye EB, DNA Marker;

Reagent preparation

Experimental Procedure

Plasmid extraction

1.1 Check whether RNase A has been added to Buffer P1.

1.2 Check whether anhydrous ethanol has been added to the Wash Solution.

1.3 Check whether buffer P2 and P3 are precipitated.

1.4 Take 1.5-5 mL of the yeastl culture overnight and centrifuge at 8,000×g for 2 minutes to collect the yeast and discard all the culture medium.

1.5 Add 250 μL Buffer P1 to the precipitate and completely suspend the yeast.

1.6 Add 250 μL Buffer P2 and gently invert the centrifuge tube 5-10 times immediately to mix. Let it stand at room temperature for 2-4 min.

1.7 Add 350 μL Buffer P3 and gently invert the centrifuge tube 5-10 times immediately to mix.

1.8 Centrifuge at 12,000×g for 10 minutes. Transfer the supernatant to the adsorption column and let it stand for 3-4 minutes. Centrifuge at 8,000×g for 30 seconds, then discard the liquid from the collection tube.

1.9 Add 500 μL Buffer DW1 and centrifuge at 9000×g for 30 seconds. Pour off the liquid in the collection tube.

1.10 Add 500 μL Wash Solution and centrifuge at 9000 x g for 30 seconds. Pour off the liquid in the collection tube. Repeat 2.10 once.

1.11 The empty adsorption column was centrifuged at 9000 ×g for 1 min.

1.12 Place the adsorption column into a clean 1.5mL centrifuge tube, add 50-100 μL Elution Buffer in the center of the adsorption membrane, and centrifuge for 1 min after room temperature standing for 1 min. Save the DNA solution in the tube.

Extraction of genomic DNA from Saccharomyces cerevisiae

2.1 Take 200μL yeast culture solution and centrifuge (4000rpm, 30s) to discard the supernatant;

2.2 Resuspend the cell precipitate in 100μL LiOAc-1%SDS solution, and incubate it in a 70℃ oven for 5min;

2.3 Add 300μL anhydrous ethanol, repeatedly blow and mix, centrifuge (15000rpm, 3min) and discard the supernatant;

2.4 Wash the precipitate with 70% ethanol solution, and discard the supernatant after centrifugation;

2.5 The precipitate was dissolved in 100μL water and stored in a-20℃ refrigerator.

PCR and agarose gel electrophoresis

3.1 Prepare PCR reaction system according to Table 1;

3.2 Set the PCR reaction program according to Table 2 and carry out PCR;

3.3 Prepare 2% agarose gel according to Table 3 and microwave heat dissolve;

3.4 Add 9 μL EB to the agarose gel and shake it well. Then pour it into the mixing tank where the comb has been placed. Place it flat in the refrigerator and wait for the gel to solidify;

3.5 Put the gel into the electrophoresis tank and pour in an appropriate amount of 1×TAE electrophoresis buffer. After sampling, electrophoresis at 1 40V constant voltage for 25min;

3.6 After electrophoresis, observe and take photos under ultraviolet lamp;

Experimental Results

Results Analysis and Discussion

1. The three target genes were detected in the positive control group PC (plasmid ①) with clear bands, indicating that the three target genes were successfully cloned into the plasmid vector

2. The positive bands of the three target genes in the target strain suggested that the three target genes were successfully transferred into yeast.

3. The expression of the three target genes in the target yeastl solution was significantly higher than that in the negative control group, indicating that the target genes were successfully overexpressed in Saccharomyces cerevisiae.

Protein extraction of yeast strain samples transferred to plasmid ② and SDS-PAGE gel electrophoresis; carboloyd staining

Experiment time: 2025.08.12

Experiment operator: Wang Jingning, Wang Quanhao, Min Yingxuan, Sun Jiayin

Experimental Objective

(1) Sample protein extraction was performed by SDS-PAGE gel electrophoresis; Coomassie Brilliant Blue staining was used to prepare for normal plasmid expression

(2) Determine whether the genes of three enzymes, MtCPR, CYP716A12 and β-AS, were transferred into Saccharomyces cerevisiae

Experimental Equipment and Reagents

Equipment

Ultra-clean workbench, centrifuge, water bath, gel plate, electrophoresis tank, vertical electrophoresis instrument, transfer electrophoresis instrument, decolorization shaker, chemiluminescence imaging system;

Reagent

NaOH, β-glycerophosphate sodium, sodium orthovanadate, NaF, EDTA, PMSF, Tris, glycine, SDS, Coomassie Brilliant Blue R-250, methanol, glacial acetic acid, 30% polyacrylamide, 1.0 MTris-HCl (pH 6 .8), 1.5MTris-HCl (pH8.8), 10%APS, TEMED

Reagent preparation

Experimental Procedure

sample preparation

1.1 Yeast lysate is prepared according to Table 1: with NaOH as the benchmark, each reagent in the table below is added to 1mL NaOH, and protease inhibitors and phosphatase inhibitors (non-essential) can be added according to specific experimental requirements;

1.2 Take 200μL yeast culture solution, centrifuge (4000rpm, 30s) and discard the supernatant;

1.3 Add the lysis solution to the obtained yeastl precipitate and lyse on ice for 10min;

1.4 Centrifuge (5000rpm, 1min), discard the supernatant;

1.5 Add 80μL 1× loading buffer to the precipitate and mix well. After metal bath (100℃,5min), store in-20℃ refrigerator for standby

Protein blotting and SDS-PAGE gel electrophoresis

2.1 Configure the SDS-PAGE gel and electrophoresis according to the method in 4.2;

2.2 Before protein loading, the sample was bathed in hot water for 5min and centrifuged at 5000rpm for 3min. The supernatant and precipitate were respectively loaded for electrophoresis. After the gel electrophoresis, remove the gel plate, carefully peel off the gel from the glass plate, and put it into the staining box/tray; rinse the surface of the gel with deionized water 1 to 2 times to remove the residual electrophoresis buffer (gentle operation to avoid tearing the gel).

2.3 Fixation (removes SDS and protein leakage, improves staining signal-to-noise ratio): Add sufficient fixative solution (methanol/acetic acid/water, typically 45% methanol + 10% acetic acid + 45% water) to the gel-containing container. Shake gently at room temperature for 30 minutes. The fixation process can be extended to 1 hour or overnight at 4℃°C to enhance staining quality.

Carma blue staining and decolorization

3.1 Discard the fixed liquid and add the newly prepared Comass blue staining solution to completely immerse the gel. Stain for 1-2 h at room temperature on a horizontal shaker; if more sensitive, stain overnight (do not exceed 24 h to avoid increased background).

3.2 After the dyeing, discard the dye and use the decolorizing solution (40% methanol, 10% acetic acid, 50% water) for the first decolorization. Shake gently at room temperature for 30 min to 1 h until the background begins to fade obviously.

3.3 Replace the decolorizing solution and continue decolorization on a horizontal shaker. Adjust the decolorizing solution based on background coloration and band development, repeating this process every 30-60 minutes until the background becomes sufficiently pale and band contrast is clear. Complete decolorization may take several hours to a couple of days (allow overnight exposure for continued decolorization the next day). For faster results, perform short decolorization cycles, capture images immediately, then proceed with detailed decolorization.

Gel imaging analysis

4.1 After decoloring to the desired extent, rapidly rinse the gel with deionized water to remove residual methanol/acetic acid. Place the gel in a photo tray and use a gel imaging system or flatbed scanner to photograph/scan it. It is recommended to use either white light transmission or reflection mode depending on the equipment, and record exposure parameters for comparison.

4.2 Take a photo/scans and measure the band intensity quantitatively with image software (e.g., ImageJ), paying attention to using the same exposure/scanning Settings and measuring within the unsaturated range.

Experimental Result

Results Analysis and Discussion

(1) In the supernatant and sediment groups (labeled as Control Sup and Control Pel), no distinct protein bands corresponding to the indicated molecular weights of 76.8 kDa, 54.3 kDa, and 43.8 kDa were observed. This indicates that in normal W303 yeast without genetic manipulation, proteins matching these target molecular weights are either absent or present only in trace amounts undetectable by conventional methods. These findings exclude the possibility of interference from endogenous yeast proteins in the experimental results.

(2)In the experimental plasmids Plasmid 2 Sup and Plasmid 2 Pel, three distinct protein bands were clearly visible at the positions indicated by the arrows. The molecular weights of these bands perfectly matched the expected target proteins (MtCPR: 76.8 kDa; CYP716A2: 54.3 kDa; β-As: 43.8 kDa). This result directly demonstrates that after plasmid transfection, yeast cells successfully transcribed and translated the foreign genes, producing substantial quantities of the target proteins.

