NOTEBOOK

Main Timeline:

Each month is depicted with a grid system. Each square in the picture below corresponds to a 3-day period.

January：

February：

March：

April：

May：

June：

July:

August:

September:

October:

Safety training is required before the experiment begins

Stage one：Construction of plasmid

Task: PCR

Goal：Obtain the target gene

Participants: Yue Cao, Yuqin Zhang, Yutong Du, Jian Tang, Zilan Qin, Jingwei Wu, Yunke Lu, Lizhuo Chen, Daoyang Zhang

Materials：Forward/Reverse Primers, Agar 2%， Template plasmid, H2O, Buffer, Enzymes， Base pairs（ATCG）

Procedure：

Put 25 μL 2*PCR mix into each PCR tubes, then add 22 μL ddwater. Then add 1 μL Forward Primer and 1 μL Reverse Primer into the tubes. After that, add 1 μL template plasmid into each PCR tubes.

Put those total 14 PCR tubes load into thermal cycle and start PCR.

The PCR cycle starts with 95°C 3 minutes Then move as 95°C for 30 seconds, 55°C for 30 seconds and 72°C for 1.5 minutes. This cycle repeats 33 times.Finally, 72°C for 10min.

Task: Verified the PCR product by electrophoresis, purified the target DNA fragment, and performed bacterial transformation using the heat-shock method.

Goal：Construction of plasmid.

Participants: Yue Cao, Yuqin Zhang, Yutong Du, Jian Tang, Zilan Qin, Jingwei Wu, Yunke Lu, Lizhuo Chen, Daoyang Zhang

Procedure：

Agarose gel electrophoresis verification

Mix agarose and ATE buffer together, with a ratio of 1：100

Sol by heat and dissolve in the microwave oven and add nucleic acid dye when it cools down to 50-60℃

Pour into the mold to cooling and solidification

Place 8μl PCR the mold cavities and then electrophoresis 180V for 20 minutes

Finally put the gel in to the Gel imager，ultraviolet irradiation to observe DNA that has reached a specific length

Column-based DNA gel Purification.

Cut out the gel block containing the target fragment from the agarose gel and weigh it.

Add Buffer B2 into the gel block and metal bath55℃.

Transfer sol solution into the adsorption column and 8000Xg centrifugation for 1 minute.

Add Wash Buffer and 9000Xg centrifugation for 1 minute, pour the solution in the tube.

Repeat the previous step.

Let empty adsorption column in the centrifuge for 1 minute.

Put adsorption column into a clean 1.5mL centrifuge tube and add 30μl Elution Buffer into the center of the adsorption film. centrifugation for 1 minute.

Finally preserve DNA solution.

DNA homologous recombination and transformation.

Prepare solutions.

Mix 5μl 2×ClonExpress Mix, 1μl aga, 1μl sall, 3μl pom together.

Heat at 50 ℃ for 15 minutes. Then ice bath for 4 minutes.

Preparation for antibiotic plates (LB+1:1000 Kanamycin)

Thaw competent E. coli cells on the ice.

Add 10μL recombinant product (plasmid) into the competent cells, gently flick vessel wall, gently mix it by using the pipette tip. Incubate on ice for 30 minutes, stationary.

42℃ heat shocks for 30s in order to create channels and pores in the cell membrane that connect the cell interior to the external environment.

Remove immediately, incubate on ice for 2 minutes to reduce the temperature.

 Add 900μL LB culture medium. Incubate at 37℃ with shaking for one hour.

Centrifuge and discard 800μL of the supernatant.

Separate the thallus in the remaining 200μL of LB medium. 

Evenly spread the sample on antibiotic plates by using a sterile cell spreader.

Task: Plasmid Verification

Goal：Verify whether the plasmid was successfully constructed.

Participants: Yue Cao, Yuqin Zhang, Yutong Du, Jian Tang, Zilan Qin, Jingwei Wu, Yunke Lu, Lizhuo Chen, Daoyang Zhang

Result:

Figure 1

Stage two：Protein expression and verification

Goal：The protein expression was induced and verified by SDS-page.

Task: Protein extraction, purification, and verification result

Participants: Yue Cao, Yuqin Zhang, Yutong Du, Jian Tang, Zilan Qin, Jingwei Wu, Yunke Lu, Lizhuo Chen, Daoyang Zhang

Procedure：

Cultivate bacterial samples for protein production:

Select the correct colonies and culture them to an OD of 0.5.

Prepare 4 conical flasks.

Add 100ml of LB culture medium in 2 flasks and 100ml of soywhey culture medium in 2 flasks.

Add IPTG(1mM) in 1 flask of LB culture and 1 flask of soywhey culture.

Add galactose(1mM) another flask of LB culture and another flask of soywhey culture.

Place it in the shaking incubator maintain 25°C, shake for 20 hours.

Collect the strains cultured last night, get 50ml from each and pour them into a tube.

