Team:HUBU-WUHAN/Procedure

Procedure

PROCEDURE

Preparation of competent cells of E. coli

  1. Streak from Escherichia coli stock on Luria–Bertani medium (LB, 10 g/L tryptone, 5 g/L yeast extract, 10 g/L NaCl) agar plate. Incubate overnight at 37 °C.
  2. Pick a single colony of E. coli, incubate in 1 L flask containing 100 mL of NZY liquid medium, and incubate at the conditions of 18 °C, 250 rpm for about 16 h (optimal OD600nm= 0.6-0.8).
  3. After incubation, divide into two 50 mL centrifuge tubes, collect cells at 4 °C, 4000 rpm for 10 min, and remove the supernatant.
  4. Resuspend the cells by adding 30 mL of pre-cooled TB buffer (4 °C) to each centrifuge tube, shake vigorously.
  5. Repeat step 4.
  6. Add 10×OD600nm pre-cooled TB buffer solution to each centrifuge tube, and slowly add DMSO to a final concentration of 7% and make it fully dispersed.
  7. The E. coli competent cells are prepared by dispensing on ice, 100-200 μL of bacterial liquid per tube, place in liquid nitrogen for quick freezing and then transfer to the -80 °C refrigerator.
  8. Test whether the competent cells is contaminated.

Calculation of Transformation Efficiency

  1. Add 0.01 ng of pEZ15A plasmid to a 1.5 mL EP tube containing 50 μL of T1 competent cells, gently spin and mix, and stay in ice for 30 min. (pEZ15A plasmid with spectinomycin resistance).
  2. Heat shock at 42 °C for 45 s and immediately place it on ice for 2 min.
  3. Add 100 μL of LB liquid medium to the EP tube and incubate at 37 °C, 250 rpm for 1 h.
  4. Apply 50 μL of the above solution to LB agar plates with spectinomycin (0.15 mg/mL). At the same time, 50 μL of T1 is applied to LB with spectinomycin (0.15 mg/mL) agar plates. If the plates do not grow on the next day, it means that the competent cells are not contaminated.
  5. On the next day, record the number of colonies on the plates for measuring efficiency and calculate the conversion efficiency. Transformation efficiency = total number of colonies/total amount of DNA = number of colonies × (500 / 50) ×103 / 0.01 cfu/μg

Preparation of competent cells of Zymomonas mobilis

  1. Streak from ZM4 (model strain of Z. mobilis) stock on rich medium (RMG2, 10 g/L yeast extract, 20 g/L glucose, 2 g/L KH2PO4) plate. Incubate at 30 °C about 2 days.
  2. Pick a single colony of ZM4 and incubate in 8-9 mL RMG2 liquid medium and incubate at 30 °C for 2 day.
  3. Transfer the appropriate amount of bacterial into 200 mL of RMG2 liquid medium to make the initial OD600nm = 0.025-0.03, and collect cells until OD600nm=0.4-0.6.
  4. Cool the bacterial solution on ice for about 10 min, then collect the cells (4 °C, 4,000 rpm, 10 min) with two 50 mL centrifuge tubes. (100 mL of bacterial solution is collected per centrifuge tube).
  5. Add 40 mL of pre-cooled sterile water (4 °C) to each tube, resuspend and wash the cells, centrifuge (4 °C, 4,000 rpm, 10 min) and discard the supernatant.
  6. Add 40 mL of pre-cooled 10% glycerol (4 °C) to each tube, resuspend and wash the cells, centrifuge (4 °C, 4,000 rpm, 10 min) and discard the supernatant.
  7. Add 1 mL of pre-cooled 10% glycerol (4 °C) to each tube and resuspend the cells. Dispense ZM4 on ice. Load 55 μL of bacteria solution per EP tubes. Store in liquid nitrogen after dispensing and transfer to the -80 °C refrigerator.
  8. Test whether the competent state is contaminated.

T5 exonuclease assisted cloning

T5 exonuclease assisted cloning method is based on the Gibson assembly, but the operation of T5 method is easier. Currently, it is a commonly used cloning method in our laboratory. The steps are as follows:

  1. Design homologous arm (15 bp).
  2. Prepare reactive system (5 μL).
    F/V: molar ratio of 3:1
    T5 exonuclease (NEB, USA): 0.5 μL
    Buffer: 0.5 μL
    ddH2O: complemented to 5 μL
    All regents are mixed and react on the ice for 5 min.
  3. Add 100 μL T1 competent cells and react on the ice 30 min.
  4. Heat shock, 42 °C for 45 s, stay in 2-3 min.
  5. Add 10 μL NZY to recovery, culture at 37 °C with 250 rpm for 1 h.
    NZY medium: 1% NZ-Amine, 0.5% yeast extract, 0.5% NaCl
    (autoclaving), 12.5 mmol/L MgCl2·6H2O, 12.5 mmol/L MgSO4·7H2O, 20 mmol/L glucose (filtration sterilization).
  6. 6. Plate cells on LB agar plates with special antibiotics.