Transfer of plasmid ② yeast product OA extraction and cell cryopreservation

Experiment time: 2025.08.14

Personnel: Sun Jiayin

Experimental Objective

(1) Oleanolic acid OA was extracted from the cells of Saccharomyces cerevisiae after 72h of cultivation to prepare for the detection of OA content by liquid chromatography;

(2) Brewers yeast with frozen chromosomal plasmid ②.

Experimental Equipment and Reagents

Equipment

Ultra-clean workbench, oscillator, ultrasonic crusher, centrifuge.

Reagent

Chloroform, methanol, 50% glycerol

Experimental Procedure

Liquid chromatography sample preparation

(1) Take 3mL of fermentation liquid in the crushing tube, centrifuge (13000 rpm/ min, 3min);

(2) Add the culture medium, add an appropriate amount of glass beads (diameter 0.5mm) and 1mL extractant (chloroform: methanol =1:1), and shake to break for 5min;

(3) After ultrasonic crushing for 30min, centrifugation (13000 rpm/min, 3min) was performed, and the supernatant was taken for later use.

Saccharomyces cerevisiae cryopreservation

(1) Take 3 EP tubes and add 750μL 50% glycerol solution to each EP tube;

(2) Add 750μL of yeastl liquid to each EP tube, mark it and put it in the-20℃ refrigerator for preservation.

Determine whether OA is present in the HPLC analysis product

(1) Gradient elution was performed using VP-ODS C18 chromatography column (5μl,150nm×4.6nm).

(2) Separation conditions: Acetonitrile (mobile phase A), purified water (mobile phase B); Column temperature 35℃, injection volume 20μl, flow rate 1.0m L/min, UV detector wavelength 203nm.

(3) Elution conditions: 0 ~ 25min, 19﹪ B, 81﹪ A, 25 ~ 50min, 29﹪ B, 71﹪ A.

Experimental Result

Results Analysis and Discussion

The absence of positive results in liquid chromatography analysis may be attributed to the degradation of OA caused by abnormal photothermal conditions during product extraction. This suggests that controlling the temperature during ultrasonic crushing or maintaining an appropriate organic phase ratio in the mobile phase is necessary, as excessive elution could prevent OA retention. Further optimization should focus on regulating the systems crushing temperature and selecting suitable mobile organic phases for elution.

matters need attention

(1) When preparing the extraction solution, the volume should be calculated in advance to prevent insufficient dosage from repeated preparation.

(2) In the first step of centrifugation, try to pour out the supernatant as far as possible, otherwise the liquid will be separated and the cell layer may float up and affect the effect of ultrasonic crushing.

(3) When shaking, pay attention to the moment to see if there is any leakage of liquid. If three tubes can not be done at the same time, the quantity can be reduced.

Product extraction and cell cryopreservation

Experiment time: 2025.08.16

Experiment operator: Sun Jiayin, Wang Quanhao, Min Yingxuan

Experimental Objective

(1) Cyclamen acid OA was extracted from yeast cells for 72h to prepare for the detection of OA content by liquid chromatography;

(2) Brewing yeast transfected by cryopreservation.

(3) The yeast was cultured and a single clone was determined to determine the OD value for plasmid transfection

Experimental Equipment and Reagents

Equipment

laminar flow hood, ultrasonic homogenizer, ultrasonic crusher, centrifuge, spectrophotometer, 96-well plate, yeastl filter

Reagent

Chloroform, methanol, 50% glycerol, ethyl acetate, YPD medium

Experimental Procedure

Liquid chromatography sample preparation

(1) Take 3mL of fermentation liquid in the crushing tube, centrifuge (1 2 000 rpm/ min, 3min);

(2) Add 1mL extractant (chloroform: methanol =1:1 or ethyl acetate) to the culture medium and mix well by blowing, add an appropriate amount of glass beads (0.5mm in diameter), and shake for 5min;

(3) After 30min ultrasonic crushing, centrifugation (12000 rpm/min, 3min) was performed, and the supernatant was taken for later use.

Saccharomyces cerevisiae cryopreservation

Take 3 EP tubes and add 750μL 50% glycerol solution to each EP tube;

Add the yeastl liquid to each EP tube until full, mark it and put it in-20℃ refrigerator for preservation.

Pick a single colony from the culture medium

(1) The ultra-clean workbench is irradiated with ultraviolet lamp 30min in advance;

(2) Take 50mL centrifuge tube, pour 5mL YPD liquid culture medium into the centrifuge tube;

(3) Pick the single clone yeast from the YXZ6 yeast culture plate and add it to the centrifuge tube. Put it into the shaker culture (30℃,200rpm) for about 7h;

(4) After 7 hours, measure OD₆₀₀. like OD600=0.3, then take 200μL liquid and add it to 50mLYPD medium.

Determine whether OA is present in the HPLC analysis product

(1) Gradient elution was performed using VP-ODS C18 chromatography column (5μl,150nm×4.6nm).

(2) Separation conditions: Methanol (mobile phase A), purified water (mobile phase B); Column temperature 35℃, injection volume 20μl, flow rate 1.0m L/min, UV detector wavelength 203nm.

(3) Elution conditions: 0 ~ 25min, 19﹪ B, 81﹪ A. 25 ~ 50min, 29﹪ B, 71﹪ A.

Experimental Result

Discussion and analysis

(1) A distinct chromatographic peak was observed in the 23-minute region when compared with the standard reference. This indicates that oleanolic acid OA is indeed present in the sample under these chromatographic conditions, flowing out around 23.5 minutes. The presence of oleanolic acid OA after transformation confirms successful plasmid transfection and normal function of the corresponding protein.

(2) The shape of the peak is basically symmetrical, without serious tailing or forward extension, which indicates that the chromatography column is in good condition, there is no serious nonspecific adsorption between the sample and the stationary phase, and the analysis conditions are relatively ideal.

(3) From the spectrum, the peak and the adjacent peaks (the small peaks around 21 minutes and 25 minutes) all achieved baseline separation. This means that the target component was well separated and not easily interfered by the adjacent peaks.

matters need attention

1. Trim the edge of the paper before shock to avoid paper offset caused by EP tube offset or leakage during shock

2. When shaking, pay attention to observe whether the EP tube position is offset and whether there is any leakage of liquid

3. OD of yeast on day 2600If it is greater than 0.8 but not more than 1.5, the next step can be carried out

4. The HPLC mobile phase should be selected as a 9:1 methanol: purified water system, which can fully elute OA without excessive elution

Multi-step glycosylation to synthesize Ginsenoside Ro

Streak Culture of Three Strains; Preparation of YPD, Sc-Trp-Leu Double-Deficient Solid Medium, and Sc-Trp Liquid Medium

Experimental time: 2025.08.07

Experimental personnel: Sun Jiayin, Wang Quanhao

Objectives of the Experiment

(1) Perform streak culture on Strain 1, Strain 2, and Strain 3 from the three cryopreservation tubes into which Plasmid ② has been transferred.

(2) Prepare YPD medium, Sc-Trp liquid medium, and Trp-Leu double-deficient solid medium.

Experimental Equipment and Reagents

Equipment

Electronic balance, magnetic stirrer, autoclave, aseptic operating bench, Petri dishes

Reagents

Glucose, peptone, yeast extract, arginine (Arg), adenine, SC-Trp, SC-Trp-Leu

Reagent Preparation

Experimental Procedure

Preparation of YPD Medium

(1) Prepare the solution according to Table 1.

(2) Place the glass bottle containing the solution into an autoclave for autoclaving (115°C, 15 minutes).

(3) The preparation of YPD medium is completed.

Preparation of Sc-Trp Liquid Medium

(1) Prepare the Sc-Trp solution according to Table 2 and the YNB solution according to Table 4.

(2) Place the glass bottle with the solution into an autoclave for autoclaving (115°C, 15 minutes).

(3) Sterilize the YNB solution by filtration through a 0.22 μm filter, then transfer it to a 100 mL autoclaved empty bottle for later use.

(4) Take 20 mL of the filter-sterilized YNB solution, add it to the Sc-Trp solution, and mix thoroughly.

Preparation of Trp-Leu Double-Deficient Solid Medium

(1) Prepare the Trp-Leu double-deficient solution according to Table 3 and the YNB solution according to Table 4.

(2) Place the glass bottle with the solution into an autoclave for autoclaving (115°C, 15 minutes).

(3) Sterilize the YNB solution by filtration through a 0.22 μm filter, then transfer it to a 100 mL autoclaved empty bottle for later use.

(4) Take 20 mL of the filter-sterilized YNB solution, add it to the Trp-Leu double-deficient solution, and mix thoroughly.

