1.Preparation
1.1 Culture medium
(1) LB medium//no-indent
Yeast extract fermentation 5 g/L
Peptone 10 g/L
NaCl 10 g/L
Adjust the pH to 7.0 with sodium hydroxide. To prepare LB solid medium, add 1.5%-2.0% agar powder.
(2) M9 medium//no-indent
Prepare a solution of M9 1× salt
Na2HPO4·12H2O 15.6g/L;
KH2PO4 3g/L;
NaCl 1g/L;
NH4Cl 1g/L ,
Each 50ml bottle is divided and autoclaved（121℃，20min），When using, add 50 μl of sterile 1M MgSO4, 5μl of sterile 1M CaCl2, 2ml of sterile 50% Glucose (w/v）to each bottle.
(3) SOC medium//no-indent
Yeast extract fermentation 5 g/L
Peptone 20 g/L
MgSO4 1.2 g/L
KCl 0.186 g/L
NaCl 0.5 g/L
Glucose 20 g/L
Adjust the pH to 7.0 with sodium hydroxide.
1.2 PCR reaction system
(1) Phanta Max Super-Fidelity DNA Polymerase Reaction system（50 μl）//no-indent
20μl ddH2O
25μl 2×Phanta Max Buffer
1μl Upstream primers
1μl Downstream primers
1μl dNTP
1μl template
1μl Phanta Max Super-Fidelity DNA Polymerase
(2) Es Taq MasterMix Reaction system（20μL）//no-indent
10μl 2×Es Taq MasterMix
1μl Upstream primers
1μl Downstream primers
A little thallus
8μl ddH2O
1.3 Strain preservation
800μl bacterial liquid + 200μl sterile glycerol store in a -80°C refrigerator.
1.4 Strain, plasmids and resistance
(1) Strain //no-indent
DGF298-103Z is kanamycin resistant.
(2) Plasmids//no-indent
pCDF-QS-GFP CmR is chlorampenicol resistant.
Plas-RFP AmpR is ampicillin resistant.
pUC-Pcon-PHB is ampicillin resistant.
pTKRED is Spectinomycin resistant.
The PACYC series are ampicillin resistant:
PACYC-Ptac-GFP-Amp;
PACYC-Ptac-pfkA-nuoF-UD;
PACYC-Ptac-pfkA-nuoF-MU;
PACYC-Ptac-pfkA-nuoF-DU;
PACYC-Ptac-pfkA-nuoF-DM.
The PACYC series are Spectinomycin resistant:
pCL-100RFP-spc
pCL-110RFP-spc
pCL-116RFP-spc
pCL-109RFP-spc
pCL-113RFP-spc
pCL-103RFP-spc
2. Main molecular experiments
2.1 Fragment purification
Use E.Z.N.A. Cycle Pure Kit Centrfugation Protocol Kit to purify Kan-FtsZ fragment. The steps
are as follows.
fragments purification protocal-1.pdf
2.2 Fragment phosphorylation
Use T4 Polynucleotide Kinase purchased from ThermoFisher Scientific，use the reaction system Protocol for Phosphorylation of DNA to perform phosphorylation. The steps are as follows.
phosphorylation protocol-1.pdf
2.3 Agarose gel nucleic acid fragment recovery
Use Omega E.Z.N.A.® Gel Extraction Kit. The steps are as follows.
2.4 Gibson assembly
(1) Mix the linear load and gene fragments in a ratio (1:1) (10 μl total).
(2) Add 10μl 2×MultiF Seamless Assembly Mix.
(3) Incubate at 50 °C for 15-60 min after mixing.
2.5 Electroporation
(1) Take 2-5 μL of the constructed plasmid and add 100 μL of electrocompetent and mix gently.
(2) Transfer the above-mentioned mixed electroporation system to 2 mm Bio-Rad electric turning cups
(3) Put the electric turning cup into the electroporator, and set the parameters of the electroporator:2.5 KV、25 μF、200 Ω。
(4) Click the “Pulse” button on the electroporator to perform electrical conversion, and after the electroporator emits a “drop” sound, the instrument interface will display the electroporation constant, and the reading is about 5.0. Then remove the electroporation cup, pipette 1 mL of LB medium into the electroporation cup on the clean bench, and transfer it to a sterile 1.5 mL centrifuge tube after repeated pipetting and mixing, and place it in a thermostat culture shaker to recover the culture at 37°C, 400 rpm for 1 h.
