Exercises in basic genetic engineering method

1. PCR

50 μL PCR: 19 μL deionized water, 25 μL Phusion High-Fidelity PCR Master Mix (Thermo Fisher, Waltham, MA, USA), 2 μL forward primer (10 μM), 2 μL reverse primer (10 μM), and 2 μL genomic DNA template.

The PCR program: a) one cycle of 98°C for 30s; b) 30 cycles of 98°C for 10s, 58°C for 30s, and 72°C for 1 min 44s; c) one cycle of 72°C for 5 min.

Electronic gel for 50ml

With 1% of agarose and 55ml of water; Put liquid gel into microwave oven for 1 min for 2 times for melting; Add 5ul gel blue into liquid gel (1/10000); All genes fluorescence is successfully seen on the electronic gel.

Cut the gel carefully to small pieces contain DNA under blue light then add 1:3 of binding buffer. Put the mixture in the warm water bath in 50℃ for 5-10 minutes until the gel is totally dissolved.

Use DNA gel recovery kit to extract genes from the gel. Adding Buffer B2 for twice for 1:3 in first time, second time with 300ul, each round put tube into centrifuge take for 8000 xg.

Then washing solution for 500ul, put tube into centrifuge take for 9000 xg, twice. For each round, clean the liquid in the collection tube and add solution to the top of the kit contain adsorption column made by resin. Then dd water for the last time for 30ul, put tube into centrifuge take for 9000xg.

2. Use restriction endonucleases to digest the vector and PCR product

Add restriction enzymes. Then put the two tubes in 37℃ warm water bath for 2 hours, for enzymes to be activated.

3. T4 DNA ligase to ligate DNA fragment and vector

Add each DNA of the 5 DNA for 2μl into a new clean centrifuge tube. Add 1.2μl T4 DNA ligase with Buffer mixture into the centrifuge tube. Add 1μl vector (the plasmids) into the centrifuge tube. Add 0.5μl T4 enzyme into the centrifuge tube. Wait for 2 hours at room temperature until all the DNA are combined and bind with the vector.

4. Transform the recombinant plasmid

The DH5α receptor cells were removed from -80℃ and quickly inserted into ice. After 5 minutes, when the bacteria had melted, the target DNA (plasmid or junction product) was added and gently mixed by tapping the bottom of the EP tube with your hand (avoiding suction with gun), and then left for 25 minutes in ice.

42℃ water bath heat shock for 45 seconds, quickly put back on the ice and stand for 2 minutes, shaking will reduce the conversion efficiency.

5. Extract the recombinant plasmid from E. coli

Move 60ml absolute ethyl alcohol into 15ml PW solution. Pipette BL solution into the spin columns, centrifuge for 12000rpm, 1min. Pipette 1ml E. coli solution into the centrifuge tubes, centrifuge for 12000rpm, 1min. remove the supernatant.

Notes & Results:

Partly of us are succeeded.

Experimenter:

Yuemeng Li, Haobin Yao, Muyan Li, Shiran Liu, Weier Qi, Yu Chen, Ruoxin Yang, Zheren Xu, Xinyi Wang, Dongyun Fan, Yichen Zhao, Yutong Tian, Jinran Xu

Recorder:

Xinyi Wang, Haobin Yao, Yu Chen

Construct pGEM - sacB-5 - Ptac – Kana - rrnB-T - sacB-3

1. PCR amplification on genes fragments sacB-5, Ptac, Kana, rrnB-T, sacB-3

Perform PCR amplification on the gene fragments to generate the necessary DNA sequences.

2. Join five fragments together into E. coli plasmid by using enzymes

Use restriction enzymes to ligate the five gene fragments into an E. coli plasmid vector.

3. Construct pGEM - sacB-5 - Ptac – Kana - rrnB-T - sacB-3

Successfully construct the plasmid with the five joined fragments: sacB-5, Ptac, Kana, rrnB-T, and sacB-3.