(5) Pour the medium into sterile Petri dishes (approximately 15-20 mL per dish). After standing and solidifying, store them upside down at 4°C (to avoid the accumulation of condensed water).

Colony Streak Culture

(1) Sterilize the biosafety cabinet with ultraviolet light for 30 minutes in advance, and wipe the experimental bench with 75% alcohol for disinfection.

(2) Use a marker pen to label the bottom of the plate with information such as strain name, date, and operator.

(3) Pass a bamboo stick through the outer flame of an alcohol lamp for sterilization, let it cool down, and then directly dip a small amount of yeast solution (a visible droplet to the naked eye is sufficient).

(4) Gently draw a "Z" pattern with the bamboo stick dipped in the yeast solution on one side of the plate (about 1/4 of the area, marked as Area A). The lines should be dense but not overlapping, and the angle of the inoculating tool (bamboo stick) should be approximately 30°-45° during streaking. Discard the bamboo stick after streaking.

(5) For the second streaking (Area B), use a cooled bamboo stick to cross 1-2 times from the end of Area A, extend to Area B (about 1/2 of the area), and draw a sparse "Z" pattern. For the third streaking (Area C/D), repeat the above steps: streak from the end of Area B to Area C (the remaining area) with even sparser lines, and finally form a gradient dilution (high yeast concentration in Area A and low yeast concentration in Area D).

Experimental Results

Analysis and Discussion

The culture medium was successfully prepared, and the streaked colonies grew normally.

Precautions

(1) Before using the biosafety cabinet, sterilize it with ultraviolet light for 30 minutes. When starting the experiment, the first step is to light the alcohol lamp. The flame of the alcohol lamp in the laboratory is easy to ignite but invisible, so be careful to avoid scalding.

(2) After opening the bottle cap, do not place anything above the bottle mouth, and place the bottle cap upside down. When taking centrifuge tubes, EP tubes, and bamboo sticks from the box, use tweezers, and sterilize the tweezers by passing them through the flame.

(3) YNB does not require autoclaving, nor does it need to be treated with a microwave oven before use; for YPD medium, perform microwave disinfection one day before the experiment.

(4) Apply gentle force with your hand during streaking to avoid scratching the agar. The lines should be dense but not overlapping, and the angle of the inoculating tool (bamboo stick) should be approximately 30°-45° during streaking.

Monoclonal Culture of Saccharomyces cerevisiae Transfected with Plasmid ② and OD Value Determination

Experimental time: 2025.08.09

Experimental personnel: Sun Jiayin, Wang Quanhao, Min Yingxuan

Objectives of the Experiment

Cultivate monoclonal colonies of Saccharomyces cerevisiae transfected with Plasmid ② and determine their OD values to prepare for plasmid transfection.

Experimental Equipment and Reagents

Equipment

Ultra-clean workbench, absorbance meter, 96-well plate

Reagents

YPD medium

Experimental Procedure

Picking Monoclonal Colonies from the Medium

(1) Turn on the ultraviolet lamp of the ultra-clean workbench for 30 minutes in advance for sterilization; prepare YPD medium and perform microwave treatment in advance for later use.

(2) Take a 50 mL centrifuge tube and pour 5 mL of liquid YPD medium into it.

(3) Pick a monoclonal colony of Saccharomyces cerevisiae (transfected with Plasmid ②) from the culture plate, add it to the centrifuge tube, and place the tube in a shaker for cultivation (30°C, 200 rpm) for approximately 9 hours.

(4) After 9 hours, determine the OD₆₀₀ value. If the OD₆₀₀ value is 0.3, take 2 mL of the yeast solution and add it to 100 mL and 50 mL of YPD medium respectively (to explore which system yields better transfection efficiency).

Experimental Results

Analysis and Discussion

After 9 hours, the OD value of the yeast reached 0.3, and its growth status was excellent.

Precautions

(1) For the initial 100 mL YPD medium, resuspend the yeast cells with 800 μL of 0.1 M LiAc/TE later.

(2) For the initial 50 mL YPD medium, resuspend the yeast cells with 400 μL of 0.1 M LiAc/TE later.

Preparation of Sc-Trp-Leu Medium: Competent Cells

Experimental time: 2025.08.11

Experimental personnel: Sun Jiayin, Wang Quanhao, Min Yingxuan

Objectives of the Experiment

(1) Prepare Sc-Trp medium and pour it into plates to lay the foundation for the cultivation of Saccharomyces cerevisiae after subsequent plasmid transfection.

(2) Prepare competent cells to get ready for plasmid transfection.

Experimental Equipment and Reagents

Equipment

Ultra-clean workbench, microplate reader, high-temperature and high-pressure autoclave, shaker

Reagents

SC-Trp-Leu, arginine, adenine, glucose, YNB (Yeast Nitrogen Base), ammonium sulfate ((NH₄)₂SO₄), agar, sterile water

Reagent Preparation

Experimental Procedure

Preparation of Sc-Trp-Leu Medium

(1) Take 0.4 g of Sc-Trp-Leu, 0.04 g of arginine, 0.2 g of adenine, and 4 g of glucose, add 60 mL of water, and place the mixture in an empty bottle.

(2) Take 4 g of agar, add 120 mL of water, place the mixture in another empty bottle, and add a magnetic stir bar.

(3) Put the two aforementioned bottles and one 100 mL empty bottle into a high-temperature and high-pressure autoclave for autoclaving (115°C, 15 minutes).

(4) Weigh 1.6 g of YNB for yeast and 5 g of ammonium sulfate, place them in a beaker, add 100 mL of autoclaved water, and add a magnetic stir bar to stir until completely dissolved.

(5) Sterilize the solution by filtration through a 0.22 μm filter, then transfer it to a 100 mL autoclaved empty bottle for later use.

(6) Take 20 mL of the filter-sterilized YNB solution and add it to the autoclaved bottle containing agar. While the mixture is still hot, pour the SC-Trp solution into the agar bottle with YNB added, and mix thoroughly using a magnetic stirrer.

(7) Quickly pour the mixed solution into sterile Petri dishes to make plates. Let them stand at room temperature for 4 hours, then store them in a refrigerator for later use.

Preparation of Competent Cells

(1) Place sterile water in a 4°C refrigerator for later use, and dilute LiAc/TE (Lithium Acetate/Tris-EDTA Buffer) to a concentration of 0.1 mol/L for later use.

(2) Determine the OD₆₀₀ value of the Saccharomyces cerevisiae (transfected with Plasmid ②) cultured the previous day. If the OD₆₀₀ value is 0.8, proceed to the next step of the experiment.

(3) Take 50 mL of the yeast solution, centrifuge it (3500 rpm, 2 minutes), and discard the supernatant.

(4) Resuspend the yeast pellet with 50 mL of cold sterile water, centrifuge again (3500 rpm, 2 minutes), and discard the supernatant.

(5) Resuspend the pellet with 1 mL of 0.1 mol/L LiAc/TE, centrifuge rapidly (4000 rpm, 30 seconds), and discard the supernatant.

(6) Resuspend the pellet with 400 μL of 0.1 mol/L LiAc/TE, mix well, and store it on ice in the refrigerator overnight.

Experimental Results

Precautions

(1) The filter-sterilized YNB solution must be quickly poured into the agar while it is still hot to prevent uneven distribution of YNB, which may cause abnormal solidification of the medium.

(2) After the medium is prepared, it should be quickly poured into plates to avoid premature solidification due to prolonged standing.

(3) Monitor the OD value of Saccharomyces cerevisiae to prevent failure to proceed to the next step of the experiment due to excessive culture time.

Plasmid③ transfection and glycerol stock plate coating

Experiment time: 2025.08.12

Experimental personnel: Sun Jiayin, Wang Quanhao, Min Yingxuan

Objectives of the Experiment

(1) Saccharomyces cerevisiae was transfected with plasmid 3 to synthesize the final product ginsenoside Ro;

(2) Glycochocera coating plate, easy to pick single colony for plasmid extraction.

Experimental Equipment and Reagents

Equipment

laminar flow hood, shaker, 30℃ incubator, water bath, shaker, oven, electronic balance, magnetic stirrer, autoclave, PCR instrument, electrophoresis system, gel imaging system, adsorption column

Reagents

Buffer P1、Buffer P2、Buffer P3、Buffer DW1、Wash Solution、ElutionBuffer; PEG, LiAc (1mol/L), salmon sperm, plasmid, yeast receptive state, YPD medium;

Experimental Procedure

Viral transfection

(1) Salmon sperm was boiled in boiling water for 5min and then rapidly placed in an ice water mixture (on ice)

(2) Add about 50 μL of yeast receptive strain that has been transplanted with plasmid ② prepared the previous day into each EP tube, and centrifuge (4000rpm, 30s) to discard the supernatant;

(3) Add 42μL plasmid, 10μL croaker sperm (10 mg/mL), 240μL PEG, and 36μL LiAc (1 mol/L) sequentially into the EP tube. After repeatedly pipetting to mix thoroughly with a 1mL pipette, incubate in a 30℃ incubator for 30 minutes.