(5) After the recovery culture is over, centrifuge at 12000 rpm for 2 min to remove 900 μL of supernatant and remove the remaining 100 μL
The bacterial solution was purged and mixed well, and then coated on LB solid plates containing antibiotics, and placed in a 37°C incubator until a single colony grows.
2.6 Chemical transformation
(1) Remove chemically competent DH5α from the -80°C freezer and place on ice to thaw slowly.
(2) In the ultra-clean bench, use a pipette to pipette 2-10 μl of the plasmid to be transformed and transfer it to DH5α competent cells, mix it with gentle pipette and mix it with gentle aspiration, and then take an ice bath for 30 min.
(3) After the ice bath, the competent cells were placed in a 42°C water bath for 90 s, and then quickly placed on ice for 3 min.
(4) Add 1 ml of liquid LB and resume culture at 37°C for 1h.
(5) Centrifuge at 13000rmp for 40s, discard the supernatant.
(6) The remaining 150~200μl is pipetted and mixed, coated on a solid LB plate, and placed in a 37°C incubator until a single colony grows.
2.7 Plasmid extraction
Use Omega E.Z.N.A. Plasmid Mini Kit to extract plasmids. Add RNase A to Solution I and store at 4℃, add 120 ml absolute ethanol to Solution I before use. The steps are as follows.
plasmids mini exyraction protocol-1.pdf
3. Others
3.1 Characterization
Single colonies of DGF-298 and DGF-298-103Z were picked and transferred to 10 mL of liquid LB medium for 12 h at 37°C, and then the seed solution was inoculated at 2% (v/v) to a 48-well plate containing 700 μL of liquid LB or M9 medium and inoculated with 2% (v/v). For phenotypic analysis of temperature, cells are cultured in liquid M9 or LB medium under the corresponding conditions, respectively. When culturing strain DGF298-103Z, 50 μg/mL kanamycin needs to be added to maintain polyploidy stabilization of the cells. During the period of cell growth to the exponential phase, at least five consecutive time points were taken and the mass doubling time (min) of the cells was calculated with the following formula: Doubling time = (tj-ti)/log2() where tj and ti represent two time points, and Cj and Ci represent two OD600 values corresponding to tj and ti.
3.2 DAPI staining
(1) Cells DGF-298 and DGF-298-103Z were cultured to exponential phase by liquid LB medium, in which DGF-298-103Z was added with 50 μg/ml kanamycin to maintain ploidy, and cells were collected by centrifugation at 12000 rpm for 2 min.
(2) Wash the collected cells three times with 1×phosphate buffer at pH 7.2. Centrifugation was followed by removal of the supernatant, followed by the addition of 1 mL of 3.7% formaldehyde and the cells were fixed at 4°C for 30 minutes. The fixed cells are then washed three times, centrifuged and the cells are collected. To increase the permeability of the cell membrane, resuspend the fixed cells with 5% Triton X-100 and let stand at 4°C for 30 minutes.
(3) Wash the cells three times again. After centrifugation and complete removal of the supernatant, resuspend the cells with 10 μL of 5 mg/L DAPI and let stand for 10 minutes at room temperature in the dark for chromosome staining. After staining, wash the cells three more times to remove excess DAPI, and finally add an appropriate volume of 1× phosphate buffer to resuspend and store in the dark.
(4) Chromosomes were observed under fluorescence microscopy at excitation at 364 nm and emission at 454 nm.