Electroporation transformation

Electroporation is used to transfer plasmids or fragments into Z. mobilis. The steps are as follows:

  1. Precool the cuvettes and prepare competent cell in the ice.
  2. Mix plasmid (0.5-10 ng) and 50 μL competent cells in the cooling cuvette.
  3. In 0.1-cm electroporation cuvettes, the cells and plasmid DNA are electroporated (1.6 KV, 25 μF, and 200 ohms) using a Bio-Rad Gene Pulser.
  4. The electroporated cells are transferred to 1 mL of mating medium (glucose 50 g/L, yeast extract 10 g/L, tryptone 5 g/L, (NH4)2SO4 2.5 g/L, K2HPO4 0.2 g/L, 1 mM MgSO4), and recovered at 30 °C for 6 to 12 h.

PCR

  1. PCR reaction:
    Reagent Volume Concentration
    Primer F 2 μL 10 μM
    Primer R 2 μL 10 μM
    Template 1 μL 20 ng
    Primer Star 25 μL 2 ×
    H2O up to 50 μL /td>
  2. Parameters:
    ...
  3. Loading samples and running an agarose gel:
    Pouring a standard 1% agarose gel, add loading buffer to each of DNA samples. Fill gel box with 1x TAE buffer until the gel is covered. Carefully load a molecular weight ladder into the first lane of the gel. Carefully load samples into the additional wells of the gel. Run the gel until the dye line is approximately 75-80% of the way down the gel.

Screening of target strain

  1. PCR reaction:
    Reagent Volume Concentration
    Primer F 2 μL 10 μM
    Primer R 2 μL 10 μM
    Template 1 μL 20 ng
    Primer Star 25 μL 2 ×
    H2O up to 50 μL /td>
  2. Parameters:
    ...

Plasmid extraction

  1. Add 250 μL BL Buffer (TSINGKE) to the adsorption column AC, centrifuge at 12000 rpm for 1 min.
  2. Collect 1-4 mL of cultures and centrifuge for 1 min at 12000 rpm, discard the supernatant.
  3. Add 20 μL of Buffer S1 (placed in a 4 °C freezer, check for RNase A before use) and resuspend the bacterial pellet.
  4. Add 200 μL Buffer S2, gently invert the tube 4-6 times to completely lyse the bacteria.
  5. Add 200 μL Buffer S3, gently invert the tube 4-6 times to mix. Centrifuge at 12000 rpm for 10 min, and white precipitate appears after centrifugation.
  6. The supernatant after centrifugation in the previous step is poured into an adsorption column. Centrifuge at 6000 rpm for 1 min and centrifuge at 12000 rpm for 1 min. The supernatant is resuspended in the column by pouring back to the adsorption column, and the liquid in the collection tube is discarded.
  7. Add 700 μL Buffer W2 to the adsorption column (please check if absolute ethanol has been added), centrifuge at 6000 rpm for 1 min, and discard the liquid in the collection tube.
  8. Repeat step 7 once again.
  9. Place the adsorption column back into the empty collection tube and centrifuge at 12000 rpm for 2 min.
  10. emove the adsorption column, place it in a clean 1.5 mL EP tube, and open it at 65 °C for 4-5 min.
  11. Add 30 μL of H2O to the middle of the adsorption membrane, let stand for 1 min at 65 °C, and centrifuge for 2 min at 12000 rpm.