(4) Add 36μL DMSO to each EP tube, mix by repeated blowing and place it in a 42℃ water bath for 20min.

Yeast culture after transfection

(1) The EP tube was taken out of the water bath, and the supernatant was discarded after centrifugation (3000rpm, 20s). The suspension was resuspended with 1 mL YPD medium and cultured in a shaker for 3 h.

(2) After centrifugation (3000rpm, 20s), the supernatant was discarded, and the yeast suspension was resuspended with 100μL water. The yeast suspension was spread on the plate (Sc-Trp) with glass beads, and cultured in a 30℃ incubator for about 3 days.

Glycozyme smear

Single colonies of glycerobacterium were picked and streaked on ampicillin LB medium

Experimental Results

Precautions

(1) Salmon sperm need to be boiled in boiling water for 5min in advance, and then quickly put into ice water mixture to play the role of protecting exogenous plasmid.

(2) All kinds of reagents required for particle transfection should be mixed evenly to maximize the permeability of cell membrane and improve the transfection efficiency.

(3) The incubation time of the titer transfection incubator and the heat shock time of the water bath must be strictly controlled to prevent the decrease of transfection efficiency after too long time.

Preparation of Sc-Trp-Leu solid medium; Preparation of Sc-Trp liquid medium; Cultivation of Saccharomyces cerevisiae (with plasmid 2); Cultivation of glycerophages; Extraction of W303 genome

Experiment time: 2025.08.13

Experimental personnel: Wang Quanhao, Min Yingxuan

Objectives of the Experiment

(1) Prepare Sc-Trp-Leu solid culture medium and Sc-Trp liquid culture medium for standby;

(2) Cultivate yeast for wine making (with plasmid 2) and measure OD value for preparation of receptive state;

(3) Cultivate glycerol stock for plasmid extraction;

(4) W303 genome was extracted for PCR negative control;

Experimental Equipment and Reagents

Equipment

High pressure sterilizer, super clean workbench, centrifuge, shaker, enzyme labeling instrument, 96 plate, conical flask;

Reagents

SC-Trp, arginine, adenine, glucose, ammonium sulfate, agar, sterile water, Sc-Trp-Leu liquid medium, YNB solution, YPD medium, LiOAc-1%SDS solution, anhydrous ethanol, 70% ethanol;

Reagent Preparation

Experimental Procedure

Culture preparation

(1) Prepare Sc-Trp liquid culture medium according to Table 1 in Section 2 and place it in an empty bottle;

(2) Take 4g agar and add 120mL water to another empty bottle;

(3) Place the two bottles into the autoclave and sterilize (115℃,15min);

(4) Prepare the solution according to Table 2 in section 2 while hot, and mix it thoroughly with a magnetic stirrer;

(5) The mixture was poured into a sterile petri dish to make a plate. After 5 hours at room temperature, it was put in the refrigerator for storage;

Brewth yeast (with plasmid 2) culture

(1) The ultra-clean workbench is irradiated with ultraviolet lamp 30min in advance;

(2) Take 50mL centrifuge tube, pour 10mL YPD liquid culture medium into the centrifuge tube;

(3) A single colony was picked from the yeast ① culture plate containing plasmid 2 and added to a centrifuge tube. The culture was placed in a shaker (30℃,200rpm) for about 7h;

(4) After 7 hours, 3 mL of yeast solution was inoculated into 100 mL YPD medium to measure whether the OD value was between 0.2 and 0.3;

Glycojugate culture

(1) Take a 50 mL centrifuge tube and add 15 mL glycerol-fatty acid liquid culture medium.

(2) Pick a single colony from plate 2 and 3 of glycerophages and add it to a centrifuge tube. Place it in a shaker culture (30℃,200 rpm);

W303 genome extraction

(1) Take 200 μL yeast culture and centrifuge to discard the supernatant;

(2) The cell was precipitated and resuspended in 100 μL LiOAc-1%SDS solution, and incubated in a 70℃ oven for 5min;

(3) Add 300 μL anhydrous ethanol, repeatedly blow and mix, centrifuge (12000rpm, 3min) and discard the supernatant;

(4) Wash the precipitate with 70% ethanol solution, and discard the supernatant after centrifugation;

(5) The precipitate was dissolved in 100 μL water and stored in a -20℃ refrigerator.

Experimental Results

Precautions

(1) Turn on the oven to warm up in advance when extracting the genome;

Yeast monoclonal culture after protomer transfection

Experiment time: 2025.08.15

Experimental personnel: Wang Jingning, Wang Quanhao

Objectives of the Experiment

Three single clone colonies of dual plasmid yeast 3 were picked from the plate, transferred to Trp-Leu double deficiency medium, and cultured on a shaker;

Experimental Equipment and Reagents

Equipment

Ultra-clean workbench, autoclave, shaker, centrifuge.

Reagents

Peptone, yeast extract, sodium chloride, glucose, sterile water.

Reagent Preparation

Experimental Procedure

Plasmid ②③ dual plasmid yeast 3 single clone double deficiency culture medium culture

(1) Take the yeast ②③ double plasmid 3 culture plate out of the yeast incubator and observe the growth of yeast;

(2) Take 350mL centrifuge tubes and pour 15 mL Trp-Leu deficiency medium into each centrifuge tube;

(3) Three single clones were picked from yeast plasmid 3 plate, transferred to the above centrifuge tube, and cultured on a shaker;

(4) Subsequently, yeast growth was monitored every 12h.

Experimental Results

Analysis and Discussion

The yeast growth was monitored every 12h, and the growth had far exceeded the logarithmic phase of the obvious yeast growth curve, and was close to the plateau phase. The monitoring interval time was too long, and the growth rate of yeast exceeded the expected rate

Precautions

(1) When transferring from the culture plate to the centrifuge tube, a single clone must be selected to obtain a strain with stable genetic background.

(2) When pouring the medium into the centrifuge tube, in order to avoid contamination by the remaining medium, part of the medium can be transferred to a 50mL centrifuge tube for reserve.

(3) The growth curve should be tested in time, and the interval time should not be too long

Determination of growth curve of yeast monoclonal culture after transfection with plasmid ② + ③

Experimental time: 2025.08.17

Experimental personnel: Wang Jingning, Wang Quanhao

Objectives of the Experiment

Every 2h, three single clone colonies of plasmid ② cultured in Trp-Leu deficient liquid medium were transferred into 96-well plate with 100ul yeast solution. The absorbance value was measured to indirectly judge the growth status of the strain. The continuous monitoring was carried out for 96h, and finally the growth curve of the single clone colony was drawn.

Experimental Equipment and Reagents

Equipment

Laminar flow cabinet, autoclave, shaker, microplate reader, 96-well plate

Experimental Procedure

(1) The ultra-clean workbench is irradiated with ultraviolet lamp 30min in advance;

(2) Take 50mL centrifuge tube and pour 15mL glycerol fatty acid liquid culture medium into the centrifuge tube;

(3) Pick three single clone yeast from the culture plate of Saccharomyces cerevisiae with the transferred plasmid ② + ③ and add it to the centrifuge tube, and put it into the shaking bed culture (30℃,200rpm);

(4) Observe the growth of yeast to see if the liquid culture medium becomes cloudy;

(5) Take one 96-well plate, add 100ul of yeast fluid into each well, set the Trp deficiency culture medium control group, and set three groups of repeated controls for three monoclonal yeast fluid and single deficiency culture medium. Measure the OD600 value with a microplate reader and calculate the average value.

Experimental Results

Analysis and Discussion

(1) Trp-Leu defect culture medium After shaking culture, the centrifuge tube was obviously turbid. The OD value of three single clone colonies measured under the microplate reader was also increased compared with the control group. The yeast growth degree of the plasmid ②+ ③ was considerable after transplantation.

(2) The yeast growth was continuously monitored, and the yeast reached the plateau period in about 30h;

Precautions

(1) Trp-leu defect medium control group was set up to better reflect the growth degree of yeast fluid.

(2) The growth OD value was measured every 2h as far as possible to monitor the growth rate and degree of yeast in time.

Glycoflavin streaking; PCR and agarose gel electrophoresis to verify the successful transfection of plasmid ② + ③ enzyme gene

Experimental time: 2025.08.18

Experimental personnel: Sun Jiayin, Wang Quanhao, Min Yingxuan

Objectives of the Experiment

(1) Monoclonal cells were prepared by glycohydrolase streaking.