3.3 Flow cytometry
Strains DGF298 and DGF298-103Z were incubated in 10 mL of liquid LB medium at 37°C for exponential phase. When culturing DGF298-103Z, 50 μg/mL kanamycin is added to maintain chromosomal stability. Cells were then collected and stained with DAPI with 1× phosphate buffer at pH 7.2 to an OD600 value of <0.1. Filter the sample with a 300 mesh filter before analyzing with flow cytometry. A flow cytometer (ImageStreamXMarkII, Amnis/Merck) was then used to analyze the fluorescence intensity of the single cells. A total of 20,000 cells were collected for analysis per sample. Blue fluorescence was detected using the UV1 channel and data were analyzed using flow cytometry analysis software (Flowjo VX10).
3.4 Continuous culture
(1) In order to analyze the chromosomal stability of polyploid E. coli, polyploid E. coli DGF298-103Z was inoculated at a 2% (v/v) inoculation ratio under shake flask fermentation conditions, and polyploid E. coli DGF298-103Z was continuously transferred every 12 h. Add 50 μg/mL kanamycin (Kan) to transshipment strain DGF298-103Z. Polyploid E. coli DGF298-103Z underwent a total of 6 transfers for a total of 72 hours.
(2) The 0th, 1st and 5th, 6th transfer samples were taken and coated on solid LB plates to isolate single colonies, and 5 single colonies were selected from each transfer for PCR amplification verification.
3.5 Scanning electron microscopy observation
(1) Inoculated with cryopreserved bacteria DGF298 and DGF298-103Z in LB-containing liquid medium, resuscitated and cultured at 37°C to logarithmic growth stage. When culturing the polyploid strain DGF298-103Z, kanamycin at a working concentration of 50 ug/ml was added to maintain ploidy.
(2) Take a large number of samples and centrifuge (rotation speed 1000r-4000r, the rotation speed is adjusted according to the respective sample, and low rotation speed is preferred) and remove supernatant, add appropriate pH (6.8-7.4) of 1X PBS and wash again, 15min, to remove the supernatant.
(3) Fixation: Add 2.5% glutaraldehyde (prepared by 1X PBS) to fix in a 4°C freezer for 3h, remove the supernatant, and then wash it three times with 1X PBS for 15min each time, and centrifuge at 3000r to remove the supernatant.
(4) Dehydration: Dehydrate the sample with ethanol aqueous solution according to the concentration gradient of 30%, 50%, 70%, 80% and 90%, and each step is about for 15 min.
The supernatant was discarded by centrifugation at 3000rmp, followed by dehydration in 100% ethanol for 15 min 2 times.
(5) Critical point desiccator drying: rinse the slides with ethanol, shake the bacteria in the suspension tube after the slides are dry, absorb the bacterial suspension droplets on the coverslips, and transfer them to the critical point desiccator after the ethanol volatilizes. Open the CO2 valve and start the critical point dryer, add ethanol to half the height of the sample tank after the preheating is complete, and place the coverslip containing the sample in the sample tank of the critical point dryer to dry.
(6) Gold spraying: After the sample is fully dried, the coverslip is adhered to the sample stage with conductive tape and plated with gold.
(7) Perform Scanning electron microscopy observation through FESEM(Quanta 250 FEG; FEI Ltd., Brno, Czech Republic).
3.6 RT-qPCR
(1) Extraction of total RNA//no-indent
Extraction was performed using the instructions of the Bacterial Total RNA Isolation Kit (DP430) purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd. The steps are as follows.
（2）RNA-cDNA reverse transcription //no-indent
Place the extracted RNA in a 65°C water bath for 5 minutes, followed by an ice bath immediately. Prepare gDNA elimination as Table1.
The reaction system was carried out at 37 °C for 5 min.
Table 1 gDNA elimination reaction (16 μL)//caption
| Component | The amount added |
|---|---|
| 4×DN Master Mix | 4 μL |
| RNA template | 0.5 pg-0.5 μg |
| Nuclease-free Water | Add to 16 μL |
Add 4 μL of 5×RT master MixII. to the reaction solution at the above steps and proceed reverse transcription with the reaction conditions in Table 2.
Table 2 Reverse transcription reaction conditions//caption
| Temperature | Time |
|---|---|
| 37℃ | 15min |
| 50℃ | 5min |
| 98℃ | 5min |
After the reaction is complete, store the resulting cDNA in a -20 °C freezer.