DNA gel extraction

  1. Under the UV lamp, cut the gel as small as possible, put it into a 2 mL centrifuge tube.
  2. Add 500 μL Buffer GL and heat at 65 °C for 4-6 min. Do not mix upside down 2-3 min, only completely melted. At the same time, 250 μL Buffer BL is added to the adsorption column, and 12000 rpm is centrifuged for 1 min to activate the silica gel membrane.
  3. Transfer the solution to the adsorption column, centrifuge at 6000 rpm for 1 min, and centrifuge at 12000 rpm for 1 min.
  4. Pour back into the column, centrifuge at 6000 rpm for 1 min, and centrifuge at 12000 rpm for 1 min.
  5. Add Buffer W2 700 μL, centrifuge at 6000 rpm for 1 min, and discard the waste.
  6. Repeat step 5 once again.
  7. Discard the waste, 12000 rpm for 2 min.
  8. Remove the adsorption column, place it in a clean 1.5 mL EP tube, and open it at 65 °C for 4-5 min.
  9. Add 30 μL of H2O to the middle of the adsorption membrane, hold for 4-5 min at 65 °C, and centrifuge for 2 min at 12000 rpm.

Extraction of crude enzyme solution

  1. Extraction of intracellular crude enzyme solution.
    • Collect 100 mL fermentation broth at 4000 rpm 5 min in 4 °C using 50 mL centrifuge tubes.
    • Use 10 mL PBS to resuspend bacteria.
    • 4 m/s 40 s in high speed grinding machine.
    • 12000 rpm centrifuge for 10 min in 4 °C.
    • Supernatant is intracellular crude enzyme solution.
  2. Extraction of extracellular crude enzyme solution.
    • Collect 100 mL supernatant of fermentation broth by centrifugation.
    • Slowly add anhydrous ammonium sulfate powder (48.1 g anhydrous ammonium sulfate per 100 mL bacterial solution) in batches on ice at a precipitation concentration of 80%.
    • Stay overnight in 4 °C.
    • Centrifuge for 13000 rpm, 5 min. Retain the precipitation.
    • Add 2 mL PBS for per 100 mL fermentation broth, resuspend and get the extracellular crude enzyme solution.

Western blot analysis

  1. Log phase and stationary phase cells are harvested to conduct the flow cytometry analysis.
  2. Cells are lysed and protein is extracted using Protein Extraction Kit (Zomanbio, China). Total protein concentrations of total cellular lysates are measured by the Bradford method with 200 ng of total protein loaded for each sample.
  3. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) is performed with a 5% stacking and a 12% running gel, followed by stain with Coomassie Brilliant Blue R-250. Molecular weight is estimated using a pre-stained protein ladder (10–170 kDa, Termo, Lithuania).
  4. For Western blot analysis, after the electrophoresis, gels are transferred to methanol-activated PVDF membranes using the Trans-Blot® Semi-Dry Electrophoretic Transfer Cell (Bio-Rad, USA) and run for 20 min at 25 V. PVDF membranes is then blocked with 5% non-fat milk in phosphate-bufered saline with Tween 20 (PBST) for 1 h at room temperature, and subsequently EGFP or opmCherry are probed with primary antibody (1:5000, Proteintech, China), respectively. Peroxidase-conjugated goat anti-Mouse IgG (1:5000, Proteintech, China) is used as secondary antibodies. Color development is performed by West Dure Extended Duration Substrate Kit (AntGene, China). All images are visualized using AI600 Imaging System (GE, USA), and analyze by ImageJ (National Institutes of Health, USA).

The DNS method of measuring enzyme activity

  1. Add 500 μL 1% CMC, 250 μL citrate buffer solution (pH=6), 250 L crude enzyme solution.
  2. Incubate at 30 °C for an hour, blank groups skip this step.
  3. Add 500 μL DNS, and incubate at 100 °C for 5 min.
  4. Measure the absorbance at 540 nm, compare with standard curve

Measurement of standard curve

Configurate the reaction system as follow, incubate at 100 °C for 5 min. Measure the absorbance at 540 nm. Take absorbance as ordinate, glucose concentration as abscissa, make glucose standard curve.

glucose concentration (mg/mL) 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4
1 mg/mL Glucose solution 0 50 100 150 200 250 300 350 400
ddH20 (mL) 500 450 400 350 300 250 200 150 100
citrate buffer solution (pH=6) (mL) 250
1% CMC (mL) 500
DNS (mL) 500

