Molecular Cloning
1.DNA Synthesis
Primer synthesis service is provided by Tsingke Biotechnology Co., Ltd. Gene synthesis service is provided by Genscript.
2.PCR
Preparation: The simple fragment amplification system is generally 50 μl, and the components of the non-50 μl system can be increased or decreased in proportion. Using different DNA polymerases, the optimal amount of template and primer may vary. 2 ×Phanta Max Master Mix(Dye Plus) is commonly used in our laboratory. The following is the general reaction system of this enzyme for PCR.
While overlapping-extension, the concentration of bridge primer is 1/20 of the normal primer’s usage.
3.Homologous recombination
After the system configuration was completed, the solution was gently sucked and mixed with a pipette gun, briefly centrifuged and collected to the bottom of the tube, and then reacted at 37°C for 30 min (generally placed in a PCR instrument). After the completion, the solution was immediately placed on ice for cooling, which could be directly used for the next experiment.
4.Colony PCR
PCR reactions were performed with bacterial colonies or bacterial solutions. The heat causes bacterial cells to burst, releasing genomic and plasmid DNA, which can be used as a template for PCR and is often used in laboratories to test the success of plasmid transformation.
5.Enzyme digestion
Steps:
1.Prepare a clean PCR tube to undergo this experiment.
2.Add the components in following order: ddH20, DNA(4℃), 10x buffer(-20℃), enzyme(-20℃).
3.After adding, pipetting the tube to mix it well.
4.Put the tube into the incubator or water bath(37°C) for 2 hr.
6.Gel purification
Gel Extraction is carried out with Gel Extraction Kit from Omega Bio-Tek biotech co., ltd and the protocol it provided.
7.Plasmid extraction
Plasmid Extraction is carried out with Plasmid Mini Kit I from Omega Bio-Tek biotech co., ltd and the protocol it provided.
Transformation
1.Remove the competent cells from -80℃ and immediately melt them in an ice water bath for 5min
2.Add the transposition DNA into 100uL competent cells, gently stir well, and incubate on ice for 30min
3. Place the centrifuge tube in the water bath at 42℃ without shaking. After the heat shock of 90s, immediately place it in the ice water bath for 2-3min
4. Add 900uLLB medium (preheated at room temperature or 37 ℃ to the centrifuge tube, and then put it in a 37 ℃ shaker for 1h
5. Take different volumes of transformation products, spread them on the correct resistant plates with sterile coating sticks, and incubate them overnight at 37℃ in an incubator
Protein
1. purification
protein purification is carried out with Ni-NTA 1 ml (Pre-Packed Gravity Column), and the corresponding reagents Binding/Wash Buffer (pH 7.9 = 8.1) for Ni-NTA or Ni-IDA Sefinose (TM) Resin, Elution Buffer (pH 7.9 = 8.1) for Ni-NTA or Ni-IDA Sefinose (TM) Resin from Sangon Biotech (Shanghai) Co., Ltd. The specific experimental steps are the protocols they provided.
2. Identification of proteins
Determination of protein molecular weight of normal size protein were carried out with SDS-PAGE Preparation kit from Sangon Biotech (Shanghai) Co., Ltd and the protocol it provided.
3. Protein quantitative test
Protein quantitative test were carried out with Bradford Protein Assay Kit from Sangon Biotech (Shanghai) Co., Ltd.
Trimethylamine dehydrogenase/Dimethylamine dehydrogenase verification
bacteria samples treatment
700 µl bacteria samples were centrifugated at 3000 × g 5 min at 4 °C, take 500µl supernatant. Then 300 µl freshly prepared 10 mM solution of FMOC-Cl in acetonitrile was added, after 1 min, 100 µl 100 mM glycine solution was added to neutralize the reaction.
HPLC
Supernatant was transferred to new tube for analysis on HPLC system. 10 µl was loaded on to C18 column equilibrated with acetonitrile-buffer (50%) at flow rate 0.75 ml/min. The column was then flushed with a gradient to 100% elutant buffer B (acetonitrile 75% v/v) within 5 min. Ultraviolet absorption of column elutant was monitored (220 nm) and DMA quantification was calculated based on ratio to standard sample peak area.
