Material

AG SteadyPure Plasmid DNA Extraction Kit
Pipettes and pipette tips
Vortex
Microcentrifuge
Nuclease-free 1.5 mL microcentrifuge tubes
Sterile deionized water
100% ethanol

Before Starting
Add the RNase A Solution included in the kit to the Resuspension Solution and mix throughly. After the additon of RNase A, the Resuspension Solution should be stored at 4 ℃.
Add the indicated volume of ethanol (96-100%) to Wash Solution II (concentrated) prior to first use.
Make sure to wear gloves.

Protocol

1.Add a 1.5 mL of bacterial culture to microcentrifuge tubes and harvest the cells by centrifugation for 10 min at 5,000 x g. Discard the supernatant.
2.Resuspend the pelleted cells in 250 uL of the Resuspension Solution. The bacteria should be resuspended completely by vortexing and no cell clumps remain.
3.Add 250 uL of the Lysis Solution and mix throughly by inverting the tube 4-6 times until the solution becomes viscous and slightly clear.
Note:Do not vortex to avoid shearing of chromosomal DNA. Do not incubate for more than 5 min to avoid denaturation of supercoiled plasmid DNA.
4.Add 350 uL of the Neutralization Solution and mix immediately and throughly by inverting the tube 4-6 times. The neutralized bacterial lysate should become cloudy.
5. Centrifuge at 12,000 x g for 5 min to pellet cell debris and chromosomal DNA.
6. Transfer the supernatant to the supplied GeneJET spin column bt decanting or pipetting. Avoid disturbing or transferring the white precipitate.
7. Centrifuge at 12,000 x g for 1 min. Discard the flow-through and place the column back into the same collection tube.
8. Add 500 uL of the Wash Solution to the spin column. Centrifuge at 12,000 x g for 1 min and discard the flow-through. Then place the column back into the same collection tube.
9. Repeat the step 8.
10.Centrifuge the empty tube for an additional 1 min to remove residual Wash Solution.
11.Transfer the spin column into a fresh 1.5 mL microcentrifuge tube. Add 50 uL of the sterile deionized water to the center of spin column membrane to elute the plasmid DNA. Incubate for 2 min at room temperature.
12.Discard the column and store the purified plasmid DNA at -20℃.

Material

Takara PrimeSTAR® Max DNA Polymerase
Colony template
Primers
Microcentrifuge
Nuclease-free 0.2 ml PCR tubes
PCR instrument

Protocol

1. The choice of reaction system:

2.The choice of PCR program:

Note: x is the annealing temperature of the reaction, usually 2℃ to 3℃ lower than Tm of the primer.
The calculation of Tm: Tm = 4(G+C) + 2(A+T)

Material

LB plates
Note: In the experiment, different engineering bacteria were added with corresponding antibiotics
1.5 mL centrifuge tube
centrifuge
Solution A
Solution B

Protocol

1.Take out the preserved strain from the -80℃ ultra-low temperature refrigerator, streak it on the LB plate, and cultivate it overnight at 37℃ to pick out a single colony to LB liquid medium for expansion.
2.Take 20 mL of logarithmic bacterial liquid with an OD600 value between 0.35 and 0.5, aliquot into a 1.5 mL centrifuge tube, and place it on ice for 10 minutes to stop culturing.
3. Use a centrifuge at 4℃ and 5000 rpm for 5 minutes to collect bacteria and discard the supernatant. Add 100 μL of low-temperature Solution A to the centrifuge tube, and tap the centrifuge tube to suspend the bacterial solution.
4. Centrifuge again at 4℃, 5000 rpm for 5 minutes to collect the cells, then add 100 μL of pre-cooled Solution B to the centrifuge tube, and flick to suspend the cells.
5. Immediately store the heat-shock competent cells prepared above at -80℃ for use in the next step.

Material

Materials for 1 l:
Yeast Extract: 5 g
Tryptone: 10 g
NaCl: 10 g

Protocol

1.Preparation of LB Liquid Culture Medium
2.Measure 10 g of Tryptone, 5 g of Yeast extract, and 10 g of NaCl. Place them in a 1,000 ml sterilized bottle.
3.Add 0.5 l of deionized water, shake the bottle until the solids completely dissolve. Then, add more deionized water until the solution has a volume of 1 l.
4.Place the bottles into the autoclave for sterilization, under 121°C for 30 minutes.
5.Let the bottles cool down under room temperature. The solution would preserve under room temperature for a maximum of 7 days.
6.15 g/L agarose can be added to form a solid medium, and after sterilization, it can be divided into solid agar plates

