Protocol
Experiment on microbiology
LB medium (for E. coli )
Tryptone: 10 g/L
Yeast extract: 5 g/L
Sodium chloride: 10 g/L
Agar: 15 g/L
RMG2/5 (for Z. mobilis ）
Glucose: 20 /50 g/L
Yeast extract: 10 g/L
Potassium dihydrogen phosphate: 2 g/L
Agar: 15 g/L
Inoculation and preservation of strains
From liquid to liquid: Use the pipette to transfer the bacterial solution into 6 mL liquid medium with special antibiotic added.
From liquid to solid: Use the tip of the pipette to streak the plate in special antibiotics solid medium.
Strain preservation: Add 60% glycerol solution and bacterial solution to the frozen storage tube with one to one volume ratio and keep in-80 oC refrigerator.
Strain culture
Culture of E. coli
Plate culture：Preserved / cultured strains are inoculated into solid LB medium with special antibiotics, and plates are incubated by inverted in a 37℃ incubator overnight.
Liquid culture:The strains are inoculated into 6 mL of special antibiotics liquid medium on a constant temperature shaker and grown overnight at 37℃, 250rpm.
Culture of Zymomonas mobilis
Plate culture：Preserved/ cultured strains are inoculated into solid RMG2 medium with special antibiotics, and the plate are sealed and placed inverted in a 30℃ incubator for two days.
Liquid culture:The preserved strains are inoculated into 200µL liquid RMG 5 medium on a constant temperature incubator and incubated for half a day.
Experiment on molecular biology
Preparation of competent cells of E. coli
1.Pick a single colony of E.coli ,incubate in 10 mL LB medium, and incubate at the conditions of 37℃, 250 rpm shaking overnight.
2.After incubation,take1-2% overnight culture media into 500 mL triangular flask containing 100 mL of LB medium and incubated at 37℃ with 250 rpm shaking for 3 h to make OD 600 to 0.4-0.6. (optimal OD600nm= 0.4-0.6).
3.After incubation,divide into two sterilized ice-cold 50 mL centrifuge tubes to ice bath cells for 30 min.
4.4℃, 4000 rpm for 10 min (after the operation is strictly on ice, 10% glycerol, EP tube and other required material should be pre-cooled). Remove the supernatant , the cells are suspended in 40 mL ice cold 0.1 M CaCl2 for 25 min, centrifuged at 4000 rpm for 10 min, remover the supernatant .
5.Repeat step 4.
6.All cells are re-suspended in pre-cooled 0.1 M CaCl2 containing 15% glycerol.
7.Divide them in EP tubes, 80-100 μ L of each EP tube.
8.Place in liquid nitrogen for quick freezing.
9.Use it immediately for transformation or keep the packed competent cells in a-80℃ refrigerator.
Primer design
Principle of primer design：
1.The primer length is generally between 15 and 30 bp. The primer length is usually 18-27 bp, but should not be greater than 38 bp, because too long will cause its extension temperature is greater than 74℃, which is not suitable for the reaction of Taq DNA polymerase.
2.The GC content of the primers is between 40% and 60%: too high or too low GC content is not conducive to trigger the reaction.The Tm value (melting temperature) of the upstream and downstream primers is the effective start temperature of the unwinding temperature of the oligonucleotide, which is generally lower than the Tm value of 5~10℃.
3.Bases should be randomly distributed: primer sequences should not have high similarity within the template, especially sequences with high similarity at the 3 ′ end, otherwise it is easy to lead to errors.
4.There should be no complementary sequence between the primer itself and the primer.
5.The 5 ′ end of the primer can be modified, but not the 3 ′ end: the 5 ′ end of the primer determines the length of the PCR product, which has little effect on the amplification specificity.
Polymerase chain reaction
1.PCR reaction:
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.
4.Overlap
1)system:50µL
Prime STAR Max:25µL
Primer F/R：2µL×2
fragment1：1~2µL
fragment2：1~2µL
H2O：up to 50µL
2）Parameters
Add the F/R primers，run again
3）Recovered the product gel
Purify the PCR products
1.Direct recovery of PCR products
Add Buffer GL according to the ratio of PCR stock solution: Buffer GL=1:3 (when PCR stock solution is lower than 50 μL, Buffer GL dosage is 150μL) and mix well (for example, in 1.5 mL centrifuge tube, 50μLPCR stock solution is mixed well after adding 150μLBuffer GL). After the activation of the adsorption column (step 1 below), add the above mixture directly to the adsorption column and proceed to step 5.