(2) The genes of UGT73P40, UGT73F3 and Pn022859 were identified to determine whether they were transferred into yeast, and the expression of plasmids in yeast strains was detected.

Experimental Equipment and Reagents

Equipment

Clean workbench, centrifuge, micropipette and gun head, PCR instrument, electrophoresis instrument, gel imaging system

Reagents

Plasmid 2, glycerol-free freeze-dried culture medium, Amoxicillin LB solid agar plates, 2x TransFast Taq PCR SuperMix (with dye), DNA templates, specific primers, TAE buffer, agarose, EB DNA stain, and DNA markers

Reagent Preparation

Experimental Procedure

Glycojugate streaking

(1) The ultra-clean workbench was sterilized with ultraviolet light 30 minutes in advance, and the experimental table was wiped and disinfected with 75% alcohol;

(2) Mark the name of the strain, date, operator and other information on the bottom of the tablet with a marker pen;

(3) The bamboo stick is burned in the outer flame of the alcohol lamp, cooled, and then dipped directly into a small amount of yeast liquid (visible drops can be seen);

(4) The bamboo stick dipped in yeast solution is gently drawn in a zigzag pattern on one side of the plate (about 1/4 area, marked as A area). The line should be dense but not overlapping. The angle of the bamboo stick is about 30°-45° when drawing the line. Discard the bamboo stick after drawing the line;

(5) The second marking step (Zone B) involves passing cooled bamboo sticks 1-2 times from the end of Zone A, extending to Zone B (approximately half of the area) to create sparse zigzag patterns. The third marking step (Zones C/D) repeats the process, extending from Zone B's end to Zone C (the remaining area), with even finer lines. This creates a gradient dilution pattern where Zone A contains concentrated yeast while Zone D has sparse yeast distribution.

Yeast genome extraction

(1) Take 200μL yeast culture solution and centrifuge (4000rpm, 30s) to discard the supernatant;

(2) Resuspend the cell precipitate in 100μL LiOAc-1%SDS solution, and incubate it in a 70℃ oven for 5min;

(3) Add 300μL anhydrous ethanol, repeatedly blow and mix, centrifuge (15000rpm, 3min) and discard the supernatant;

(4) Wash the precipitate with 70% ethanol solution, and discard the supernatant after centrifugation;

(5) The precipitate was dissolved in 100μL water and stored in a-20℃ refrigerator.

PCR

(1) PCR reaction system was prepared according to 3.1 in Section 2;

(2) Set the PCR reaction program according to 3.2 in Section 2 and carry out PCR;

Agarose gel electrophoresis

(1) Prepare 2% agarose gel according to 3.3 in Section 2 by microwave heating;

(2) Add 9 μL EB and shake well, then pour it into the mixing tank where the comb has been placed. Place it flat in the refrigerator and wait for the gel to solidify;

(3) Put the gel into the electrophoresis tank and pour in an appropriate amount of 1x TAE electrophoresis buffer. After sampling, electrophoresis for 25min under constant voltage of 140V;

(4) After electrophoresis, observe and take photos under ultraviolet lamp;

Experimental Results

Analysis and Discussion

(1) The three target genes were not detected in the negative control group NC, indicating that the primer amplification was specific;

(2) The three target genes were detected in the positive control group PC (plasmid ② + ③ ) with clear bands and normal molecular weight, indicating that the three target genes were successfully cloned into the plasmid vector;

UGT73P40

In this experiment, the UGT73P40 gene showed obvious bands in yeast 2 and yeast 3, indicating tthat he gene was successfully transferred in these two colonies; the band was not obvious in yeast 1, indicating that the gene was successfully transferred, and the results of this experiment may be caused by the increase of amplified fragment length or insufficient sample addition during electrophoresis;

UGT73F3

In this experiment, the UGT73F3 gene was clearly visible in three monoclonal yeast strains, which was strongly positive and basically consistent with the previous results, indicating that the gene was successfully transferred.

Pn022859

The experimental results showed weak positive for yeast 1, positive for yeast 2 and yeast 3, indicating that the gene may have been successfully transfected in yeast 1, but the transfection amount was low. The negative result in this study may be caused by the increase of amplification fragment length and the decrease of PCR efficiency.

Precautions

(1) The tail of the bamboo stick is picked out with tweezers and then roasted.

(2) Start the line from the end of the last line. The lines should be dense but not overlapping.

(3) The PCR instrument should stop working first, then open the lid, and finally shut down.

(4) Set the electrophoresis instrument to constant voltage.

Protein extraction of yeast strain samples transferred to plasmid ② + ③ ; SDS-PAGE gel electrophoresis; Coomassie Brilliant Blue staining

Experimental time: 2025.08.22

Experimental personnel: Wang Jingning, Wang Quanhao, Min Yingxuan, Sun Jiayin

Objectives of the Experiment

(1) Sample protein extraction was performed by SDS-PAGE gel electrophoresis; Coomassie Brilliant Blue staining was used to prepare for normal plasmid expression.

(2) Determine whether the genes of three enzymes, MtCPR, CYP716A12 and β-AS, were transferred into Saccharomyces cerevisiae.

Experimental Equipment and Reagents

Equipment

Ultra-clean workbench, centrifuge, water bath, gel plate, electrophoresis tank, vertical electrophoresis instrument, transfer electrophoresis instrument, decolorization shaker, chemiluminescence imaging system;

Reagents

NaOH, β-glycerophosphate sodium, sodium orthovanadate, NaF, EDTA, PMSF, Tris, glycine, SDS, Coomassie Brilliant Blue R-250, methanol, glacial acetic acid, 30% polyacrylamide, 1.0M Tris-HCl (pH6.8),1.5M Tris-HCl (pH 8.8),10% APS, TEMED.

Reagent Preparation

Experimental Procedure

Sample preparation

(1) Prepare yeast lysate according to Table 1: with NaOH as the benchmark, add each reagent in the table below to 1mL NaOH, and add protease inhibitors and phosphatase inhibitors (non-essential) according to specific experimental requirements;

(2) Take 200μL yeast culture solution, centrifuge (4000rpm, 30s) and discard the supernatant;

(3) Add the lysis solution to the obtained yeast precipitate and lyse on ice for 10min;

(4) Centrifuge (5000rpm, 1min), discard the supernatant;

(5) Add 80μL 1x loading buffer to the precipitate and mix well. After metal bath (100℃,5min), store in-20℃ refrigerator for standby

Protein blotting and SDS-PAGE gel electrophoresis

(1) Configure the SDS-PAGE gel and electrophoresis according to the method in 4.2;

(2) Before protein loading, the sample was bathed in hot water for 5min and centrifuged at 5000rpm for 3min. The supernatant and precipitate were respectively loaded for electrophoresis;

(3) After the gel electrophoresis, remove the gel plate, carefully peel off the gel from the glass plate, and put it into the staining box/tray; rinse the surface of the gel with deionized water 1 to 2 times to remove the residual electrophoresis buffer (gentle operation to avoid tearing the gel);

(4) Fixation (removes SDS and protein leakage, improves staining signal-to-noise ratio): Add sufficient fixative solution (methanol/acetic acid/water, typically 45% methanol + 10% acetic acid + 45% water) to the gel-containing container. Shake gently at room temperature for 30 minutes. The fixation process can be extended to 1 hour or overnight at 4℃°C to enhance staining quality.

Carma blue staining and decolorization

(1) Discard the fixed liquid and add the newly prepared Comass blue staining solution to completely immerse the gel. Stain for 1-2 h at room temperature on a horizontal shaker; if more sensitive, stain overnight (do not exceed 24 h to avoid increased background).

(2) After the dyeing, discard the dye and use the decolorizing solution (40% methanol, 10% acetic acid, 50% water) for the first decolorization. Shake gently at room temperature for 30 min~1 h until the background begins to fade obviously.

(3) Replace the decolorizing solution and continue decolorization on a horizontal shaker. Adjust the decolorizing solution based on background coloration and band development, repeating this process every 30-60 minutes until the background becomes sufficiently pale and band contrast is clear. Complete decolorization may take several hours to two days (allow overnight exposure for continued processing). For faster results, perform short decolorization cycles, capture images immediately, then proceed with detailed processing.

Gel imaging analysis

(1) After decoloring to the desired extent, rapidly rinse the gel with deionized water to remove residual methanol/acetic acid. Place the gel in a photo tray and use a gel imaging system or flatbed scanner for photography/scanning. It is recommended to use white light transmission or reflection mode depending on the equipment, and record exposure parameters for comparison;

(2) Take a photo/scans and measure the band intensity quantitatively with image software (e.g. ImageJ), paying attention to using the same exposure/scanning Settings and measuring within the unsaturated range.

