Experiments

PCR

Supplies

• 2×Phanta Flash Master Mix
• Bio-rad T100 Thermal Cycler
• PCR tubes

Materials

• \(\ce{ddH2O}\)

Protocol

PCR of Atox1, BpfA, AggC

1. In 3 PCR tubes, mix in the following order on ice :

2. In 3 PCR tubes, mix in the following order on ice :

3. In 3 PCR tubes, mix in the following order on ice :

The total volume of reagent assembled in each of the PCR tubes is 50μl.

4. Place the 9 tubes into the thermal cycler and close the lid.
5. Amplify the acids using the denaturation, annealing, and polymerization times and temperatures listed below.

6. Withdraw 5μl from the PCR product and analyze them by agarose gel electrophoresis.

PCR of Agbp2,bpfA,aggC

Same with the PCR protocol of Atox1,bpfA,aggC.

ClonExpress MultiS

Material

• Template for fragment amplification
• Primer
• Linearization carrier
• pfu ultra: Phanta® Max Super-Fidelity DNA Polymerase
• Competent cell
• \(\ce{ddH2O}\)
• PCR Tubes
• PCR Amplifier

Protocol

1. Preparation of linearized carrier

• The appropriate cloning site was selected and the vector was linearized. It is recommended that the region with no repetitive sequence and uniform GC content be selected as far as possible. When the GC content in the upstream and downstream 20bp region of the vector cloning site is between 40% Mel 60%, the recombination efficiency will reach the maximum.
• Vector linearization: you can choose restriction endonuclease digestion or reverse PCR amplification.

1. Insert fragment to get

• By introducing the homologous sequence at the 5' end of the primer, there are identical sequences (15-20bp, excluding restriction sites) among the amplification products and between the amplification products and the linearized cloning vector.
• The inserted fragment was amplified by PCR.

1. Usage of linearized vectors and inserts

• Concentration determination
• Calculation of the usage of carriers and fragments

1. Recombination reaction

• The amount of DNA needed for the recombination reaction was calculated according to the formula. In order to ensure the accuracy of the sample addition, the linearized vector and the insert can be diluted properly before preparing the recombination system, and the sample volume of each component is not less than 1 μl.
• Prepare the following reaction system on ice

• Use the pipette to gently suck and mix well (please do not oscillate and mix well), and briefly centrifuge to collect the reaction solution to the bottom of the tube.
• Reaction at 37 ℃ for 30 min; drop to 4 ℃ or immediately cool on ice.

1. Transformation of recombinant product

• Thaw chemical competent cells for cloning on ice.
• Add 10 μl recombinant product to 100 μl competent cells, mix the tube wall evenly (please do not shake and mix well), and place 30min on the ice.
• After being heated in a water bath at 42 ℃ for 45 sec, it was immediately placed on ice to cool for 2-3 min.
• Add 900ul SOC or LB medium (without antibiotics) and shake bacteria at 37 ℃ for 1 h (rotation speed 200-250rpm).
• The corresponding resistant LB plate solid medium was preheated in 37 ℃ incubator.
• The supernatant of 900 μl was discarded by 5000rpm centrifugation 5min. The bacteria were re-suspended with the remaining medium and gently smeared evenly on the plate with correct resistance with a sterile coating stick.
• The cells were cultured upside down in an incubator at 37 ℃ for 12-16 h.

1. Identification of recombinant products

Agarose Gel Electrophoresis

Supplies

• SUb-Cell GT Agarose Gel Electrophoresis System
• ChemiDoc Touch Gel Imaging System
• Microwave oven
• Electronic scale
• 200ml erlenmeyer flask
• 50ml and 1000ml graduated cylinders
• Power source

Materials

• Agarose
• GelRed nucleic acid dyes
• DL2000 DNA Marker
• DL5000 DNA Marker
• PCR products
• Double digestion products

Protocol

Agarose gel electrophoresis of PCR products

1. Add 1.2g of agarose and 80ml of 1×TAE buffer to a Erlenmeyer flask,and swirl to suspend the agarose powder in the buffer.
2. Place the gel solution into the microwave.Using a low to medium setting,set the timer for a minimum of 5 minutes,stopping the microwave oven every 30 seconds and swirling the flask gently to suspend the undissloved agarose.
3. Boil and swirl the solution until all of the small translucent agarose particles are dissolved.
4. Set aside the flask to cool to 60℃ before adding 2μl GelRed nucleic acid dyes.
5. Place the comb into the 15×7 cm slot of the tray and pour the molten agarose into the gel tray.
6. Allow 30-40 minutes for the gel to solidify at room temperature.
7. Carefully remove the comb from the solidified gel and remove the tape from the edges of the gel tray.Place the tray onto the leveled Sub-Cell so that the sample wells are near the cathode.
8. Submerge the gel beneath 2 to 6 mm of 1× TAE buffer.
9. Withdraw 2.5μl from the PCR products as loading samples. Load the samples as the following arrangement:

10. Close the lid of gel tank and apply a voltage of 75V.
11. When the DNA samples or dyes have migrated a suffcient distance through the gel,turn off the electric current and remove the leads and lid from the gel tank.
12. Place the gel on the UV transilluminator for nucleic acid visualization and analysis.