2.DNA recovery in the agar gel
1)Activate the silica gel membrane：Place the adsorption column in the collection tube，add 250μLBufferBL, centrifuge at 12000rpm for 1min,discard the waste.
2)Under the UV lamp, cut the gel as small as possible, put it into a 2 mL centrifuge tube.
3)Add 500μLBuffer GL (If the gel is too large, increase the amount of Buffer GL appropriately)；
4)65℃ water bath for 4 to 6 min, mix upside down every 2 to 3 min until the gel is completely melted, the solution is light yellow (if the solution is lilac, add an appropriate amount of Buffer GL to the solution to light yellow);
5)Transfer the solution to the adsorption column,centrifuge at 12000rpm for 1 min,discard the waste,place the adsorption column back to the empty collection tube.
6)Add 700μL Buffer W2 to the adsorption column (please check whether the specified volume of absolute ethanol has been added), centrifuge at 12,000rpm for 1min, and discard the waste .
7)Repeat step 6 once.
8)Placed the adsorption column back into an empty collecting tube and centrifuged at 12,000rpm for 2min.
9)Remove the adsorption column, place it in a clean 1.5 mL EP tube, and open it at 65 °C for 4-5 min to dry the left ethanol.Add 30μL 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.
T5 exonuclease assisted cloning
1.Prepare reaction system(5µL)
F/V:molar ratio of 3:1
T5 Exonulease: 0.5 μL
Buffer: 0.5 μL
ddH2O: complemented to 5 μL
All regents are mixed and react on the ice for 5 min.
2. Add 50μL E. coli DH5α competent cells and react on the ice 30 min.
3.Heat shock at 42℃ for 45s and then immediately put in an ice bath for 3 min.
4.Add 1 mL of LB medium and then incubate at 37℃ for 1 hour.
5.Collect cells at 1,2000 rpm for 1 min, remove the supernatant and the cells resuspended with residual medium, then Plate all cells on soild LB medium with special antibiotics.
6.The plates are inverted and incubated in incubator at 37℃.
Screening of target strain（colony PCR）
1.Prepare reaction system(10µL）
Pick a single colony in pcr tubes containing 10µLH2O,take 1µL mixed bacterial solution. Primer F/R: 1μL
T5 Super PCR mix:5μL
H2O：up to 10µL
2.Parameters（refer to PCR）
Plasmid extraction
1.(Optional) Activate the silica gel membrane: place the adsorption column in the collection tube, add 250µL Buffer BL, centrifuge at 12,000rpm for 1min, and discard the waste .
2.Take 1 ~ 6 mL of overnight culture solution and centrifuge at 12,000rpm for 1min, collect the cells and remove the supernatant Precipitation of resuspended bacteria.
3.Add 250uL Buffer PA (Please check for RNase A and TSINGRed have been added),suspensed cells ,vortex until there are no obvious clot.
4.Add 250 μL Buffer PB, gently invert the tube 4-6 times to completely lyse the bacteria（ This step requires gentle invert and should not be violently shaken.After mixing, the bacteria should become clear and viscous. If it is not clear, it may be due to the excessive amount of bacteria and insufficient lysis, so the amount of bacteria should be reduced. After using TSINGRed, if the bacteria is fully cracked, the solution should be completely changed from cloudy pink to clear purple; if insufficient, the purple solution is mixed with cloudy pink. At this point, continue to flip up and down and mix until the solution becomes clear purple).
5.Add 350 μL Buffer PC, gently invert the tube 4-6 times to mix，centrifuge at 12000rpm for 10min（After using TSINGRed, if the neutralization is sufficient, the solution should completely change from clear purple to light yellow, with white flocculent precipitation）.
6.The supernatant after centrifugation in the previous step is poured into an adsorption column，centrifuge at 12,000rpm for 1min，place the adsorption column back to the empty collection tube.
7.Add 500L Buffer PWA to the adsorption column, centrifuge at 12,000rpmfor 1min,discard the liquid.
8.Add 600 μL Buffer PWB to the adsorption column (please check if absolute ethanol has been added), centrifuge at 12000 rpm for 1 min, and discard the liquid in the collection tube.
9.Repeat step 8.
10.Remove the adsorption column, place it in a clean 1.5 mL EP tube, and open it at 65 °C for 4-5 min to dry the left ethanol.Add 30μL 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.
