1. Preparation of Experimental Reagents and Media

Each group will prepare the corresponding volume/quantity of reagents/media.

1.1 Experimental Objective

To prepare reagents needed for subsequent experiments.

Genetic Engineering:

• LB Liquid Medium: 20 ml/tube * 5
• LB Liquid Medium: 20 ml/bottle * 10
• CaCl2 0.1M: 50 ml
• LB Solid Medium (1.5% Agar): 200 ml
• TB Liquid Medium: 50 ml/bottle * 70

1.2 Experimental Procedure: Preparation of Reagents for Genetic Engineering

1.2.1 Preparation and Sterilization of Solutions

LB Medium

A total of 300 ml is needed, but 400 ml will be prepared to ensure sufficient volume during aliquoting.

Preparation Method: [Tryptone: 4g, Yeast Extract: 2g, NaCl: 4g] + [400 ml pure water], stir well in a beaker, aliquot 20 ml into 10 conical flasks, and the remainder into a blue-cap bottle, and autoclave (loosen the cap for the blue-cap bottle).

LB Solid Medium (1.5%)

[Tryptone: 2g, Yeast Extract: 1g, NaCl: 4g] + [200 ml pure water] + [Agar powder: 3g], prepare directly in a 500 ml conical flask, do not stir, cover, and autoclave (loosen the cap for the blue-cap bottle).

Calcium Chloride (for competent cell preparation)

[0.55g CaCl2] + [50 ml pure water], prepare directly in a 100 ml blue-cap bottle, and autoclave.

TB Medium

A total of 3500 ml is needed, but 4000 ml will be prepared to ensure sufficient volume during aliquoting.

Preparation Method: [47.2 g tryptone, 94.4 g/L yeast extract, 37.6 g K2HPO4, 8.8 g KH2PO4, 16 ml glycerin] + [4000 ml pure water], stir well in a beaker, aliquot 50 ml into 70 conical flasks, and the remainder into a blue-cap bottle, and autoclave (loosen the cap for the blue-cap bottle).

Plastic Consumables

1.5 ml centrifuge tubes, 20 pcs, packaged in a plastic bag, and autoclaved.

1.2.2 Pouring Plates

Add Ampicillin (1 ml + 1 μl, final concentration 50 μg/ml) to the sterilized and cooled LB solid medium below 60°C, mix well, and pour plates into 90 mm plastic petri dishes, approximately 10-15 ml per plate, at least 10 plates needed.

2. Inoculation (Preparation for Plasmid Extraction)

Add 15 ml of autoclaved LB medium from 1.2.1 to a sterile 50 ml centrifuge tube, add 15 μl of Ampicillin stock solution (1 ml + 1 μl, final concentration 50 μg/ml), mix well. Add 150 μl of DH5α bacterial culture.

Cap and remove from the biosafety cabinet, incubate in a shaker at 220 rpm, 37°C, overnight.

3. Fundamental Experiments in Genetic Engineering/Molecular Biology

3.1 Objective

The molecular biology/genetic engineering experiments on the 11th are conducted individually. Each student must perform the basic molecular biology/genetic engineering experiments. The experiments on the 12th will be conducted in groups.

3.2 Experimental Procedures

3.2.1 Plasmid Miniprep and Concentration Measurement

3.2.2 PCR

3.2.2.1 Fragment Preparation

PCR Conditions for Fragment

Estimated Time: 1 hour (Morning Session)

PCR Conditions for pet28a Fragment

Estimated Time: 1 hour (Noon Break)

3.2.2.2 Purification of PCR Products

Due to the complexity of the PCR system, which contains templates, primers, enzymes, and other impurities that may hinder seamless cloning, purification is necessary to obtain the target gene fragment.

After purification, measure DNA concentration again and calculate the yield of the purification process.

3.2.3 Agarose Gel Electrophoresis

Prepare the gel during the PCR amplification of the fragment. In the afternoon, add the loading buffer and then run the gel electrophoresis.

(1) Prepare the gel (do this during the morning PCR session; due to the limited number of gel trays, prepare two small gels per group).

Agarose Gel Electrophoresis

• Purpose: Used for the separation, identification, and extraction of nucleic acid fragments.
• Principle: Fragments of nucleic acids (DNA or RNA) are separated based on their size. After applying an electric field, the negatively charged DNA or RNA fragments migrate through the agarose gel.Smaller nucleic acid fragments migrate faster. Higher agarose concentration in the gel reduces pore size, affecting the separation. Higher agarose concentrations result in smaller pores, making it suitable for separating smaller fragments.

Our target gene and vector backbone are approximately 700 bp and 6000 bp, respectively, so prepare a 1% agarose gel.

Dissolve 1 g of agarose in 100 ml TAE buffer (not pure water!) in a 250 ml Erlenmeyer flask, cover with foil, and heat in a microwave until dissolved. Cool to approximately 60°C, add nucleic acid dye, mix, and pour into the gel tray. Cover with foil and allow to solidify in the dark.