Experimental Results

Analysis and Discussion

(1) In the supernatant and sediment groups (labeled as Control Sup and Control Pel), no distinct protein bands corresponding to the indicated molecular weights of 81.5 kDa, 61.1 kDa, and 55.2 kDa were observed. This indicates that in normal W303 yeast without genetic manipulation, proteins matching these target molecular weights are either absent or present only in trace amounts undetectable by conventional methods. These findings exclude the possibility of interference from endogenous yeast proteins in the experimental results.

(2) In the experimental plasmids Plasmid 3 Sup and Plasmid 3 Pel, three distinct protein bands were clearly visible at the positions indicated by the arrows. The molecular weights of these bands perfectly matched the expected target proteins (Pn022859:81.5 kDa, UGT-73P40:61.1 kDa, and UGT73F3: 55.2 kDa). This result directly demonstrates that after plasmid transfection, yeast cells successfully transcribed and translated the foreign genes carried on the plasmids, synthesizing substantial amounts of the target proteins.

The final product of transplanted plasmid ② + ③ yeast was ginsenoside Ro extraction and cell cryopreservation

Experimental time: 2025.08.24

Experimental personnel: Sun Jiayin, Wang Jingning, Wang Quanhao, Min Yingxuan

Objectives of the Experiment

(1) Ginsenoside Ro was extracted from the cells of Saccharomyces cerevisiae at 72h to prepare for the detection of Ro content by liquid chromatography;

(2) Brewer's yeast with frozen chromosomal plasmid ② + ③ .

Experimental Equipment and Reagents

Equipment

Ultra-clean workbench, oscillator, ultrasonic crusher, centrifug

Reagents

Chloroform, methanol, 50% glycerol

Experimental Procedure

Liquid chromatography sample preparation

(1) Take 3mL of fermentation liquid in the crushing tube, centrifuge (13000 rpm/min, 3min);

(2) Add the culture medium, add an appropriate amount of glass beads (diameter 0.5mm) and 1mL extractant (chloroform: methanol =1:1), and shake to break for 5min;

(3) After ultrasonic crushing for 30min, centrifugation (13000 rpm/min, 3min) was performed, and the supernatant was taken for later use.

Saccharomyces cerevisiae cryopreservation

(1) Take 3 EP tubes and add 750μL 50% glycerol solution to each EP tube;

(2) Add 750μL of yeast solution to each EP tube, mark it and put it in the-20℃ refrigerator.

HPLC analysis to determine whether Ro is present in the product

(1) Gradient elution was performed using VP-ODS C18 column (5μl,150nmx4.6nm).

(2) Separation conditions:

Acetonitrile (mobile phase A), purified water (mobile phase B); Column temperature 35℃, injection volume 20μl, flow rate 1.0mL/min, UV detector wavelength 203nm.

(3) Elution conditions:

0 ~ 25min, 19﹪B, 81﹪A。25 ~ 50min, 29﹪B, 71﹪A。

Experimental Results

Analysis and Discussion

The liquid chromatography analysis showed a positive result.

Precautions

(1) When preparing the extraction solution, the volume should be calculated in advance to prevent insufficient dosage from repeated preparation.

(2) In the first step of centrifugation, try to pour out the supernatant as far as possible, otherwise the liquid will be separated and the cell layer may float to affect the effect of ultrasonic crushing.

(3) When shaking, pay attention to the moment to see if there is any leakage of liquid. If three tubes can not be done at the same time, the quantity can be reduced.

Rough enzyme extraction and in vitro transformation of active components IVa and R1

Experimental time: 2025.09.01

Experimental personnel: Min Yingxuan, Wang Jingning

Objectives of the Experiment

(1) The crude enzyme was extracted from the yeast cells that were transferred to plasmid ② + ③ to prepare for in vitro experiments;

(2) Using R1 and IVa as substrates and UDPG as cofactor, crude enzymes were used to transform the active ingredients;

(3) To detect whether the product of the in vitro transformation experiment contains the target product Ro.

Experimental Equipment and Reagents

Equipment

Ultra-clean workbench, ultrasonic crusher, centrifuge, constant temperature water bath, vortex oscillator, liquid chromatograph

Reagents

Methanol, ethyl acetate, Panax notoginseng saponin R1, ginseng saponin IVa, ginsenoside Ro, uridine diphosphate glucose UDPG, dimethyl sulfoxide DMSO, Tween 80, tris hydrochloride trimethylolammonium salt Tris-Hcl (PH=8.5), sterile water.

Reagent Preparation

Experimental Procedure

Extraction of target protein from engineered yeast

(1) Pick the W303 strain on YPD medium and the single colony with good plasmid ② + ③ transfection in Sc-Trp-Leu double deficiency medium respectively, and carry out liquid culture in YPD medium and Sc-Trp-Leu double deficiency medium respectively.

(2) Cultivation After 20h, when OD600 reaches 1.8~2, take 1ml of the yeast liquid with a pipette and re-inoculate it into the liquid culture medium to cultivate until OD600 reaches 1.8~2. Collect 15ml of the yeast liquid, centrifuge at 4000rpm for 10min, and collect the precipitate.

(3) Wash the obtained yeast with 1mlPBS once, centrifuge at 10000rpm for 2min, and then suspend the precipitate with 1m IPBS.

(4) Finally, the yeast cells were broken with an ultrasonic cell crusher. The temperature was controlled in an ice bath at a low temperature (0-4℃) and ultrasonic crushing was performed for 20min.

(5) After cell suspension is broken, it becomes obviously clear. The obtained cell suspension is the whole protein solution and can be directly used for enzymatic reaction.

Construction of enzymatic reaction system

(1) As shown in Table 1, Dissolution of R1 and IVa: Dissolve notoginsenoside R1 and chikusetsusaponin IVa in DMSO (dimethyl sulfoxide) solution. Specifically, 9.33 mg of notoginsenoside R1 is dissolved and made up to 0.1 mL with DMSO, and 7.95 mg of chikusetsusaponin IVa is dissolved and made up to 0.1 mL with DMSO, thus preparing 100 mM system solutions.

(2) As shown in Table 2, System preparation (total system volume 300 μL) Experimental group: (Crude enzyme extract of strains transfected with plasmids ② + ③ ) Add 251.85 μL of crude enzyme solution, 15 μL of Tris-HCl (pH 8.5), 30 μL of 50 mM UDPG, 0.15 μL of 100 mM substrate (R1 or IVa), and 3 μL of Tween 80 in sequence, then gently pipette to mix well. Control group (crude enzyme extract of w303 strain): Replace the crude enzyme solution of the experimental group with the crude enzyme solution of w303 Saccharomyces cerevisiae, while other components and their dosages are exactly the same.

(3) Control of reaction conditions: All reaction tubes are placed in a 20°C constant temperature water bath for reaction in the dark for 20 hours. During this period, gently invert and mix once every 5 hours to prevent substrate precipitation.

Reaction product treatment

(1) Enzyme inactivation and centrifugation: After the reaction is completed, place the reaction tube in a 99°C water bath and heat for 10 minutes to inactivate the enzyme activity, then immediately cool to room temperature in an ice bath. Centrifuge at 12000 rpm for 15 minutes, and transfer the supernatant (containing reaction products) to a new centrifuge tube.

(2) Extraction and concentration: Add an equal volume of ethyl acetate, vortex and oscillate for 1 minute, let it stand for phase separation (about 5 minutes), and pipette the upper ethyl acetate phase (containing saponin products) to a new tube. Repeat the extraction twice, combine all ethyl acetate phases, place them in a 50°C water bath, evaporate to dryness slowly, and dissolve the residual solid with 300 μL of methanol.

(3) Filtration and purification: Filter the methanol solution through a 0.45 μm organic phase filter membrane, collect the filtrate into a high-performance liquid chromatography injection vial, store at 4°C, and wait for detection.

Product detection and result analysis

High-performance Liquid Chromatography (HPLC) Injection: Inject 20 μL of the filtrates from the experimental group, control group, and the standard solutions (R1, IVa, Ro) respectively into the liquid chromatography injection valve, and record the chromatograms.

Experimental Results

Analysis and Discussion

When compared with the reference standard, the sample group exhibited a sharp and symmetrical main chromatographic peak within 59-60 minutes, showing high consistency with the standard. This indicates that the transplanted enzyme demonstrated significant activity in vitro experiments, capable of converting substrates IVA and R1 into the final product Ro. In vitro experiments demonstrated that the transplanted glycosyltransferase into Saccharomyces cerevisiae exhibited enzymatic activity. Meanwhile, the peak area of the sample group derived from R1 conversion was observed to decrease compared to that from IVA conversion. Our results suggest that the conversion efficiency from R1 to Ro is significantly lower than that from IVA to Ro. To address this, we aim to optimize enzyme selectivity through directed evolution to reduce intermediate product R1 formation.