Preparation of competent cells of Zymomonas mobilis
1.Prepare sterile water, RMG 5 medium, and 10% glycerol solution. Streak the Zymomonas mobilis soltion on RMG 2 plate, and cultured in 30 °C constant temperature incubator for activation, for 2 days long (pick a single colony for verification if necessary).
2.Pick activated single colonies of Zymomonas mobilis into bacterial vials containing about 10 mL of RMG 5 liquid medium and culture for 30 °C overnight (different volumes of RMG 2 can be set to ensure that the next day activated fluid is in logarithmic phase). Transfer an appropriate amount of logarithmic solution into a 500 mL triangle containing 200 mL RMG5 liquid medium to set the initial OD600nm at at 0.025-0.05. 100 rpm, 30 °C until OD600 nm is between 0.3 and 0.4.The angles are incubated on a 30 °C shaker at 100 rpm until the OD600 nm is between 0.3 and 0.4.
3.Collect the cells (25 °C, 4,000 rpm, 10 min) with four 50 mL centrifuge tubes. (50 mL of bacterial solution is collected per centrifuge tube),after centrifugation, carefully discard the liquid in the horizontal flow clean bench.
4.Add 40 mL sterile water to each tube, resuspend and wash the cells, centrifuge (25 °C, 4,000 rpm, 10 min) and discard the supernatant.
5.Add about 5 mL of sterile water to each tube, resuspend the cells, concentrate the 4 tubes in a centrifuge tube, fill with sterile water to 40 mL, resuspend and wash the cells; prepare another centrifuge tube and centrifuge together with it; Centrifuge (25 °C, 4,000 rpm, 10 min)and carefully discard the supernatant in the horizontal flow clean bench.
6.Add 40 mL 10% glycerol to each tube, resuspend and wash the cells, centrifuge (25 °C, 4,000 rpm, 10 min) and discard the supernatant in the horizontal flow clean bench.
7.Repeat step 6.
8.Add 200 µ L of 10% glycerol and resuspend the cells,Load 55 μL of bacteria solution per EP tubes. Store in liquid nitrogen after dispensing and transfer to the -80 °C refrigerator.
Electroporation transformation
Electroporation is used to transfer plasmids or fragments into Z. mobilis. The steps are as follows:
1.Pre-cooled electric cup, put the competent cell, plasmid on ice.
2.Mix plasmid and competent cell into the cooling electric cup.
3.Carefully dry the outside of the cup and press the start button,the cells and plasmid DNA are electroporated (1.6 KV, 25 μF, and 200 ohms) using a Bio-Rad Gene Pulser.
4.Put the G5 medium into the electric cup, mix, absorb, and repeat several times.
5.The mixed medium is put into the EP tube, plastic-sealed and placed in the 30 °C incubator.
Vector construction (pL2R-gRNA)
1.The gRNA is synthesized as a primer. It is designed to bear the entire 32-bp sequences containing a 5′-CCC-3′ PAM with a 4 bp restriction site added at the 5′ end.
2. pL2R digestion (Digestion of pL2R with BsaI generated a linearized plasmid having protruding repeat sequences of 4 nt at both ends)
System: 50 µL
pL2R: 2 µg
BsaI: 1 µL
10×Buffer: 5 µL
H2O: up to 50 µL
Add the mixture in a 2 mL EP tube and place it in a 37 °C water bath for 12 h (overnight)
3.gRNA annealing (The spacer fragments are designed to carry 4 nt protruding ends complementary to those in the linearized pL2R)
System: 10 μL
Primer F/R: 1 μL
H2O: up to 10 µL
Add the mixture above in a 200 μL EP tube, heat it to 95 °C for 5 minutes and then cool gradually down to room temperature.
4. Ligation by T4 DNA Ligase (T4 DNA ligase links the linearized plasmid with the annealed double-stranded gRNA into a circular plasmid)
5.Add 50 μL E. coli DH5α competent cells and react on the ice 30 min
6.Heat shock at 42 oC for 45 s and then immediately put in an ice bath for 3 min
7.Add 1 mL of LB medium and then incubate at 37 oC for 1 h
8.Collect cells at 12,000 rpm for 1 min, remove the supernatant and the cells resuspended with residual medium, then plate all cells on soild LB medium with special antibiotics.
9.The plates are inverted and incubated in incubator at 37 oC.
10.Colony PCR, and then select the correct bacterial solution with correct gene size and raise it.
11.Extract thr correct plasmid pL2R-gRNA，and PCR amplify with appropriate primers to get the vector fragment.
12.Recovery the vector fragmentin the agarose gel.