(2) Preparation of Electrophoresis Samples

Mix a small amount of the purified PCR product with loading buffer (6X), in a 1:5 ratio. Suggested: Take 10 µl each of the fragments in a 0.2 ml EP tube, add 2-3 µl loading buffer, and mix well.

(3) Loading Samples

(4) Electrophoresis and Analysis

Run at 90V. After verifying the molecular weight, store the samples at -20°C for use on the 12th.

4. Competent Cells, Seamless Cloning, and Transformation

4.1 Experimental Procedures

4.1.1 Preparation of Competent Cells

Upon arriving at the laboratory, first, pre-cool the calcium chloride solution prepared and sterilized on the 10th in an ice bath.

Transfer 1.25 ml of the bacterial culture into 1.5 ml centrifuge tubes (distributed into 8 tubes) and place them on ice for 10 minutes to cool to 0°C. Centrifuge the bacterial culture at 4°C, 4000 rpm for 10 minutes, and discard the supernatant (invert the tubes to drain the medium thoroughly).

Add 150 μL of ice-cold 0.1 M CaCl₂ to resuspend, combine two tubes into one, and place on ice for 30 minutes (resulting in 4 tubes total). Centrifuge again at 4°C, 4000 rpm for 10 minutes, discard the supernatant, and add 100 μL of ice-cold 0.1 M CaCl₂ to resuspend to obtain BL21 competent cells for immediate transformation. If not used immediately, add an equal volume of 50% glycerol, rapidly freeze in liquid nitrogen, and store at -80°C.

4.1.2 Seamless Cloning

Design the seamless cloning system according to the manual (classroom exercise; concentrations vary per individual, so each person's table will differ).

1. Concentration Determination of Linearized Vectors and Insert Fragments

If the linearized vector and insert fragments have been purified through a gel recovery kit, and the absorbance reading shows no apparent bands or smears remaining, you can use Nanodrop or similar spectrophotometers to measure concentration. However, the A260/A280 ratio should only be considered reliable between 1.8 and 2.0. Instruments like Nanodrop, Qubit, and PicoGreen are recommended for concentration measurement. When sample concentrations are below 10 ng/μl, different instrument models may yield significantly varying readings based on A260.

2. Calculation of Vector and Insert Fragment Usage:

For single-fragment source recombination, the minimum recommended vector usage is 0.03 pmol, and the minimum recommended insert fragment usage is 0.06 pmol (with a vector-to-insert molar ratio of 1:2).

For multi-fragment source recombination, the minimum DNA usage for each fragment is 0.03 pmol (with a vector-to-insert molar ratio of 1:1).

The amount of DNA used in these calculations can be approximated using the following formulas:

• For Single-Fragment Source Recombination:
• Optimal Cloning Vector Usage=[0.02*Number of Base Pairs in the Cloning Vector]ng(0.03pmol)
• The optimal amount of insert fragment=[0.04* The number of base pairs in the insert fragment]ng(0.06pmol)
• For Multi-Fragment Source Recombination:
• Optimal Cloning Vector Usage=[0.02*Number of Base Pairs in the Cloning Vector]ng(0.03pmol)
• The optimal usage amount for each fragment=[0.02* The number of base pairs in each fragment]ng(0.03pmol)
• For single-fragment source recombination, the insert fragment usage should not exceed 20 ng. When the insert fragment is larger than the vector, it is advisable to calculate the appropriate mass conversion, treating the insert fragment as the vector and vice versa.
• For multi-fragment source recombination, each fragment should not exceed 10 ng. If the minimum recommended usage is below this value, directly use 10 ng/μl.
• In single-fragment source recombination, to avoid non-specific bands in PCR, do not use direct linearized DNA. Add no more than 1/5 of the total reaction volume (2 μl), or directly clone after gel extraction.

3. Prepare the Following Reaction Systems on Ice:

1. X/Y is calculated based on the calculated vector and insert fragment amounts. To ensure amplification accuracy, vectors and insert fragments should be concentrated before reaction setup and used in no less than 1 μl volumes.
2. Positive Controls-1 and -2 employ plasmids as templates, with amplification lengths <3 kb. Primer Tm values should be >60°C.
3. c.Positive Control-3 uses genomic DNA as a template. Use high-fidelity polymerases for amplification.
4. d.Fragment length, GC content, and sequence complexity affect reaction time. High GC content in insert fragments can hinder the reaction; consider adding DMSO or betaine to enhance amplification.

4. Gently mix using a pipette (avoid vortex mixing) and briefly centrifuge the reaction mixture to the bottom of the collection tube.

5. Recombination Reaction Conditions:

Single-Fragment Recombination: Incubate at 50°C for 5 minutes; cool to 4°C or place on ice immediately.