Material

Electrocompetent cells
Assembly product
LB plates
Note: In the experiment, different engineering bacteria were added with corresponding antibiotics
Electroporation cuvette
Electroporation machine
Pipette with tips
Incubator cabinet

Protocol

1.Take glycerol strains to LB plate, pick out monoclonal colony and expand the culture overnight on a shaker at 30℃ and 180 rpm.
Note: Shewanella was cultured at 30℃, E. coli at 37℃.
2. Take 1 mL of bacterial solution into a centrifuge tube, centrifuge at 15,000 rpm for 2 min to collect cells, and discard the supernatant.
3.Use 1 mL of 0.3 mol/L sterilized sucrose solution to resuspend the cells by pipetting up and down, centrifuge again at 15,000 rpm for 2 min, and discard the supernatant.
4. Repeat step 3.
5.Add 100 μL of sucrose solution again to resuspend, add about 100 ng of recombinant plasmid to the competent cells, and mix well. The mixture was added to the electro-rotating cup, electric shock was performed at 2.5 KV for 5 ms, and 900 μL of LB liquid medium was quickly added to resuspend the bacteria.Then placed on a shaker at 200 rpm and activated at 30 ℃ for 2 h.
6. Centrifuge at 5000 rpm for 5 min and discard the supernatant. Use 100 μL of LB liquid medium, pipette to resuspend, spread on LB plate by dilution plate coating method, and incubate upside down in a 30℃ bacterial incubator for 12-20 h until colonies appear.
7. Pick monoclonal colonies for PCR verification, screen out strains with correct bands for sequencing, and strains with correct sequencing for glycerol storage.
Note:Antibiotics can be determined according to the plasmid.

Material

Ethanol 96-100%
Isopropanol
3M sodium acetate, pH 5.2 (may be necessary)
Microcentrifuge
1.5 or 2 mL microcentrifuge tubes
Heating block or water bath
GeneJET PCR Purification Kit (produced by Thermo Fisher Scientific®)

Before Starting

Examine the Binding Buffer for precipitates before each use. Re-dissolve any precipitate by warming the solution to 37 ℃ and cooling to 25 ℃ .

Protocol

1.Add a 1:1 volume of Binding Buffer to completed PCR mixture (e.g. for every 100 µL of reaction mixture, add 100 µL of Binding Buffer). Mix thoroughly. Check the color of the solution. A yellow color indicates an optimal pH for DNA binding. If the color of the solution is orange or violet, add 10 µL of 3 M sodium acetate, pH 5.2 solution and mix. The color of the mix will become yellow.
2.Optional: if the DNA fragment is ≤ 500 bp, add a 1:2 volume of 100% isopropanol (e.g., 100 µL of isopropanol should be added to 100 µL of PCR mixture combined with 100 µL of Binding Buffer). Mix thoroughly.
Note:If PCR mixture contains primer-dimers, purification without isopropanol is recommended. However, the yield of the target DNA fragment will be lower.
3.Transfer up to 800 µL of the solution from step 1 (or optional step 2) to the GeneJET purification column. Centrifuge for 30-60 s. Discard the flow-through.
Notes: If the total volume exceeds 800 µL, the solution can be added to the column in stages. After the addition of 800 µL of solution, centrifuge the column for 30-60 s and discard flowthrough. Repeat until the entire solution has been added to the column membrane. Close the bag with GeneJET Purification Columns tightly after each use!
4.Add 700 µL of Wash Buffer (diluted with the ethanol as described on p. 3) to the GeneJET purification column. Centrifuge for 30-60 s. Discard the flow-through and place the purification column back into the collection tube.
5.Centrifuge the empty GeneJET purification column for an additional 1 min to completely remove any residual wash buffer.
Note.:This step is essential as the presence of residual ethanol in the DNA sample may inhibit subsequent reactions. 6
6.Transfer the GeneJET purification column to a clean 1.5 mL microcentrifuge tube (not included). Add 50 µL of Elution Buffer to the center of the GeneJET purification column membrane and centrifuge for 1 min.
Note:For low DNA amounts the elution volumes can be reduced to increase DNA concentration. An elution volume between 20-50 µL does not significantly reduce the DNA yield. However, elution volumes less than 10 µL are not recommended.
If DNA fragment is >10 kb, prewarm Elution Buffer to 65℃ before applying to column.
If the elution volume is 10 µL and DNA amount is ≥5 µg, incubate column for 1 min at room temperature before centrifugation.
7.Discard the GeneJET purification column and store the purified DNA at -20℃.