Donor DNA preparation
1.The (US) upstream sequence and downstream sequence (DS) of the knockout or replaced target gene are designed as homology arms.
2. PCR amplify the US and DS .
3. Construct the donor DNA through overlapping the target gene with US and DS fragment.
4. Ligate the donor DNA with the vector fragment by T5 exonuclease.
Construction and screening of mutants
1.Electrotransferred the plasmid into Zymomonas mobilis.
2.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.
3.Mutant candidates are checked by colony PCR.
Curing of genome engineering plasmids
1.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.
2. 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.
Fermentation of Zymomonas mobilis
1.Glycerol preservation bacteria activation :Take the glycerol bacteria from the -80 °C refrigerator, 100 μL are added to a cryopreservation tube containing 2 mL liquid RMG5 culture with special antibiotics, then put it in the incubator to cultivate to turbidity.
2.Transfer to a large system for expanded culture as seed liquid :Add the bacteria from the whole cryopreservation tube to a centrifuge tube containing 20 mL/30 mL liquid RMG5 with special antibiotics and incubate overnight in a 30 °C incubator.
3.Prepare medium for fermentation: 40 mL RMG5 medium，the second day，clean the horizontal flow clean bench and fully irradiate and sterilize with ultraviolet rays.
4.Measure the OD value of seed liquid (seed liquid should be blown evenly):The initial OD value of the control fermentation culture medium is about 0.10. The OD value of seed liquid is measured for one night of culture, and the algorithm is 0.1*40=OD2*x.xμL is the bacterial solution to be added. Add all media to a certain concentration of the resistance of the corresponding bacteria, and set the tetracycline gradient (generally set the tetracycline gradient for each parallel group).
5.After collecting x ml of seed solution, resuspend it with medium into a triangular flask containing 40 ml of medium of interest (RMG5) and mix well.
6.Connect the medium label of the seed liquid to mark, suck and beat evenly and take samples, put them in the spectrophotometer to measure the 0 h OD value, measure the OD in one bottle, and retained one bottle.
7.Place the fermentation medium in a 30 °C -shaker culture.After that: 0 h, 3 h, 6 h, 9 h, 12 h, 24 h, 30 h, 36 h, 48 h ( according to the specific situation to adjust the sampling time ) sampling OD value, sample retention, generally for 48 h fermentation test.
Strain growth curves
1.Take the cryopreserved bacteria and transfer 100 µm into 200 mL medium, and put in a 30 °C incubator overnight to grow the bacteria until mid-log phase.
2.The medium was sterilsterile and divided into 50 mL triangular flasks.
3.Collect the seed liquid and centrifuge to remove the background culture medium. After washing twice with fermentation medium, adjust the initial OD600 nm to 0.1 and inoculate it into the corresponding fermentation medium, placing it on a shaker for cultivation.
4. Sample collection is performed every 3 h, measuring the OD600 nm using a spectrophotometer. Additionally, 1 mL is taken in a sterile EP tube, and after centrifuging to separate the bacterial cells, the supernatant is collected. After passing it through a sterile 0.22 µm filter, it is stored at -20 °C. This will be used later for glucose and ethanol analysis.
5. Continue this process until sugar depletion in the sample. After obtaining the data, perform data processing with time on the x-axis and OD600 nm on the y-axis, using GraphPad Prism 8.0 for plotting and conducting significance analysis.
Determination of isobutanol using HPLC
1. Prepare the mobile phase: prepare sulfuric acid solution with a concentration of 5 mmol/L, and use a filter film with a pore diameter of 0.22 μm to defilter the mobile phase.
2. Ultrasonic degassing of the mobile phase: ultrasonic degassing of the mobile phase after filtration 20-30 min, and then used after returning to room temperature.
3. Standard product configuration.
4. Sample processing: After high-speed centrifugation, add 500 μL supernatant sample into the sample bottle through a 0.22 μm filter.
5. Sample analysis: concentrations of isobutanol, glucose, and ethanol in the supernatant was determined by a HPLC system (HPLC Prominence, Shimadzu, Japan) equipped with a Bio-Rad Aminex HPX-87H column (Bio-Rad, Hercules, CA, USA) and a refractive index.
Detection by flow cytometry
With a fluorescent tag on the recombinant plasmid, different inducer concentrations can be set, and the optimum inducer concentration can be found according to the fluorescence intensity.
1.Remove the frozen bacteria in the-80 °C refrigerator,dashed on the special antibiotics plates,and culture for activation in a constant-temperature incubator.