2-3 Fragment Recombination: Incubate at 50°C for 15 minutes; cool to 4°C or place on ice immediately.

4-5 Fragment Recombination: Incubate at 50°C for 30 minutes; cool to 4°C or place on ice immediately.

• It is recommended to perform the reaction on instruments such as a PCR machine that offers precise temperature control.
• This protocol accommodates 0.01 - 0.25 pmol input of vectors and fragments. When the total vector and insert volume exceeds 5 μl, a lower input amount is acceptable, but avoid exceeding the recommended reaction time.
• Store the recombination product at -20°C for up to a week. Purify only when necessary for further transformation.

4.1.3 Transformation

Recombinant Product Transformation

1. Thaw chemically competent cells on ice (e.g., Fast-T1 Competent Cell, Vazyme #C505).
2. Add 5-10 μl of the recombinant product to 100 μl of competent cells. Gently flick the tube to mix (avoid vortexing) and incubate on ice for 30 minutes.
• The volume of recombinant product should not exceed 1/10 of the volume of competent cells used.
3. Heat shock the cells at 42°C for 30 seconds, then immediately place them back on ice for 2-3 minutes.
4. Add 900 μl of SOC or LB medium (without antibiotics) and incubate at 37°C with shaking for 1 hour (rotation speed 200-250 rpm).
5. Pre-warm the required LB agar plates containing the appropriate antibiotic at 37°C in an incubator.
6. Centrifuge at 5,000 rpm (2,500 x g) for 5 minutes and discard 900 μl of the supernatant. Resuspend the bacterial pellet in the remaining medium and spread it gently on the pre-warmed agar plate with the antibiotic.
7. Invert the agar plates and incubate at 37°C for 12-16 hours.

Each experimental group will have four sets of samples (AC, AD, BC, BD), with each set plated on 1-2 LB plates and incubated overnight at 37°C.

5. Growth curve,qPCR and Western Blot

5.1 Determination of growth curve

Take nine 50 mL Erlenmeyer flasks and divide them into three groups:
Group 1: Engineered bacteria + LB + antibiotics
Group 2: Wild-type bacteria + LB
Group 3: Empty vector bacteria + LB + antibiotics
Ensure the initial OD600 of each group is 0.2 during inoculation. Cultivate in LB medium for 24 hours. Samples are collected every 4 hours for growth curve analysis.

5.2 Inoculation

Distribute the fermentation seed culture prepared on the LB into the remaining three 250 ml sterilized culture bottles, adding at least 5 ml of seed culture to each (using sterile Pasteur pipettes). Incubate at 37°C, 220 rpm for 5-10 hours.

5.3 Induction

Add 125 μl of IPTG and L-arabinose stock solution to each bottle (the stock is 1 M, with a final concentration of 1 mM). Incubate overnight at 22°C, 200 rpm for induction.

5.4 Total RNA extraction

Materials
Lysozyme (prepare fresh):
Gram-positive: 400 µg/mL Gram-negative: 3 mg/mL
Buffer Rlysis-B (RNase-free)
DEPC-treated ddH₂O (RNase-free water)
Chloroform (molecular biology grade)
Absolute ethanol and 75% ethanol (made with DEPC-H₂O)
RNase-free 1.5 mL tubes, ice, microcentrifuge (4 °C)
Procedure:

1. Harvest cells

 Collect 1 mL late-log culture into a 1.5 mL RNase-free tube. Centrifuge 8,000 rpm, 4 °C, 1 min and discard the supernatant. Add 100 µL lysozyme solution, vortex briefly to resuspend, and incubate 5-10 min at room temperature.

 Lysozyme concentration: 400 µg/mL for Gram-positive bacteria; 3 mg/mL for Gram-negative bacteria.

2. Chemical lysis

 Immediately add 900 µL Buffer Rlysis-B, mix thoroughly (quick vigorous inversion or brief vortex), and incubate on ice for 3 min.

3. Phase separation

 Add 200 µL chloroform, cap tightly, shake vigorously for \~30 s (or invert 15x), incubate 3 min at room temperature, then centrifuge 12,000 rpm, 4 °C, 5 min. Carefully transfer the upper aqueous phase to a new RNase-free tube.

4. Ethanol addition / precipitation

 To the collected aqueous phase, add 1.3 volumes of absolute ethanol, mix gently, incubate 5 min at room temperature, then centrifuge 12,000 rpm, 4 °C, 5 min. Carefully decant the supernatant.

5. Pellet wash

 Wash the pellet with 500 µL of 75% ethanol (prepare with DEPC-H₂O). Centrifuge 12,000 rpm, 4 °C, 3 min and discard the supernatant.

6. Repeat the wash once

 Perform one additional 75% ethanol wash under the same conditions.