Experimenter:

Yuemeng Li, Haobin Yao, Muyan Li, Shiran Liu, Weier Qi, Yu Chen, Ruoxin Yang, Zheren Xu, Xinyi Wang

Recorder:

Xinyi Wang, Muyan Li, Shiran Liu, Ruoxin Yang, Yuemeng Li

Notes & Results:

The first time we succeeded in cloning only sacB-5, sacB-3 and rrnB-T. After readjusting the protocol, kana and Ptac gene fragments were obtained. Finally, succeeded.

Construct pGEM - sacB-5 - Ptac – pgi - rrnB-T - sacB-3 and pGEM - gdh-5- Ptac – Kana - rrnB-T - gdh-3

1. PCR amplification on genes fragments: pgi, vector (pGEM - sacB-5 - Ptac – rrnB-T - sacB-3)

Perform PCR amplification on the pgi gene fragment and the vector for further ligation.

2. Use Gibson to connect fragments together, construct pGEM - sacB-5 - Ptac – pgi - rrnB-T - sacB-3

Mix 2.5μl of the pgi fragment and 2.5μl of the vector in a tube. Add 5μl of 2× Gibson enzyme mix, and incubate the mixture at 50℃ for 30 minutes.

3. PCR amplification on 4 gene fragments: pGEM, gdh-5, Ptac – Kana- rrnB-T, gdh-3

Perform PCR amplification on the 4 gene fragments necessary for the construction of the second plasmid.

4. Use Gibson to connect 4 gene fragments together, construct pGEM - gdh-5- Ptac – Kana - rrnB-T - gdh-3

Use Gibson assembly to connect the 4 gene fragments to construct the second plasmid.

5. Cultivate Komagataeibacter xylinus

Prepare HS media with the following components: Glucose 40g/L, Yeast extract 5g/L, Peptone 5g/L, Citric acid 1.5g/L, Anhydrous disodium hydrogen phosphate 2.7g/L, pH 6. Sterilize at 115℃ for 30 minutes. For solid media, add 15g/L agar. For producing bacterial cellulose (BC), add 1% ethanol after sterilizing. For transformation, add 0.1% cellulase after sterilizing.

Experimenter:

Yuemeng Li, Haobin Yao, Muyan Li, Shiran Liu, Weier Qi, Yu Chen, Ruoxin Yang, Zheren Xu, Xinyi Wang

Recorder:

Xinyi Wang, Zheren Xu, Weier Qi, Haobin Yao, Ruoxin Yang

Notes & Results:

Succeeded.

Construct gene-edited K. xylinus ΔsacB and gdh KO

1. Transform pGEM-sacB-5-Ptac-Kana-rrnB-T-sacB-3 and pGEM-gdh-5-Ptac-Kana-rrnB-T-gdh-3 into K. xylinus WT

After a failed transformation attempt, below are our optimized transformation methods:

• Take the competent cells out of the -80°C freezer and immediately place them on ice.
• Once the competent cells have thawed, add 3-4 µL of plasmid or DNA fragment (approximately 0.8-1 pg of DNA) and gently mix.
• Transfer the DNA and competent cell mixture to a 0.1 cm sterile electroporation cuvette and let it sit on ice for 20 minutes.
• Remove the electroporation cuvette from the ice box, dry the surface, and place it in the electroporator for electroporation (voltage: 1.8 kV, can be shocked twice, program name: Ec1).
• After electroporation, resuspend the cells in 500 mL of antibiotic-free medium for the corresponding strain, and transfer to a 1.5 mL sterile EP tube.
• Recover the cultured cells (K. xylinus at 30°C, cultured for 4 hours at 180 rpm).
• Centrifuge (8000 rpm, 2 minutes) to collect the cells, discard the supernatant (retain 100 µL of liquid), and plate the resuspended cells on agar plates with the appropriate antibiotics (amphotericin/kanamycin) and medium (K. xylinus cultured at 30°C in an incubator).

2. Screen positive colonies using antibiotic resistance

Screen colonies based on their resistance to antibiotics, indicating successful gene transformation.

3. Genotype identification of ΔsacB and gdh KO

Perform genotyping to confirm the presence of the ΔsacB and gdh KO mutations.

