Protocols

 

DNA Gel Electrophoresis

1. Add 0.4g agarose with 400 mL 1×TAE buffer solution (after dilution) into a flask, blend completely, and heat for 1min to melt the agarose by a microwave oven.

2. Cool the flask down at room temperature till it can be handled by hands, add 2.5 μl SYBR green Ⅰ dye into the melted agarose, and blend completely.

3. Pour the agarose into sealed chamber with comb in position, wait until it solidifies then remove the comb.

4. Place the gel in the electrophoresis chamber with TAE buffer solution in it (wells facing negative electrode).

5. Mix samples with loading buffer (1:1), and load all sample solutions in the gel wells.

6. Add DL5000 DNA marker and DL2000 DNA marker in the side wells of gel.

7. Attach power supply and begin electrophoresis.

8. After the process is finished, remove gel from the chamber, photograph the agarose gel under UV illumination.

 

Heat Shock Transformation

1. Unfreeze the competent cells (DH5α) in an ice box.

2. Add 10 μl of plasmid in 30 μl of competent cell solution into a centrifuge tube, mix them by knocking the tube gently, place the tube on ice and wait for 30 min.

3. Take a water bath for 90sec at 4 2℃ and cool it down instantly on ice for 2-3 min.

4. Add 1ml of LB culture medium and rotate the tube at 200 rpm and 37 ℃ for 1 h.

5. Warm a solid LB plate with antibiotics (100 mg/mL Ampicillin or 50 mg/ml Kanamycin) at 37℃.

6. Centrifuge the tube at 5000rpm for 5min, and suck out 900μl of the supernatant. Use the rest supernatant to resuspend the germs and spread them on the solid LB plate with antibiotics. The antibiotics will kill the germs that failed to transform.

7. Put the plate in a 37 ℃ incubator upside down for 10 to 12 hours.

 

Electrotransformation

1. Unfreeze the competent cells, E. coli Nissle 1917(pKD46)on ice.

2. Add 10 μl of plasmid into 100 μl of cell suspension and mix them by knocking the centrifuge tube gently. Suck the mix out and add it into a shock cup. Put the cup into an electroporator, set the voltage at 1.8 kV. After the shock, add 1ml of LB medium and resuspend the bacteria. Move them into a centrifuge tube and culture the bacteria at 200rpm for 1 h.

3. Warm a solid LB plate with antibiotics (100 mg/ml Ampicillin or 50 mg/ml Kanamycin) at 37 ℃.

4. Centrifuge the tube at 5000 rpm for 5 min, and suck out 900 μl of the supernatant. Use the rest supernatant to resuspend the germs and spread them on the solid LB plate with antibiotics. The antibiotics will kill the germs that failed to transform.

5. Put the plate in a 37 ℃ incubator upside down for 10 to 12 h.

 

Preparation of competent cells (Step 3)

1. Take out the bacteria from the cryogenic refrigerator, thaw on ice,spread them in LB solid culture, and put them in an incubator at 37 ℃ for activation overnight.

2. Single colonies were selected and cultured to logarithmic growth stage in liquid medium 37 ℃ or 30 ℃(temperature sensitive plasmid).

3. The above bacteria were obtained by centrifugation at 5000-8000 rpm 5-10 min, and the supernatant was discarded (Repeat this step 5-6 times).

4. Add pre-cooled 0.1 mol/L calcium chloride solution to resuspend the cells, and place the tube on ice.

 

Colony PCR

1. Prepare the following reaction system:

Constituent	Volume(μl)
ddH2O	up to 50
2×Phanta Max Master Mix	25
Upstream primer (10μM)	2
Downstream primer (10μM)	2
Template	1

 

2. Place the configured PCR sample in the PCR instrument and set up the following PCR system: 

Cycling procedure	Temperature	Time	Cycle number
Pre-degeneration	95℃	5min	1
Degeneration	95℃	30s
Annealing	Tm℃	15s	X
Elongation	72℃	30-60s/kb
Completely Elongation	72℃	10min	1

 

Plasmid Extraction UE Plasmid Miniprep Kit made in China.

1. Add 2-5 mL of overnight cultured bacterial solution into 1.5 mL centrifuge tube, centrifuge at 12000×g for 1min, discard the supernatant, add 250 μL Buffer S1 (confirm that RNAase has been added), and resuspend the cells.