Agarose gel electrophoresis of double digestion products

1. Add 1.2g of agarose and 80ml of 1×TAE buffer to a Erlenmeyer flask,and swirl to suspend the agarose powder in the buffer.
2. Place the gel solution into the microwave.Using a low to medium setting,set the timer for a minimum of 5 minutes,stopping the microwave oven every 30 seconds and swirling the flask gently to suspend the undissloved agarose.
3. Boil and swirl the solution until all of the small translucent agarose particles are dissolved.
4. Set aside the flask to cool to 60℃ before adding 2μl GelRed nucleic acid dyes.
5. Place the comb into the 15×7 cm slot of the tray and pour the molten agarose into the gel tray.
6. Allow 30-40 minutes for the gel to solidify at room temperature.
7. Carefully remove the comb from the solidified gel and remove the tape from the edges of the gel tray.Place the tray onto the leveled Sub-Cell so that the sample wells are near the cathode.
8. Submerge the gel beneath 2 to 6 mm of 1× TAE buffer.
9. Withdraw 2.5μl from the PCR products as loading samples. Load the samples as the following arrangement:

10. Close the lid of gel tank and apply a voltage of 85V for nearly 50 minutes.
11. When the DNA samples or dyes have migrated a suffient distance through the gel,turn off the electric current and remove the leads and lid from the gel tank.
12. Place the gel on the UV transilluminator for nucleic acid visualization and analysis.

Extract the plasmid

Material

• Bacterial solution
• Buffer P1
• Buffer P2
• Buffer P3
• FastPure DNA Mini Columns
• Collection Tube 2 ml
• Buffer PW1
• Buffer PW2
• Elution Buffer

Protocol

1. Take 1-5 ml overnight cultured bacterial solution, add it to a centrifuge tube, and centrifuge at 10,000 rpm for 1 min. Discard the medium and invert it on absorbent paper to absorb the residual liquid.
2. Add 250 μl of Buffer P1 to the centrifuge tube with the bacterial pellet left, and mix with a pipette or vortex.
3. Add 250 μl of Buffer P2, and gently mix up and down 8-10 times to fully lyse the cells.
4. Add 350 μl of Buffer P3, and immediately gently invert 8 - 10 times to completely neutralize Buffer P2. At this point, a white flocculent precipitate should appear. Centrifuge at 12,000 rpm for 10 min.
5. Place the FastPure DNA Mini Columns in a Collection Tube 2 ml collection tube. Transfer the supernatant carefully to the adsorption column with a pipette, taking care not to aspirate the pellet, and centrifuge at 12,000 rpm for 30 - 60 sec. Discard the waste liquid in the collection tube and put the adsorption column back into the collection tube.
6. Add 500 μl Buffer PW1 to the adsorption column. Centrifuge at 12000 rpm for 30-60 sec. abandoned waste liquid, put the adsorption column back into the collection tube.
7. Add 600 μl Buffer PW2 (diluted with absolute ethanol) to the adsorption column. 12000rpm centrifuge for 30 - 60 sec. Discard the waste liquid and put the adsorption column back into the collection tube.
8. Repeat the previous step
9. Put the adsorption column back into the collection tube. Dry the adsorption column by centrifugation at 12,000 rpm for 1 min to completely remove the residual rinse solution in the adsorption column.
10. Place the adsorbent in a new sterile 1.5 ml centrifuge tube. Add 30-100 ul Elution Buffer to the center of the membrane of the column adsorption column. Incubate at room temperature for 2 min, and centrifuge at 12,000 rpm for 1 min to elute DNA.
11. The adsorption column was discarded and the DNA product was stored at -20°C to prevent DNA degradation.