Construction of the CRISPR-Cas9 system of Z. mobilis for genome engineering

  1. Construction of Interference plasmids
    • First, a DNA block composed of the leader sequence of the chromosomal CRISPR2 as a promoter and two CRISPR repeats spaced by two BsaI restriction sequences in opposite orientation is synthesized from GenScript (Nanjing, China), and used as a template for PCR amplification with the primer pair of L2R-XbaI-F/L2R-EcoRI-R.
    • Then, the PCR product is digested with XbaIand EcoRI and subsequently inserted into the pEZ15Asp vector at the same sites, giving pL2R. Digestion of pL2R with BsaI generated a linearized plasmid having protruding repeat sequences of 4 nt at both ends. Double-stranded spacer DNAs are prepared by annealing two spacer oligonucleotides through heating to 95°C for 5 minutes and then cooling gradually down to room temperature. The spacer fragments are designed to carry 4 nt protruding ends complementary to those in the linearized pL2R.
    • Subsequently, donor DNA fragments each containing a mutant allele of a target gene are generated by PCR and individually cloned onto their cognate pST plasmids by T5 exonuclease assisted cloning
  2. Construction and screening of mutants
    • The plasmids used for genome engineering are individually introduced into Z. mobilis cells.
    • Electroporated cells are spread on RGM agar plates containing spectinomycin at a final concentration of 200 µg/mL (RGMSp) and incubate at 30 °C until colonies are observed.
    • Mutant candidates are checked by colony PCR.
  3. Curing of genome engineering plasmids
    • Cells of genome engineering plasmid transformants are grown up in an RMG2 broth with the supplement of spectinomycin.
    • A few single colonies are picked and individually suspend in 10 µL of sterilized ddH2O, and 1 µL of each cell suspension is spotted on an RMG agar plate with or without spectinomycin.
    • 3) Cells from the same suspension grow up on the plate without spectinomycin but not on that with spectinomycin are regarded as those lost the genome engineering plasmid.

Determination of PHB using HPLC

  1. Add 10 mg PHB (sigma:363502-10G) into colorimetric tube with 5 mL 98% sulfuric acid.
  2. Heat it in 90 °C water for 30 min.
  3. Take out and cool it fully to indoor temperature.
  4. Dilute the solution 100 times with 5 mmol sulfuric acid. Then use the 5 mL volumetric flask to determine the volume. The mother liquor concentration is 20 μL/mL now.
  5. Dilute the mother liquor with 5 mmol sulfuric acid into 0.5, 1, 2, 3, 4, 5, 6, 7, 8 μL/mL.
  6. Filter the samples through a 0.2-μm syringe filter and add 400 μL filtrates into HPLC vials.
  7. The filtered 95% sulfuric acid is used as a blank control to measure.
  8. Add 10 mg dry bacteria into the colorimetric tube with 5 mL 98% sulfuric acid.
  9. Heat it in 90 °C water for 30 min.
  10. Take out and cool it fully to indoor temperature and dilute it 100 times with 5 mmol sulfuric acid.
  11. Filter the samples through a 0.2-μm syringe filter and add 400 μL filtrates into HPLC vials.
  12. Use an HPLC system (HPLC Prominence, Shimadzu, Japan) equipped with a Bio-Rad Aminex HPX-87H column (Bio-Rad, Hercules, CA, U.S.A.) and a refractive index detector (RID) to determine the concentration of crotonic acid in acid solution.

The conditions of HPLC

  1. Mobile phase: 5 mmol sulfuric acid.
  2. UV detection wavelength: 210 nm.
  3. Flow rate: 0.6 mL/min.
  4. Column temperatue: 60 °C.

Sample preparation for FACS

  1. Absorb 200 μL bacterial solution into 2 mL centrifuge tube.
  2. Collect cells at 4000 rpm for 1 min, and remove the supernatant.
  3. Resuspend the cells by adding 400 μL of PBS buffer to centrifuge tube.
  4. shake to mix and ensures no cell clumps.
  5. Repeat step 2 and 3 for two times.
  6. Measure the sample in 30 minutes.

Measure fluorescence use CytoFLEX

  1. Open CytoFLEX and then open the control software CytExpert.
  2. Set “Acq. Setting”:
    Gain:FSC:98, SSC:86, FITC:50, PE:136, PC5.5:309, PC7:484, APC:487, APC-A750:520
    Threshold:SSC-H>2000.
  3. Initialize and load sample, click “Record” to start measurement.
  4. Load next sample and click “Next Tube”.
  5. After measurement completed, do “Daily Clean” with LiAC and Ultrapure water.
  6. Export data as CSV file, calculate the ratio of GFP to mCherry.
  7. Close computer and CytoFLEX.

Liquid nitrogen grinding

  1. Collect fermentation broth at 4000 rpm in 4 °C.
  2. After centrifugation the supernatant is discarded.
  3. Dry the wet bacteria.
  4. Put the dry bacteria in the precooled mortar. Add appropriate liquid nitrogen and carefully grind the bacteria until it is fine enough.
  5. Add the powder into the neutralized hydrolysate.