4. Extract bacterial cellulose from HS media

Steps for extracting bacterial cellulose:

• Wash the CNF particles with water overnight.
• Soak in 0.1 M NaOH at 80ºC overnight to remove any bacterial cells that may be attached to the CNF particles.
• Wash the particles several times with deionized water to completely remove NaOH and bring the particles to a neutral pH.
• Dry the purified cellulose at 60ºC for 12 hours until the mass of the CNF no longer changes.

(Three samples were prepared for quantifying the CNF particles, and the average was calculated.)

5. Transplant six sea-buckthorn trees

Transplant the trees with and without bacterial cellulose as follows:

• Place the same weight of moist soil in each pot.
• Add 200 g of wet bacterial cellulose to the soil of the BC group seedlings, while the control group soil does not contain BC.
• The soil distribution: the bottom layer consists of soil with about 10% moisture, and the middle and upper layers are divided into those with and without BC, with soil moisture maintained at around 68%.

Experimenter:

Yuemeng Li, Haobin Yao, Muyan Li, Shiran Liu, Weier Qi, Yu Chen, Ruoxin Yang, Zheren Xu, Xinyi Wang, Dongyun Fan, Yichen Zhao

Recorder:

Xinyi Wang, Yu Chen, Dongyun Fan, Yuemeng Li

Notes & Results:

Succeeded.

Construct gene-edited K. xylinus pgi OE

1. Transform pGEM-sacB-5-Ptac-pgi-rrnB-T-sacB-3 into ΔsacB

Perform transformation of the plasmid into ΔsacB K. xylinus cells.

2. Screen positive colonies using non-kanamycin/kanamycin antibiotic resistance

Screen colonies based on their resistance to kanamycin to identify successful transformants.

3. Genotype identification of pgi OE

Perform genotyping to confirm the presence of the pgi overexpression (OE) mutation.

Experimenter:

Xinyi Wang, Dongyun Fan, Yichen Zhao, Yutong Tian, Jinran Xu

Recorder:

Xinyi Wang, Yutong Tian, Jinran Xu

Notes & Results:

Succeeded.

Construct gene-edited K. xylinus pgi-OE gdh-KO double mutant and water retention performance assay of BC

1. Transform pGEM-gdh-5-Ptac-kana-rrnB-T-gdh-3 into pgi OE

Perform transformation of the plasmid into pgi OE K. xylinus cells.

2. Screen positive colonies using kanamycin antibiotic resistance

Screen colonies for kanamycin resistance to identify successful double mutants.

3. Genotype identification of pgi-OE gdh-KO

Perform genotyping to confirm the pgi overexpression and gdh knockout mutations.

4. Measurement of water content of soil after 7 days of drought

Use a soil moisture meter to measure the moisture content, taking three measurements for each pot and calculating the average. Each group will have three biological replicates.

Experimenter:

Dongyun Fan, Yichen Zhao, Yutong Tian, Jinran Xu

Recorder:

Dongyun Fan, Yichen Zhao

Notes & Results:

Succeeded.

Growth curves and BC production of gene-edited K. xylinus. Optimization of fermentation culture conditions

1. Growth curves test

Ferment and culture four different strains under the same medium conditions for 24h, 48h, 72h, 96h, and 120h. Measure the OD600 value of the medium using a Nanodrop.

2. BC production test

After culturing the four strains for 7 days, isolate and purify the BC from the medium, then weigh it after overnight air drying.

3. Optimize the fermentation conditions of pgi-OE gdh-KO

Optimization is done by adjusting the rotation speed and the amount of xanthan gum added. Experimental gradients:

• Rotation speed: 50 rpm, 100 rpm, 150 rpm, 200 rpm
• Xanthan gum addition: 0, 0.02%, 0.03%, 0.05%

After 7 days of culture, isolate and purify the BC, then weigh it after overnight air drying.

4. Observation and statistical analysis

Observe and perform statistical analysis of the sea buckthorn tree status, along with mathematical modeling analysis.

Experimenter:

Dongyun Fan, Yichen Zhao, Xinyi Wang

Recorder:

Xinyi Wang, Dongyun Fan, Yichen Zhao

Notes & Results:

Succeeded.