Double enzyme digestion - EcoR I and Kpn I

Material

• DNA
• QuickCut Kpn I
• QuickCut EcoR I
• 10X QuickCut Buffer
• 10X QuickCut Green Buffer
• Enzyme storage solution
• Sterilized water

Protocol

1. Prepare the reaction solution according to the following table:

2. Mix gently and centrifuge instantly.
3. Heat preservation at 37 ℃ for 5 min.

Preparation of competent Shewanella

Materials

• Sucrose
• 1.5mL microfuge tubes
• The S. oneidensis MR-1 cultured overnight
• \(\ce{ddH2O}\)

Protocol

1. Prepare 300mM sucrose solution by dissolving 4.11g of sucrose in 40ml of \(\ce{ddH2O}\).
2. Add 1.5ml of cell suspension into each 1.5ml microfuge tube.
3. Centrifuge at a speed of 10000rpm for 2 minutes.
4. Remove the supernatant by aspiration.
5. Add 1ml of sucrose solution into each microfuge tube to suspend the cells gently.
6. Repeat the steps 3,4 for 2 times.
7. Centrifuge the cell suspension at a speed of 10000rpm for 2 minutes.
8. Remove the supernatant by aspiration.
9. Add 100μl of sucrose solution into the center of the microfuge tube and suspend the cells gently.

Electroformation

Supplies

• MicroPulser Electroporator
• MicroPulser Electroporation cuvettes(0.1cm)
• 2ml centrifuge tubes
• Thermostatic water bath cauldron(30℃)

Materials

• LB medium
• LB agar with Kanamycin(20μg/L)
• Competent S. oneidensis MR-1
• Plasmid DNA

Protocol

1. Add 1ml of LB medium into a 2ml centrifuge tube and put it into a 30℃ water bath cauldron.
2. Turn the electroporator and set up the voltage to 1.2kv.
3. Transfer 100μl of competent cell suspension and DNA(approximately 2.4μg) to a cold electroporation cuvette by micropipette.
4. Place the cuvette in the chamber slide. And push the slide into the chamber until the cuvette is seated between the contacts in the base of the chamber.
5. Press the "Pulse" button to complete a pulse.
6. Remove the cuvette from the chamber and immediately add 1mL of LB medium to the cuvette. Gently resuspend the cells.
7. Transfer the cell suspension to a 2ml centrifuge tube and incubate at 30℃ for 2 hr.
8. Centrifuge at 10000rpm.
9. Plate on the LB agar with Kanamycin(20μg/L) and incubate at 30℃.

Biofilm formation assay

Supplies

• 96-well plate
• Microplate reader

Materials

• S.oneidensis (wild type[WT])
• S.oneidensis (mutant)
• Crystal violet(1% aqueous solution)
• Crystal violet(1% aqueous solution)
• LB medium
• MM1 medium

Protocol

1. Mix about 40μl of the overnight LB cultures(OD600~1.0) with 200μl MM1 medium on a 96-well plate.
2. Incubate the cells at 30℃ for 24 or 48 hours.
3. Discard the planktonic cells.
4. Add 100μl of crystal violet to each well. Strain the surface-associated cells for 10 minutes.
5. Remove the excess CV from each well and dry the plate at room temperature for 40 minutes.
6. Add 100μl of 95% ethanol into each well for 10 minutes to resuspend the cells.
7. Quantify the amount of biofilm biomass in each well by measuring the OD260 in the Microplate reader.

Half-cell microbial fuel cell experiment

Material

• LB Liquid medium
• Shewanella recombinant
• Ammonium lactate
• Silver nitrate
• Three-electrode system:
• Working electrode: Carbon paper, Carbon paper-rGO, Carbon paper-rGO-AgNPs (chemical synthesis), Carbon paper-rGO-AgNPs (biosynthesis in this study)
• Counter electrode: platinum electrode
• Reference electrode: Ag/AgCl
• M9 buffer

Protocol

1. The recombinant Shewanella selected after electroporation was added to 10 ml LB liquid medium for overnight culture for 12 hours.
2. The 600nm absorbance (OD600) of the bacterial liquid was determined, and the value was recorded as x, and the bacterial liquid with a volume of 40 ml*1.2/x was taken from the centrifuge tube. (the bacterial liquid concentration is expressed by optical density and defined as the common logarithm of the ratio of incident light intensity to transmitted light intensity, OD=lg (1/trans), where trans is the light transmission value of the detector).
3. The bacterial solution was centrifuged with 7000-8000 rpm 2 min, and the supernatant was taken and re-suspended with appropriate amount of M9 buffer.
4. Repeat step 3 1 to 2 times for washing precipitation.
5. To prepare the working solution of 40 ml electrolytic cell: add 2 ml LB culture solution to the bacterial liquid obtained from the last re-suspension, fill the volume to 40 ml with M9, add sodium lactate to 19 mmol/L (40 ml additive 85mg ammonium lactate), and use nitrogen 15min before use to remove dissolved oxygen.
6. Set up a three-electrode test device and prepare four different electrodes, in which the experimental group needs to add silver nitrate (1 mmol/L) to the working solution.
7. The current intensity was measured at a constant voltage of 0.2 V and the I-t curve was recorded.