Precautions

(1) The temperature of ultrasonic crushing was controlled at 0-4℃ to prevent excessive temperature from damaging the structure of crude enzyme protein and reducing enzyme activity.

(2) When preparing the substrate reaction system, the amount of DMSO and Tween 80 should be strictly controlled. High concentration of DMSO may cause denaturation of protein spatial structure. Tris-Hcl can be added to control the PH of the reaction system.

(3) In HPLC liquid chromatography detection, methanol was often evaporated at room temperature to prevent excessive temperature and saponin Ro desaccharification and degradation.

Enhancing glucose supply to improve product yield

Cultivation and Cryopreservation of Yeast

Experimental time: 2025.09.01

Experimental personnel: Zhao Xiaoqing, Sun Jiayin

Objectives of the Experiment

1. Cultivate and freeze-store Saccharomyces cerevisiae to prepare for subsequent plasmid transfection in the production of competent cells.

2. Prepare the plasmid as a solution for subsequent transfection.

Experimental Equipment and Reagents

Equipment

Shaker, refrigerator, laminar flow hood, spectrophotometer

Reagents

YPD medium, plasmid powder, sterile water, glycerol

Reagent Preparation

Experimental Procedure

Cultivation of Saccharomyces cerevisiae

(1) Irradiate the ultra-clean workbench with ultraviolet light for 30 minutes beforehand. Prepare YPD medium and microwave-treat it in advance for later use;

(2) Take a 50 mL centrifuge tube and pour 5 mL of YPD liquid medium into it;

(3) Pick a single colony of Saccharomyces cerevisiae W303 from the culture plate and inoculate into the tube. Incubate on a shaking incubator (30°C, 200 rpm);

(4) After approximately 7 hours, measure the OD600. If OD600 = 0.3, add 2 mL of the culture to 100 mL of YPD medium.

Prepare the plasmid

(1) Retrieve the plasmid powder tube and centrifuge (4μg);

(2) Add 30μL sterile water to the tube;

(3) Transfer 10μL to an EP tube, then add 32μL sterile water for later use;

(4) Store the remaining 20μL plasmid at -20°C.

Freeze-preserve experimental strains

Take an EP tube, add 750μL bacterial suspension and 750μL glycerol (50%) in a 1:1 ratio, mix, and store at -80°C. Replenish the remaining bacterial suspension in the centrifuge tube with appropriate medium.

Experimental Results

Analysis and Discussion

Yeast exhibited robust growth and is suitable for preparing competent cells for subsequent experiments.

Precautions

(1) Maintain aseptic technique when isolating single-colony yeast to prevent contamination by extraneous microorganisms.

(2) When preparing YPD medium, avoid excessive temperatures during autoclaving. Typically, autoclave at 115°C for 15 minutes; higher temperatures may cause glucose to darken.

(3) Ensure culture duration does not exceed recommended limits. If optical density (OD) exceeds 1.2, the strain is unsuitable for subsequent procedures.

Preparation of Sc-Trp medium and competent cells

Experiment time: 2025.09.04

Experiment operator: Zhao Xiaoqing, Sun Jiayin

Objectives of the Experiment

1. Prepare Sc-Trp medium and plate it to prepare for the subsequent cultivation of Saccharomyces cerevisiae after plasmid transfection.

2. Prepare the competent cells for plasmid transfection.

Experimental Equipment and Reagents

Equipment

Ultra-clean workbench, absorbance meter, high temperature and high pressure sterilizer, shaker

Reagents

Ultra-clean workbench, absorbance meter, high temperature and high pressure sterilizer, shaker

Reagent Preparation

Experimental Procedure

Sc-Trp medium configuration

(1) Take 0.4g SC-Trp, 0.04g arginine, 0.2g adenine and 4g glucose, add 60mL water, and put it in an empty bottle;

(2) Take 4g agar and add 120mL water into another empty bottle, and add a rotor;

(3) Put the above two bottles and an empty 100mL bottle into the autoclave for high-pressure sterilization (115℃,15min);

(4) Weigh 1.6g yeast with YNB, 5g ammonium sulfate, place it in a beaker, add 100mL autoclaved water, put in a rotor to stir until completely dissolved;

(5) After sterilization by filtration through 0.22μm filter, the solution was transferred to a 100mL empty high-pressure sterilized bottle for standby;

(6) Take 20mL of filtered YNB solution and add it to the high-pressure agar bottle. Pour SC-Trp solution into the agar bottle with YNB while hot, and use a magnetic stirrer to mix thoroughly;

(7) Pour the mixture into a sterile petri dish to make a plate. After 4 hours at room temperature, put it in the refrigerator for storage.

Preparation of competent cells

(1) Place sterile water in a 4℃ refrigerator for standby, and dilute LiAC/TE to 0.1mol/L for standby;

(2) Determine the OD600 of yeast culture the previous day, if OD600=0.8, then proceed to the next experiment;

(3) Take 50mL of bacterial liquid, centrifuge (3500rpm, 2min) and discard the supernatant;

(4) Resuspend in 50mL cold sterile water, centrifuge (3500rpm, 2min), and discard the supernatant;

(5) Resuspend with 1mL 0.1mol/L LiAC/TE, and centrifuge rapidly (4000rpm, 30s), then discard the supernatant;

(6) Resuspend in 400μL 0.1mol/L LiAC/TE, mix well, and overnight in the refrigerator on ice.

Experimental Results

Analysis and Discussion

(1) The filtered and sterilized YNB solution should be poured into the agar quickly while hot to prevent abnormal solidification of the culture medium caused by uneven distribution of YNB.

(2) After the culture medium is prepared, it should be poured into the plate quickly to prevent the culture medium from solidifying in advance for too long.

(3) Observe the OD value of yeast to prevent the culture time from being too long to enter the next experiment.

Plasmid ④transfection; yeast culture after transfection

Experiment time: 2025.09.05

Experiment operator: Wang Quanhao

Objectives of the Experiment

1. The prepared competent cells were used for plasmid transfection;

2. Transfection of plasmids ④ into yeast to utilize fatty acids;

3. Prepare YPD medium and Trp single deficiency solid medium for standby.

Experimental Equipment and Reagents

Equipment

Laminar flow hood, shaker, 30℃ incubator, water bath, shaker, oven, electronic balance, magnetic stirrer, autoclave

Reagents

PEG, LiAc (1mol/L), salmon sperm, plasmid, yeast receptive state, YPD culture, PEG, DMSO Glucose, peptone, arginine, adenine, SC-Trp;

Reagent Preparation

Experimental Procedure

Plasmid transfection

(1) Salmon sperm was boiled in boiling water for 5 minutes, and then quickly placed in an ice water mixture (on ice);

(2) Add about 50 μL yeast receptive strain prepared the previous day into each EP tube, and centrifuge (4000rpm, 30s) to discard the supernatant;

(3) Add 42μL plasmid, 10μL croaker sperm (10mg/mL), 240μL PEG, and 36μL LiAc (1mol/L) into the EP tube in sequence. After repeatedly mixing by pipetting with a 1mL pipette, incubate at 30℃ incubator for 30min;

(4) Add 36μL DMSO to each EP tube, mix by repeated blowing and then put it into a 42℃ water bath for 20min.

Yeast culture after transfection

(1) Take the EP tube from the water bath, centrifuge (3000rpm, 20s) and discard the supernatant. Resuspend in 1 mL YPD medium and culture in a shaker for 3h;

(2) Centrifugation (3000rpm, 20s) After discarding the supernatant, resuspend with 100μL water, spread the bacterial liquid with glass beads (Sc-Trp), and cultivate in a 30℃ incubator for about 3 days.

Preparation of Trp single deficiency solid medium

(1) Prepare Trp single solution according to (2) in "II", and prepare YNB solution according to (3);

(2) Put the glass bottle with solution into the autoclave and sterilize (115℃,15min);

(3) After sterilization of YNB solution through 0.22μm filter, it was transferred to a 100 mL empty high-pressure sterilized bottle for standby;

(4) Take 20 mL of filtered YNB solution and add it to the Trp double deficiency solution and mix thoroughly;

(5) Pour the medium into a sterile petri dish (about 15-20mL per plate). After solidification, invert it and store at 4°C (avoid condensation water accumulation).

Preparation of YPD medium

Prepare PYD medium according to 3.1 in Section 2, and the other steps are the same as 4. in Section 3. Sterilize with ultraviolet light as a liquid culture medium.