2. Add 250μl Buffer S2, turn the tube up and down 4-6 times, and let it stand until the solution is transparent (no more than 5 min).

3. Add 350μl Buffer S3 and gently turn it up and down for 12-15 times to produce white precipitate, centrifuge at 12000×g for 15min, take the supernatant into a preparation tube (2ml Preparation tube, centrifuge at 12000×g for 1min, and discard the filtrate.

4. Add 500μl Buffer W1 into the preparation tube, centrifuge at 12000×g for 1min, and discard the filtrate.

5. Add 700μl Buffer W2 (add 56 ml absolute ethanol in advance), centrifuge at 12000×g for 1min, and discard the filtrate. Repeat this step once.

6. 12000×g centrifuge for 1 min.

7. The preparation tube was transferred to a new 1.5 mL EP tube and placed in a metal bath at 65 ℃ for 5min to volatilize the ethanol, then add 30μl ddH2O (preheated at 65℃ to improve elution rate), centrifuge at12000×g for 1min, and collect the filtrate. Plasmid DNA is obtained in the filtrate.

8. Label the obtained plasmid DNA, measure concentration by the ultra-micro ultraviolet spectrophotometer and purity and record them on the EP tube.

 

 

One step-cloning   

ClonExpress® II One Step Cloning Kit, Vazyme Biotech Co., Ltd.

 

1. Obtain linearized plasmid vector by PCR. The purified linearized cloning vector was diluted to 50 ng/μL.

2. Get the target fragment by PCR. The insert was diluted to 25.08 ng/μL.

3. The amount of DNA required for recombination reaction was calculated according to the following formula.

Optimal amount of the linearized cloning vector = (0.02 × number of linearized cloning vector base pairs) ng

Optimal amount of the insert = (0.06 × number of the insert’s base pairs) ng

4. The following reaction systems were prepared on ice:

 

 

5. Gently suck and mix the reaction solutions with pipette (do not shake and mix), briefly centrifuge and collect the reaction solution to the bottom of the tube. Conduct the reaction at 37 ℃ for 30 min, reduce to 4 ℃ or cool immediately on ice. (The recombinant product can be stored at -20℃ for a week, and then thawed and transformed when needed.)

 

MTT

1. Weigh 50 mg of thiazolyl blue tetrazolium bromide powder, dissolve in 10 mL of PBS. Pass through a 0.22-micron membrane as MTT storage solution (5 mg/mL).

2. Remove the complete medium in the petri dish, add 2 mL PBS to wash 3 times, add 1 mL of trypsin to digest the cells for 2 min, pipette the cells to detach from the bottom of the petri dish.

3. Collect CT26 cells and put them in a 15 mL centrifuge tube, centrifuge at 1000 rpm for 5 min. After centrifugation, add complete medium to resuspend the cells.

4. Take a hemocytometer and count the cells.

5. CT26 cells were seeded in 96-well plates at a density of 1×10⁴ cells per well. After 12 h incubation, the medium was replaced with arginine at different concentrations, further incubated at 37 °C for 1 h and 24 h respectively.

6. The cells were washed with PBS and MTT working solution (Dilute the MTT storage solution with DMEM medium to 0.5 mg/mL) were added at 1h and 24h. Kept at 37 °C for 4 h.

7. Add 150 mL DMSO to dissolve formazan per well, incubate at 37°C for 20 min.

8. Record the absorbance with the microplate reader at 490nm.

 

ELISA

Mouse lactate ELISA kit, JM-12169M1, jingmei, China

 

 

1. Take the required detachable lath of the ELISA plate from the aluminum foil bag that has been equilibrated to room temperature for 20 min, and seal the remaining laths with the aluminum foil bag and put it back to 4 ℃.

2. Set standard wells and sample wells, and add 50 μL standard diluent of gradient concentrations to each standard well.

3. Add 10μL of the sample to the sample wells, and then add 40 μL of sample dilution to make a five-fold sample dilution (do not add to the blank wells).

4. 100 μL of horseradish peroxidase (HRP)-labeled detection reagent was added to each well of the standard and sample wells including the blank wells, and the reaction wells were sealed with plate sealing membrane and incubated for 60min at 37 ℃ in a water bath or thermostat.

5. Remove the liquid, pat dry on the absorbent paper, fill each well with the washing buffer (20× washing buffer diluent with ddH₂O to 1× washing buffer), hold on for a minute, pat dry on the absorbent paper. Repeat this washing step for 5 times.