2.Pick a single colony in an appropriate volume of liquid medium and culture until the late of logarithmic phase and preserved with 60% glycerol.
3.For growth testing, take glycerol bacteria and inoculate them into the culture medium until the mid-log phase period and then inoculate.
4.The cells were collected using the EP tube and the above cells are inoculated to the liquid medium containing different concentrations of tetracycline in the deep well plate, so that the initial OD600 nm is 0.1 with three biological repeats for each concentration. 30 °C, 100 rpm for Zymomonas mobilis, and 37 °C, 250 rpm for E. coli.
5.Culture until 8 h and 16 h,then collect cells and preserved.
6.The collected bacterial solution is loaded into 2 mL EP tubes and centrifuged at 12,000 rpm for 1 min, discard the supernatant and cells were resuspended and centrifugation using PBS. Cells were washed three times and finally resuspended to 107 cell number / mL and configured as sample solution.
The ultrasonic cleaning experiment will use the nozzle, and exhaust the air bubbles. Disinfect the working environment and instruments with 75% alcohol.
7. The nozzle to be used in the ultrasonic cleaning experiment is designed to expel air bubbles. Disinfect the working environment and instruments with 75% alcohol.
8. Utilize sorting quality control microspheres and adjust them to the optimal value. Set the excitation wavelength to 506 nm and the FITC channel.
9. Adjust the voltage of the deflection electrode plate for the side liquid flow window, setting an appropriate deflection angle to ensure clear fluid stream separation for ease of sorting.
10. After cleaning the tubing with a 0.5% sodium hypochlorite solution for 5 minutes, place the samples on the sample rack and proceed to detect fluorescently labeled cells.
Carboxysome characterization by fluorescence microscopy
1.Take the cryopreserved bacteria and activated for 30 °C 8 h in RMG 5 medium with special antibiotics to logarithmic phase.
2. Subsequently, perform a 10 mL macro system activation in bacterial bottles containing RMG5 medium supplemented with antibiotics. Incubate at 30 °C for 8 hours until the logarithmic phase is reached, and then measure the OD600 nm.
3.Transfer into a 200 mL RMG5 culture medium system, maintaining an initial OD600 nm of 0.1. Incubate at 30 °C for 12 h with a Tc (tetracycline) induction concentration of 0.8 μg/mL. Measure the OD600 nm.
4. Transfer the collected bacterial culture into 2 mL EP tubes, wash the cells three times with PBS, and prepare it as a sample solution.
5.Prepare a 1% agarose solution, heat and shake it until the agarose solution becomes completely clear with no suspended particles. Allow it to cool. The prepared agarose gel serves the purpose of replacing cover slips to reduce bacterial movement during microscopic observation.
6.Add 2 μL of the prepared bacterial culture onto a glass slide, and then cover it with the prepared agarose gel. Make appropriate labeling next to the glass slide for microscopy observation.
7.Turn on the computer power, microscope control box, camera switch, and laser control box switch in sequence. Open the LAS X software on the computer desktop.
8.Place the sample (with the cover slip facing down) on the specimen holder and switch to bright-field (BF) using the microscope touchscreen to locate the bacteria. Choose DIC as the observation mode, press the TL-shutter button to activate the transmitted light, and adjust the focus directly under the microscope. Use the intensity button on the touchscreen or the small black knob on the left side of the microscope to adjust the light intensity.
9.Utilize the LAS X software for automatic exposure and autofocus to capture images. Typically, for bright-field images, exposure times are set between 5-20 ms, while for fluorescence images, exposure times are generally set between 50 ms to 500 ms. Exposure times for each channel can be adjusted independently.
10. Save and label the images.
Carboxysome characterization by transmission electron microscopy
The initial OD600 nm of the seed liquid for inoculation was 0.1. After culturing in the appropriate inducer concentration corresponding to the resistance medium for 8 hours, bacterial cells were collected by centrifugation at 1,500 rpm for 2 minutes, and an appropriate amount of bacterial cells was collected and deposited at the bottom of an EP tube. They were washed twice with a pH 7.2 phosphate solution, and the fixative was placed on ice for pre-cooling. Subsequently, the fixative was added to the EP tube, and it was fixed overnight at a low temperature. The fixative solution consists of 40% distilled water, 10% ethylene glycol aqueous solution (25% concentration), and 50% 0.2 M phosphate buffer. Finally, the prepared samples were sent to Wuhan ShuoSiBai Company for transmission electron microscope sample preparation and observation.