Experimental Results

Analysis and Discussion

(1) Salmon sperm need to be boiled in boiling water for 5min in advance, and then quickly put into ice water mixture to play the role of protecting exogenous plasmids.

(2) All kinds of reagents required for particle transfection should be mixed evenly to maximize the permeability of cell membrane and improve the transfection efficiency.

(3) The incubation time of the titer transfection incubator and the heat shock time of the water bath must be strictly controlled to prevent the decrease of transfection efficiency after too long time.

PCR and agarose gel electrophoresis after plasmid ④ transfection

Experiment time: 2025.09.07

Experiment operator: Wang Quanhao, Min Yingxuan

Objectives of the Experiment

1. Prepare competent cells for plasmid transfection;

2. 2/3 of the extracted plasmid was used for PCR and plasmid transfection;

3. To determine whether the genes of FBP-ase1, PGM2 and UGP1, which were expressed, were transferred into Saccharomyces cerevisiae, and to detect the expression of plasmids in the engineered bacteria.

Experimental Equipment and Reagents

Equipment

Clean workbench, enzyme label instrument, electronic balance, centrifuge, fume hood, oscillator, ultrasonic crusher, PCR instrument, oscillator, electrophoresis tank, microwave oven, gel imaging system;

Reagents

LiAC/TE solution, sterile water;

Buffer P1、Buffer P2、Buffer P3、Buffer DW1、Wash Solution、ElutionBuffer;

Glass beads, chloroform, methanol;

2xTransFast Taq PCR SuperMix (+dye), DNA template, specific primers, 1xTAE buffer, agarose, nucleic acid dye EB, DNA Marker;

Reagent Preparation

Experimental Procedure

Protein extraction

(1) Check whether RNase A has been added to buffer P1.

(2) Check whether anhydrous ethanol has been added to the Wash Solution.

(3) Check for precipitation of buffer P2 and P3.

(4) Take 1.5-5 mL of the overnight culture, centrifuge at 8,000 ×g for 2 minutes to collect the bacteria, and discard all the culture medium.

(5) Add 250 μL Buffer P1 to the precipitate and resuspend the bacteria thoroughly.

(6) Add 250 μL Buffer P2 and gently invert the centrifuge tube 5-10 times immediately to mix. Let it stand at room temperature for 2-4 minutes.

(7) dd 350 μL Buffer P3 and gently invert the centrifuge tube 5-10 times immediately to mix.

(8) Centrifuge at 12,000 ×g for 10 minutes. Transfer the supernatant to the adsorption column and let it stand for 3-4 minutes. Centrifuge at 8,000 ×g for 30 seconds, then discard the liquid from the collection tube.

(9) Add 500 μL Buffer DW1 and centrifuge at 9000 ×g for 30 seconds. Pour off the liquid in the collection tube.

(10) Add 500 μL Wash Solution and centrifuge at 9000 ×g for 30 seconds. Pour off the liquid in the collection tube.

(11) Repeat (2)(10) once

(12) The air adsorption column was centrifuged at 9000 ×g for 1 min.

(13) Place the adsorption column into a clean 1.5mL centrifuge tube, add 50-100 μL Elution Buffer in the center of the adsorption membrane, and centrifuge for 1 min after room temperature standing for 1 min. Save the DNA solution in the tube.

PCR and agarose gel electrophoresis

(1) The PCR reaction system was prepared according to Table 1;

(2) Set the PCR reaction program according to Table 2 and carry out PCR;

(3) Prepare 2% agarose gel according to Table 3 and dissolve it by microwave heating;

(4) Add 9 μL EB to the agarose gel and shake it well. Then pour it into the mixing tank where the comb has been placed. Place it flat in the refrigerator and wait for the gel to solidify;

(5) Put the gel into the electrophoresis tank and pour in an appropriate amount of 1x TAE electrophoresis buffer. After sampling, electrophoresis for 25min under constant voltage of 140V;

(6) After electrophoresis, observe and take photos under ultraviolet lamp.

Experimental Results

Analysis and Discussion

(1) The three target genes were detected in the positive control group PC (plasmid ④) with clear bands, indicating that the three target genes were successfully cloned into the plasmid vector.

(2) The positive bands of the three target genes in the target strain suggested that the three target genes were successfully transferred into yeast

(3) The three target genes were not detected in the negative control group NC, indicating that the primer amplification was specific.

Protein extraction of bacterial strain samples transferred to plasmid ④

Experiment time: 2025.09.09

Experiment operator: Wang Jingning, Wang Quanhao, Min Yingxuan, Sun Jiayin

Objectives of the Experiment

1. Sample protein extraction was prepared for western blot test of normal plasmid expression;

Experimental Equipment and Reagents

Equipment

Ultra-clean workbench, centrifuge, water bath, gel plate, electrophoresis tank, vertical electrophoresis instrument, transfer electrophoresis instrument, decolorization shaker, chemiluminescence imaging system;

Reagents

NaOH, β-glycerophosphate sodium, sodium prontovantanate, NaF, EDTA, PMSF

Reagent Preparation

Experimental Procedure

Sample preparation

(1) Prepare yeast lysate according to the following table: with NaOH as the base, add each reagent in the table below to 1mL NaOH, and add protease inhibitors and phosphatase inhibitors (non-essential) according to specific experimental requirements;

(2) Take 200μL yeast culture solution, centrifuge (4000rpm, 30s) and discard the supernatant;

(3) Add the lysis solution to the obtained bacterial precipitate and lyse on ice for 10min;

(4) Centrifuge (5000rpm, 1min), discard the supernatant;

(5) Add 80μL 1x loading buffer to the precipitate and mix well. After metal bath (100℃,5min), store in-20℃ refrigerator for standby

Western Blot to detect successful expression of protein

Experiment time: 2025.09.11

Experiment operator: Wang Quanhao, Min Yingxuan

Objective of the Experiment

Determine whether the genes of FBP-ase1, PGM2 and UGP1 were transferred into Saccharomyces cerevisiae.

Experimental Equipment and Reagents

Equipment

Ultra-clean workbench, centrifuge, water bath, gel plate, electrophoresis tank, vertical electrophoresis instrument, transfer electrophoresis instrument, decolorization shaker, chemiluminescence imaging system;

Reagent Preparation

Experimental Procedure

Protein blotting and gel electrophoresis

(1) Prepare the 5% concentration gel and 12% separation gel according to Tables 4 and 5. Allow the separation gel to chill at room temperature for 20-30 minutes, while the concentration gel should remain at room temperature for approximately 5-10 minutes. Place both gels into an electrophoresis tank filled with electrophoresis buffer. During loading, add 10 μL of protein sample to each well, and include 4 μL marker in one well. Handle carefully to avoid bubble formation during the process;

(2) Electrophoresis: Electrophoresis conditions: 80 V for the upper layer of concentrated gel for 30min; 120 V for the lower layer of separation gel for 60 ~ 80 min;

(3) Blotting: After electrophoresis is complete, remove the gel plate and gently lift the gel. Cut the corresponding PVDF membrane and filter paper according to the required dimensions. Prior to blotting, activate the PVDF membrane with methanol for approximately 20-30 seconds. Perform the blot using an ice-cold water mixture at a constant current of 200mA for 2 hours;

(4) Milk sealing and hybridization: Take out the PVDF film, wash it with 1x TSBT solution for 3 times in a horizontal shaker, each time for 10 min; seal it with 5% skim milk and seal it for 2h in a horizontal shaker at room temperature;

(5) After 2-hour incubation, wash with TBST three times (5 minutes each). Incubate with diluted primary antibody (dilution ratio follows the antibody manufacturer's instructions) using TBST solution. Place in a 4°℃ fridge overnight. The next day, perform three TBST washes (10 minutes each) on a shaker before secondary antibody incubation for 1 hour. Finally, conduct three TBST washes (10 minutes each);

(6) AECL Colorimetry: As specified in Table 7, follow the ECL Colorimetry Manual (Thermo Fisher Scientific) to prepare Solution A and Solution B in a 1:1 volume ratio. After mixing both solutions, allow them to stand at room temperature under light protection for 1-2 minutes. Capture images using the gel imaging system and analyze the grayscale values of each band through dedicated software. Finally, perform statistical analysis on the processed data.

Experimental Results

Analysis and Discussion

Plasmid ④ contains and can express the three genes FBP-ase1, PGM2 and UGP1 with clear bands. The positive results show a significant contrast with the negative controls, indicating reliable results. The strains transformed with plasmid ④ can effectively utilize UDPG, providing an efficient gluconeogenesis pathway that facilitates normal growth and carbon source utilization.