7. Dry and dissolve RNA

 Air-dry the pellet at room temperature for \~10 min (do not overdry). Remove any remaining droplets with a pipette tip. Dissolve the RNA in 30-50 µL DEPC-H₂O. Use immediately or store at -80 °C.

5.5 cDNA acquisition

Reagents
Total RNA or poly(A)+ RNA (or target RNA)
Oligo(dT)15-20 (0.5 µg/µL) or Random hexamer (0.2 µg/µL) or Sequence-specific primer (20 pmol/µL)
RNase-free ddH₂O
5x Reaction Buffer
RNase Inhibitor (40 U/µL)
dNTP Mix (10 mM each)
AMV Reverse Transcriptase (10 U/µL)

1. Initial mix on ice (bring to 11 µL)


Mix gently, quick-spin 3-5 s.
Heat 65 °C for 5 min, then ice 30 s, quick-spin again (3-5 s).

2. Add the following on ice (final volume 20 µL)


Mix gently, quick-spin 3-5 s.

3. Reverse-transcription program
   cDNA synthesis: 42 °C, 30-60 min
   Enzyme inactivation: 95 °C, 5 min (denature RT), then hold on ice

5.6 qPCR

10 μL mix, 0.4 μL each of forward and reverse primers, 7.2 μL of DEPC water, and then 2 μL of template were added to make a 20 μL system. The qPCR conditions were a constant temperature section of 95°C for 1 min, a cycle section of 95°C for 10 s, and a cycle of 60°C for 30 s. Fluorescence was read and the number of cycles was 40.

5.7 Western Blot

1. Materials

Cell lysis buffer: RIPA or B-PER (+ protease inhibitor cocktail; phosphatase inhibitors if needed)
BCA protein assay kit
4x Laemmli Sample Buffer (final 1x: 62.5 mM Tris-HCl pH 6.8, 2% SDS, 10% glycerol, 0.002% bromophenol blue; add 100 mM DTT or 5% β-ME as reducer)
SDS-PAGE gels (10-12% resolving + 4-5% stacking)
1x Running Buffer (25 mM Tris, 192 mM Glycine, 0.1% SDS)
PVDF membrane (0.45 µm for >20 kDa; 0.2 µm for small proteins) or NC membrane
PVDF requires 1 min activation in 100% methanol
Transfer Buffer (25 mM Tris, 192 mM Glycine, 20% methanol; add 0.03-0.05% SDS for large proteins)
TBST (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.1% Tween-20)
Blocking solution: 5% non-fat milk/TBST (general) or 5% BSA/TBST (phospho-antibodies)
Primary antibody (target-specific or anti-tag His/Flag/Strep) and HRP-conjugated secondary antibody
ECL substrate

2. Sample preparation
• Lysis

 Harvest 1-5 mL induced E. coli, wash once with PBS. Add 200-500 µL lysis buffer (+ inhibitors), lyse on ice (sonication or gentle mixing). Spin 12,000 xg, 4 °C, 10 min; collect supernatant.

• Quantify & denature

 Measure protein by BCA. Add 4x sample buffer (to 1x), heat 95 °C, 5 min (or 70 °C, 10 min for aggregation-prone proteins).

• Loading amount

 Load 10 µg total protein per lane.

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3. SDS-PAGE

Assemble gel, load samples and ladder. Run at 120-160 V until the dye front reaches the bottom (~45-70 min, gel-dependent).

4. Transfer

Activate PVDF in methanol (1 min) → rinse in water → equilibrate in transfer buffer 5-10 min.
Build stack: cathode - sponge - filter - gel - membrane - filter - sponge - anode; remove bubbles.
Wet transfer: 100 V, 60-90 min, 4 °C (or 300-350 mA, 60 min).
Semi-dry: 18-25 V, 30-45 min (instrument-specific).

5. Blocking & antibody incubation
• Block: 5% milk/TBST, RT 1 h.
• Primary antibody: dilute per datasheet (1:2000).

4 °C overnight.

• Wash: TBST 3 x 10 min.
• Secondary antibody : 1:5,000-1:10,000, RT 45-60 min.
• Wash again: TBST 3 x 5-10 min.
6. Detection & imaging

Cover membrane with ECL substrate, incubate 3 min.
Image on a chemiluminescence system.

6. Fermentation

6.1 Inoculation

Distribute the fermentation seed culture prepared on the TB into the remaining three 250 ml sterilized culture bottles, adding at least 5 ml of seed culture to each (using sterile Pasteur pipettes). Incubate at 37°C, 220 rpm for 5-10 hours.

6.2 Induction

Add 125 μl of IPTG and L-arabinose stock solution to each bottle (the stock is 1 M, with a final concentration of 1 mM). Incubate 3 days at 22°C, 200 rpm for induction.

7. Response surface analysis

Ferment according to the conditions in the table below. The operation is the same as step 6.