Through a process of centrifugation and pouring out the supernatant, obtain a sediment of E.coli.

Add 4ml of lysis buffer and 40 μL of lysozyme. Place the tube within ice or keep it under a low temperature for 30 min.

Due to an ultrasonication to the bacteria formula and once again.

Place it in the centrifuge and preserve the supernatant to extract specifically the protein.

Then with pipette transfer the Nickel column in to the tube with the protein.

Purify the protein mixture:

For each tube of formula, prepare 8 of 2ml tubes, a 10ml tubes and a gravity flow column, respectively note the eight 2ml tubes as w1 w2 w3 w4 e1 e2 e3 e4, and it should be noted with the type of formula dealing with the bacteria a day before.

While the volve remain closed, transfer the formula into the gravity flow column.

Remaining under a low temperature, open the volve and collect them in a 10ml tube.

While the liquid part is totally leaked within, pick up the gravity flow column, place it respectively on the related first four 2ml tubes (w1-4). Wash 4 times with washing buffer.

Wash the same column four more times using the elution buffer, and collect the filtered liquid in four other EP tubes.

Verification

Into a PCR tube, add 20μl protein liquid

Load the sample in the protein gel.

Run electrophoresis: run at 80V for 20 minutes, then switch to 180V and run for an additional 40 minutes.

Observed by Coomassie brilliant blue staining.

Result:

Figure 2

Stage three：Functional Verification of the Carbohydrate Utilization Module.

Task: Determination of strain growth curve after introducing carbohydrate utilization module

Participants: Yue Cao, Yuqin Zhang, Yutong Du, Jian Tang, Zilan Qin, Jingwei Wu, Yunke Lu, Lizhuo Chen, Daoyang Zhang

Procedure：

Wild-type strain and S1 strain were cultured in LB medium for 24h.

The cultured bacteria were collected by centrifugation, and the supernatant was discarded.

Use sterile water to resuspend the strain, determine the OD₆₀₀, and position the final concentration of the strain as OD600=0.2.

The two strains were cultured in LB and soy whey media respectively, and OD600 was determined after 24 hours.

Stage four：Construction of galactose self-induction system.

Task: Checked GFP brightness to find the best promoter.

Participants: Yue Cao, Yuqin Zhang, Yutong Du, Jian Tang, Zilan Qin, Jingwei Wu, Yunke Lu, Lizhuo Chen, Daoyang Zhang.

Procedure：

PgalP-a-EGFP, PgalP-b-EGFP, PgalP-c-EGFP strain were cultured in LB medium for 24h.

The cultured bacteria were collected by centrifugation, and the supernatant was discarded.

Use sterile water to resuspend the strain, determine the OD₆₀₀, and position the final concentration of the strain as OD600=0.2.

The fluorescence intensity of the strain was measured at 0, 2, 4, 24, 36 and 48 hours after culture. (The excitation wavelength of GFP is 488 nm and the emission wavelength is 507 nm.)

Stage five：Construction and verification of 3'-SL synthesis module and combine it with the above two systems.

Task: Verified 3-module co-transformation.

Participants: Yue Cao, Yuqin Zhang, Yutong Du, Jian Tang, Zilan Qin, Jingwei Wu, Yunke Lu, Lizhuo Chen, Daoyang Zhang.

Procedure：

Bacterial Colony Selection and Preliminary Culture

-Configuration of the PCR system(5 µL mix，water,0.1 µL primer F and R,template)

-Set PCR procedure.

-Select individual colonies for colony PCR.

-Prepare the bacterial solution.

-Pour 1 µL of the bacterial solution into a 10 µL PCR tubes.

Stage six：The synthetic amount of 3'-SL was determined by HPLC

Task: Tested if the three modules worked

Participants: Yue Cao, Yuqin Zhang, Yutong Du, Jian Tang, Zilan Qin, Jingwei Wu, Yunke Lu, Lizhuo Chen, Daoyang Zhang.

Procedure：

After streaking and activating the fermentation strain, a single colony was picked and inoculated into 5 mL of LB liquid medium (with 5 μL Chl and Amp) and then cultured at 37°C and 200 r/min for 8-12 hours. The culture was transferred to 100 mL of Soybean Yellow Pulp Water at an initial OD₆₀₀ of 0.1 and cultured at 37°C and 200 r/min for approximately 8 hours. The culture was centrifuged at 4°C and 5000 rpm for 10 minutes. The bacterial cells were resuspended in 24 mL of Tris-HCl buffer (50 mmol/L, pH 7.0, containing 250 mg Neu5Ac). A substrate solution with a concentration of 10 g/L (1 mL of 250 g/L lactose) was added to start the reaction.

HPLC analysis method for the product: differential refractive index detector, detector temperature 35°C; Rezex ROA-Organic Acid H+ (8%) chromatographic column, column temperature 60°C; mobile phase: 5 mmol/L H2SO4 solution, flow rate 0.6 mL/min; injection volume 10 μL.

Figure 3