6. Add 50 μL of substrate A and B to each well and incubate at 37 ℃ for 15 min in the dark.

7. Add 50 μL of stop solution to each well, and measure the OD value of each well at 450 nm wavelength within 15 min.

 

RT-qPCR( reverse transcription-quantitative PCR ) 

Step 1: Reverse transcription (HiScript II Q RT SuperMix for qPCR, Vazyme)

1. Detection of RNA concentration and make each PCR (0.2 mL) tube containing 1 μg RNA.

2. Each PCR add 4 μL 5× HiScript II qRT SuperMix II.

3. Add RNase-free ddH₂O to 20 μL.

4. Incubate the reaction mixture at 50℃ for 15min and then incubate it at 85℃ for 5s to inactivate the reverse transcriptase.

Step2: Quantitative PCR ( ChamQ Universal SYBR qPCR Master Mix, Vazyme)

1. Use 0.2ml PCR tubes to prepare the reaction system as follows, and 3 tubes were prepared for each reverse transcription product.

Reaction system:

2.ChamQ Universal SYBR qPCR Master Mix 5 μL

Primer1 0.4 μL

Primer2 0.4 μL

Template cDNA 1 μL

ddH₂O 3.2 μL

2. PCR amplification protocol is shown below.

Predenaturation: 95 ℃, 10 min

Cycle (40 times): 95 ℃, 15 s → 60 ℃, 30 s

Preparation of complete medium

1. Add 5ml of fetal calf serum in a 50 mL centrifuge tube.

2. Add 500μl penicillin (100 μg/mL)-streptomycin (0.1 mg/mL).

3. Add DMEM medium to 50 mL.

 

Cell plating

1. Take a petri dish with cells grown overnight, suck out all liquid in it.

2. Add 8-10 mL buffer to wash away the impurities, and dump the buffer.

3. Add 1 mL trypsin, and incubate the dish at 37 ℃ for 5 min.

4. Add 10 mL of complete medium to neutralize the trypsin.

 

Cell counting

1. Clean the counting chamber with alcohol, and put a coverslip on it.

2. Use a pipette gun to add a drop of cell suspension in the fillister on the chamber. Put the fillister under a microscope and count the cells in the 16*25 area. There are roughly 10000 times of the counted cell numbers in 1ml of suspension.

 

Cell RNA Extraction

1. Remove all liquid from the 6-well plate containing cell culture, 1ml PBS use to clean the impurities, add 600 μL Biozol and blow the cells off the well bottom.

2. Transfer the suspension to a 1.5ml centrifuge tube (previously labeled), oscillate the tube with a vortex oscillator for 10-15s, and stand for about 5min.

3. Add 250 μL chloroform to separate RNA from the suspension, oscillate the tube for 10-15 s, and stand about 5 min.

4. Centrifuge the tube at 12000 rpm, 4 °C for 15 min.

5. Transfer 300 μL supernatant to a new 1.5 mL centrifuge tube.

6. Add 300 μL isopropanol to the new tube to lower the solubility of RNA and make it separate out, turn the tube up and down 10 times, stand it at 4 °C in a centrifugal machine for 10 min.

7. Centrifuge at 12000 rpm, 4°C for 10 min, and abandon the supernate.

8. Centrifuge at 12000 rpm for 30 s, and remove all the residual liquid in the tube.

9. Add 600 μL 75% ethanol, turn the tube up and down 5 times to clean left isopropanol.

10. Centrifuge the tube at 12000 rpm, 4 °C for 7 min, and abandon the supernate.

11. Centrifuge at 12000 rpm for 30 s, and remove all the residual liquid in the tube.

12. Open the centrifuge tube’s lid and stand the tube for 5~8 min (Avoid impurities from entering and polluting the tube)

13. Add 10μl ultra-pure water (UPW) to dissolve the RNA.

14. Store in -80 ℃ refrigerator.

 

L-Arginine detection

L-Arginine was detected by Hitachi amino acid analyzer. The 24- and 48-hour fermentation broth of engineering bacteria were collected and broken by ultrasonic crusher (100ml fermentation broth: ultrasonic 5 seconds / interval 5 seconds; 70 times; power 300 W), centrifugation (12000 g 30min) to discard cell fragments, filtered by 0.22 μm pore diameter filter, and detected by automatic amino acid analyzer.