Formaldehyde dehydrogenase verification
1.Pick the bacteria from the transformed plate and culture them in a bacterial bottle for 12-16h to test the colony OD600.
2.Transfer 100 μ L of bacterial liquid to 10mlLB liquid medium for about 7h.
3.Add different gradients of 40% saturated formaldehyde solution (0,0.25, 1,2.5, 7.5, 25 μL，the corresponding gradients are 0.000%，0.001%，0.004%，0.01%，0.03%，0.1%respectively).
4.200 μL of the mixture was added to the 96-well plates.
5.The microplate was set, the temperature was 37°, the absorbance was 600, and the overnight culture was detected every half hour.
6. The sodium sulfite fuchsin solution was prepared 2.5g/L anhydrous sodium sulfite and 0.5g/L basic fuchsin.
7. Add 500μL of sodium sulfite fuchsin solution to each bottle of bacteria solution to determine formaldehyde concentration.
Nattokinasse verification
IPTG induction and fermentation conditions
When the optical density of the cultured E.coli BL21(DE3) with pET-28a(+)-aprN(with His-tag) was between 0.7 and 0.8, IPTG was added to the final concentration of 0.7mmol/L at 20℃ and 0.1mmol/L at 37℃. And the bacterium were separately induced at 20℃ for 6 hours and at 37℃ for 6 hours.
Fibrin plate
Preparation:
1. solvent: 1×PBS
2. 2% Agar solutionAgar, 55°C
3. 0.3%Fibrin solution, 55°C
4. Thrombin solution of 0.9% NaCl (1000U/ml): 60ul
5. Petri dish: 5.5 cm in diameter
Steps:
1. Prepare a 55°C water bath.
2. Prepare the fibrinogen solution and put it in the 55℃ water bath for 10 min to dissolve fibrinogen.
3. Dissolve agar and let it cool down to 55°C.
4. Mix all solution and pour into a petri dish.
5. Let the petri dish stand for 1 hour in refrigerator.
6. Add 20 ul of sample and put it in 37°C incubator for 24 hours to see the lytic circle.
Butyric acid verification
IPTG induction and fermentation conditions
1.When the optical density of the cultured E.coli BL21(DE3) with pET-28a(+)-Tes4(with His-tag) was between 0.6 and 0.8, IPTG was added to the final concentration of 1mM. And the bacteria were induced at 18℃ overnight,
2.Samples from the induced cultures were transferred to a 35 ° C water bath for 36 hours for fermentation
supernatant was collected at 8000rpm for 2min in a fourth-degree centrifuge.
GS
1.Gas chromatography instrument: Agilent 7890A
2.capillary chromatographic column: DB-FFAP(30mX0.250mmX0.25μm)
3.Injection port heater:240℃, gas pressure: 17.154psi, gas flow rate:19.656mL/min
4.FID heater temperature: 250℃, Carrier gas: H2 at 40mL/min, air at 400mL/min, N2 at 30mL/min
Column Oven:60℃for 1 min followed by ramping up to 220℃(at 10℃/min) and holding at 220℃ for 2min.
Suicide module verification
Methods of molecular cloning and transformation are described above. Transform this plasmid into E. coli DH5α. Then spread it onto a LB medium plates with 170 μg/mL chloramphenicol and incubate overnight at 37 ℃ in an incubator.
Pick four colonies from the same plate as parallel repeats. Each colony is inoculated on two identical media with 5 ml LB medium containing 170 μg/mL chloramphenicol and cultured at two temperatures(37 ℃ and 28 ℃) respectively while shaking at 200 rpm overnight. Treated the same with the experimental groups, control bacteria are also cultured at the two temperatures.
Put all the media into the 37 ℃ orbital shaker and culture them at 200 rpm for 12 hours.
Plot the OD600 value curves of the resuspending culture media over time in an automatic microplate reader (SynergyH1 hybrid multimodal reader). Incubate the cultures for 12 hours at 28℃ while shaking at 200 rpm. Samples are taken from each group once an hour and measured by UV spectrophotometer. And then convert the raw data into OD600. Compare the data of experimental groups and control group and compare curves in two schemes with each other.
All experiments above are carried out in three biological repeats.