(2) Fluorescent quantitation PCR（qPCR）reaction system //caption
| Component | Amount |
|---|---|
| SYBR Green Realtime PCR Master Mix | 12.5μL |
| ddH2O | 8μL |
| Upstream primers | 1μL |
| Downstream primers | 1μL |
| cDNA template | 2.5μL |
At least 3 parallels in each experimental group.
4. Fermentation experiments
4.1 PHB shake flask fermentation
Single colonies of strain DGF298 and DGF298-103Z were picked from solid LB plates and incubated at 37°C for 12 h in 5 mL LB medium to obtain seeds for fermentation, and then the seeds were transferred to a 300 mL shaker containing 50 mL of M9 medium with 40 g/L glucose as carbon source at a ratio of 1% (v/v) and fermented at 37 °C in a 220 rmp shaker for 48 h. During fermentation for 9h, 24h and 36h, the pH of the fermentation broth was measured with pH test paper, and an appropriate amount of ammonia was added to adjust the pH to 7.0. During fermentation, all strains were supplemented with 100 μg/mL ampicillin, while strain DGF298-103Z was additionally supplemented with 25 μg/mL kanamycin to maintain polyploidy. Each strain does three independent biological replicates.
4.2 Cell growth assays
During the fermentation process, 1 mL of fermentation broth was taken, 20 μL was taken out of it into a 1.5 mL centrifuge tube after vortexing and mixed, and 980 μL of ddH2O was added to dilute. Cell growth density was then detected with a UV-Vis spectrophotometer at 600 nm. The SBA-40D biosensing analyzer was used to detect the sugar concentration in the fermentation broth.
4.3 Nile red dyeing
When the sugar consumption was basically stable in the later stage of fermentation (36 h), 10ul of bacterial solution was stried on an LB plate containing 0.5 μg/mL Nile red dye, 2% glucose, and 100 μg/mL ampicillin to separate single colonies, and incubated at 37°C until a single colony grew. An additional 25 μg/mL of kanamycin is added while culturing DGF298-103Z to maintain polyploidy. A blue light detector (wavelength 460~470nm) was used to observe the excitation light intensity of single colonies on different plates.
4.4 Gas chromatography
After the fermentation is over, take a 50mL centrifuge tube, weigh and record the weight of the centrifuge tube, add the fermentation broth to centrifuge to collect the bacteria for freeze-drying, and weigh and record the dry weight of the bacteria. Weigh 20mg of lyophilized bacterial powder and add it to the oil bath bottle and record the specific data. After adding 150 μL of concentrated sulfuric acid, 850 μL of methanol and 1 mL of chloroform in turn, the cap of the reaction bottle was tightened and the oil bath was held at 100°C for 1h. After the oil bath is completed, 1mL of distilled water is added to each reaction flask, mixed well, and allowed to stand for 3~4h. After the samples were stratified, 600 μL of the lowest organic phase was added to the gas chromatography flask for gas chromatography analysis. After the measurement, the peak area-PHB concentration standard curve was plotted, and the PHB titer of the shake flask was calculated by substituting the data.
4.5 Liquid chromatography
(1) Sample pretreatment//no-indent
After the fermentation was completed, the culture medium was centrifuged at 12000rmp for 2min, and the supernatant was taken as a sample. Samples were diluted with trichloroacetic acid for 10× or 20 ×, mixed and allowed to stand for 1h. Centrifuge at 12000rmp for 30min, take the supernatant and filter it to a liquid vial with a 0.22 μm organic filter membrane.
(2) Amino acid yield analysis//no-indent
The content of amino acids in the fermentation broth was detected by L-8900 automatic amino acid automatic analyzer. Determination method: mobile phase A was sodium acetate (10%, g/100mL, pH 7.2), tetrahydrofuran (0.5%, V/V), triethylamine (0.0225%, V/V), mobile phase B was sodium acetate (2%, g/100Ml, pH 7.2), acetonitrile (0.5%, V/V), methanol mixture (0.022%, V/V), and the free amino acids in the samples were analyzed with the retention time and peak area of the free amino acid standard as the control.