Material

Cwbio 2x Es Taq MasterMix(Dye)
Template DNA
ddH₂O
Primers
Nuclease-free PCR tubes
PCR imstrument

Protocol

1.Set up a reaction system:

2.Choose the PCR program based on the reaction:

Note: If the template is from a colony, add 1 μL ddH₂O and add a few templates colony using pipette tips.

Material

Ethanol 96-100%
Isopropanol
3M sodium acetate, pH 5.2 (may be necessary)
Microcentrifuge
1.5 or 2 mL microcentrifuge tubes
Heating block or water bath
GeneJET Gel Extraction Kit (produced by Thermo Fisher Scientific®)

Before Starting

Examine the Binding Buffer for precipitates before each use. Re-dissolve any precipitate by warming the solution to 37 ℃ and cooling to 25 ℃ .

Protocol

1.Excise gel slice containing the DNA fragment using a clean scalpel or razor blade. Cut as close to the DNA as possible to minimize the gel volume. Place the gel slice into a pre-weighed 1.5 mL tube and weigh. Record the weight of the gel slice.
Note:If the purified fragment will be used for cloning reactions, avoid damaging the DNA through UV light exposure. Minimize UV exposure to a few seconds or keep the gel slice on a glass or plastic plate during UV illumination.
2.Add 1:1 volume of Binding Buffer to the gel slice (volume: weight) (e.g., add 100 µL of Binding Buffer for every 100 mg of agarose gel).
Note: For gels with an agarose content greater than 2%, add 2:1 volumes of Binding Buffer to the gel slice.
3.Incubate the gel mixture at 50-60 ℃ for 10 min or until the gel slice is completely dissolved. Mix the tube by inversion every few minutes to facilitate the melting process. Ensure that the gel is completely dissolved. Vortex the gel mixture briefly before loading on the column. Check the color of the solution. A yellow color indicates an optimal pH for DNA binding. If the color of the solution is orange or violet, add 10 µL of 3 M sodium acetate, pH 5.2 solution and mix. The color of the mix will become yellow.
4.Optional: use this step only when DNA fragment is ≤500 bp or >10 kb long.
If the DNA fragment is ≤500 bp, add 1 gel volume of 100% isopropanol to the solubilized gel solution (e.g. 100 µL of isopropanol should be added to 100 mg gel slice solubilized in 100 µL of Binding Buffer). Mix thoroughly.
If the DNA fragment is >10 kb, add 1 gel volume of water to the solubilized gel solution (e.g. 100 µL of water should be added to 100 mg gel slice solubilized in 100 µL of Binding Buffer). Mix thoroughly.
5.Transfer up to 800 µL of the solubilized gel solution (from step 3 or 4) to the GeneJET purification column. Centrifuge for 1 min. Discard the flow-through and place the column back into the same collection tube.
Note: If the total volume exceeds 800 µL, the solution can be added to the column in stages. After each application, centrifuge the column for 30-60 s and discard the flow-through after each spin. Repeat until the entire volume has been applied to the column membrane. Do not exceed 1 g of total agarose gel per column. In addition ,Close the bag with GeneJET Purification Columns tightly after each use!
6.Optional: use this additional binding step only if the purified DNA will be used for sequencing. Add 100 µL of Binding Buffer to the GeneJET purification column. Centrifuge for 1 min. Discard the flow-through and place the column back into the same collection tube.
7.Add 700 µL of Wash Buffer (diluted with ethanol as described on p. 3) to the GeneJET purification column. Centrifuge for 1 min. Discard the flow-through and place the column back into the same collection tube.
8.Centrifuge the empty GeneJET purification column for an additional 1 min to completely remove residual wash buffer.
Note:This step is essential to avoid residual ethanol in the purified DNA solution. The presence of ethanol in the DNA sample may inhibit downstream enzymatic reactions.
9.Transfer the GeneJET purification column into a clean 1.5 mL microcentrifuge tube (not included). Add 50 µL of Elution Buffer to the center of the purification column membrane. Centrifuge for 1 min.
Note:For low DNA amounts the elution volumes can be reduced to increase DNA concentration. An elution volume between 20-50 µL does not significantly reduce the DNA yield. However, elution volumes less than 10 µL are not recommended.
If DNA fragment is >10 kb, prewarm Elution Buffer to 65 °C before applying to column.
If the elution volume is 10 µL and DNA amount is ≤ 5 µg, incubate column for 1 min at room temperature before centrifugation.
Discard the GeneJET purification column and store the purified DNA at -20 °C

Material

50 ×TAE concentrate Solution
Agarose (produced by Biowest®) —DNA dye (TransGen® GelStain)
100mL flask
Distilled water
Microwave oven
DNA samples
10 ×Loading buffer (produced by New England Biolabs ®)
DNA marker (produced by TsingkeBiotechnologyCo.,Ltd.®)
Electrophoresis instrument (produced by Bio-Rad®)

Before Starting

Dilute 50 ×TAE concentrate Solution to 1 ×TAE buffer with distilled water.
Add 10 ×loading buffer into marker and DNA samples. Loading buffer should occupy 10% of total volume.
Note:If the Taq enzyme used in PCR already has a premixed DNA dye, there is no need to add loading buffer

Protocol

1.Weigh 0.3 g agarose in a flask.
2.Add 30 mL 1 × TAE buffer into the flask from Step 1.
3.Melt the agarose in the microwave oven (medium-high heat, about 3 minutes).
Note: In order to make the gel more even, reheating the agarose is recommended.
4.Add 3 uL TransGen® GelStain, mix by shocking.
5.Insert slots to assemble gel pouring apparatus through the gate.
6.Pour agarose gel into the gel tray.
7.Cool for 40 minutes to FULLY solidify the DNA agarose gel.
8. Remove the pouring apparatus, place the gel into an electrophoresis instrument.
9. Pipet marker and DNA samples which have been mixed with loading buffer into the slots.
10. Turn on the electrophoresis instrument, set the working electric current at 120-150 mA and the working voltage at 120 V.
11. Electrophoresis for 20-35 minutes.
Note: When the blue strips move to two-thirds of the gel, the electrophoresis could be stopped.
12.Turn off the instrument and place the gel in the gel formatter to take and save the photo.

Table1 TAE running buffer (50x)

Material

LB medium
Note: In the experiment, different engineering bacteria were added with corresponding antibiotics
Colony template
PBS buffer
96-well plate
Micro plate reader (produced by gene5®)

Protocol

1. Pick a single colony from the plate, inoculate it into 3-5ml LB liquid medium, and inoculate it at 30℃ for about 12 h with shaking. Incubate overnight.
2. The next day, inoculate 50 μl of the overnight culture into 5 ml of fresh LB medium. After culturing to 6 h, 12 h and 24 h, take 200 μL of bacterial liquid from each sample.
Note: If necessary, add different concentrations of IPTG (0, 10-5, 10-4, 10-3, 10-2, 10-1 mM)
3. Centrifuge at 4000 rpm for 10 min and discard the supernatant. Resuspend in 1 ml of PBS, centrifuge at 4000 rpm for 10 min, and discard the supernatant. Resuspend in 1 ml of PBS, centrifuge at 4000 rpm for 10 min, and discard the supernatant. Resuspend in 200 μl PBS, and measure the fluorescence intensity in a 96-well plate. Micro plate reader parameters are shown below.

Table 1 PBS buffer formulation(1L)

Material

LB plates
Note: In the experiment, different engineering bacteria were added with corresponding antibiotics
1.5 mL centrifuge tube
competent cells
recombinant plasmid
ice box
incubator

Protocol

1. Take out the frozen competent cells from the -80℃ ultra-low temperature freezer and thaw on ice.
2. Take 10 μL of the recombinant plasmid and add it to 100℃ of heat-shock competent cells, mix them together carefully, and place in an ice box for about 30 minutes;
3. Heat in a water bath at 42℃, heat shock for 90 s, and then immediately cool on ice for 2-3 minutes;
4. Add 900 μL of LB liquid medium prepared in advance to the centrifuge tube, and then place the bacterial solution at 150 rpm, Incubate at 37℃ for 1 h.
5. The LB liquid culture was preheated in a 37℃ incubator, and the activated bacterial solution was centrifuged at 5000 rpm.
6. Discard 900 μL of the supernatant, resuspend with the remaining 100 μL, spread the bacterial solution onto LB plates using the dilution plate coating method, and invert overnight in a 37℃ incubator.
7. Pick monoclonal colonies for PCR verification, screen out strains with correct bands for sequencing, and strains with correct sequencing for glycerol storage.

Material

high pressure steam sterilizer
glycerin

Protocol

1.A 40% glycerol solution was prepared in a volume ratio of 2:3 for strain preservation.
2.After the preparation is completed, sterilize with high pressure steam at 121 °C for 20 min, and store at room temperature.
3.When using, the bacteria should be preserved according to the volume ratio of glycerol and bacterial liquid to 1:1.