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Experiments

1. Extract the plasmid

Vazyme | FastPure Plasmid Mini Kit (DC201)


1.1 Materials

RNase A, Buffer P1, Buffer P2, Buffer P3, Buffer PW1, Buffer PW2, Elution Buffer, FastPure DNA Mini Columns, Collection Tubes 2 ml


1.2 Protocol

  1. Transfer $1-5 \mathrm{mL}$ of overnight ( $ 12 - 16 h$ ) culture to a centrifuge tube, and centrifuge at $10,000 \mathrm{rpm}$ $(11,500 \times \mathrm{g})$ for $1 min$ . Discard the culture medium and place the tube inverted on a blotting paper to drain the liquid.
  2. Add $250 \mu L$ of Buffer P1 (make sure that RNase A has been added) to the centrifuge tube containing the precipitated bacterial cells, and mix thoroughly by pipetting or vortexing.
  3. Add $250 \mu L$ of Buffer P2 to the mixture from Step 2. Mix by gently inverting the tube $8-10$ times to completely lyse the cells.
  4. Add $350 \mu L$ of Buffer P3 to the mixture from Step 3. Immediately invert the tube gently $8 - 10$ times to fully neutralize Buffer P2. At this time, white flocculent precipitates should form. Centrifuge at $12,000 \mathrm{rpm}(13,400 \times \mathrm{g})$ for $10 min$ .
  5. Place FastPure DNA Mini Columns into a Collection Tube $2 mL$ . Carefully transfer the supernatant from Step 4 to the FastPure DNA Mini Columns with a pipette. Taking care not to disturb the precipitates. Centrifuge at $12,000 \mathrm{rpm}(13,400 \times \mathrm{g})$ for $30-60 \mathrm{sec}$. Discard the filtrate and place the FastPure DNA Mini Columns back into the Collection Tube.
  6. Add $500 \mu L$ of Buffer PW1 to the FastPure DNA Mini Columns. Centrifuge at $12,000 \mathrm{rpm}(13,400 \times \mathrm{g})$ for $30 - 60 sec$ . Discard the filtrate and place the FastPure DNA Mini Columns back into the Collection Tube.
  7. Add $600 \mu L$ of Buffer PW2 (make sure that absolute ethanol has been added) to the FastPure DNA Mini Columns. Centrifuge at $12,000 \mathrm{rpm}(13,400 \times \mathrm{g})$ for $30-60 \mathrm{sec}$. Discard the filtrate and place the FastPure DNA Mini Columns back into the Collection Tube.
  8. Repeat Step 7.
  9. Place the FastPure DNA Mini Columns back into the Collection Tube. Centrifuge the empty column at $12,000\ \mathrm{rpm}(13,400 \times \mathrm{g})$ for $1 min$ to completely remove the residual wash buffer.
  10. Place the FastPure DNA Mini Columns in a new sterile $1.5 mL$ centrifuge tube. Add $30-100 \mu L$ of Elution Buffer to the center of the spin column membrane. Leave the system at room temperature for $2 min$ , and centrifuge at $12,000 \mathrm{rpm}(13,400 \times \mathrm{g})$ for $1 min$ to elute the DNA.
  11. Discard the FastPure DNA Mini Columns and store the extracted DNA at $-20^{\circ}$ C to prevent degradation.

2. Plasmid double digestion

2.1 Materials

QuickCut${ }^{\text {TM }}$ $EcoR I$ , QuickCut${ }^{\text {TM }}$ $Not I$ , $10 \times$ QuickCut Green Buffer, Plasmid, ddH₂O


2.2 Protocol

  1. Take $1-2\mu g$ of plasmid and prepare the reaction system on ice as follows:
    Reagent name Reagent dosage
    QuickCut${ }^{\text {TM }}$ $EcoR I$ $1 \mu L$
    QuickCut${ }^{\text {TM }}$ $Not I$ $1 \mu L$
    10×QuickCut Green Buffer $5 \mu L$
    Plasmid $1\mu L -2 \mu L$
    ddH₂O to$50 \mu L$
  2. React in $37^{\circ} \mathrm{C}$ for $1-2 \mathrm{~h}$.

3. Gel DNA Purification

3.1 Materials

Tubes, Buffer GDP, FastPure DNA Mini Columns-G adsorption column, Buffer GW, Elution Buffer.


3.2 Protocol

  1. After the DNA electrophoresis, quickly cut the gel containing the target DNA fragment under the UV lamp, and it is recommended to use a paper towel to absorb the gel surface solution and try to remove excess gel. Weigh the gel (remove the weight of empty tubes), $100 mg$ gel is equivalent to $100 \mu L$ volume, as gel volume.
  2. Add an equal volume of Buffer GDP. Take a water bath at $50 \sim 55^{\circ} \mathrm{C}$ for $7-10 \mathrm{~min}$, and adjust the time appropriately according to the size of the gel to ensure the glue block dissolves completely. Invert and mix 2 times during the water bath to accelerate the sol.
  3. Collect droplets from the tube wall by brief centrifugation. Place the FastPure DNA Mini Columns-G adsorption column in Collection Tubes. Transfer $700 \mu L$ of sol solution to the adsorption column in a $2 mL$ collection tube and centrifuge at 12,000 rpm $(13,800 \times \mathrm{g})$ for $30 \ to \ 60 \ sec$ .
  4. Discard the filtrate and place the adsorption column in the collection tube. Add $300 \mu L$ of Buffer GDP to the adsorption column. Let stand for 1 min. $12,000 \mathrm{rpm}(13,800 \times \mathrm{g})$ centrifugation for $30-60 \ sec$.
  5. Discard the filtrate and place the adsorption column in the collection tube. Add $700 \mu L$ of Buffer GW (absolute ethanol added) to the adsorption column. $12,000 rpm$ ( $13,800 \times \mathrm{g}$ ) centrifugation for $30-60 \ sec$.
  6. Repeat step 5 .
  7. Discard the filtrate and put the adsorption column back into the collection tube. Centrifuge at $12,000 \mathrm{rpm}$ $(13,800 \times \mathrm{g})$ for 2 min.
  8. Place the adsorption column in a $1.5 mL$ sterilized centrifuge tube, add $20-30 \mu L$ of Elution Buffer to the center of the adsorption column, and leave for $2 min$. Centrifuge at $12,000 rpm$ ( $13,800 \times \mathrm{g})$ for $1 min$. Discard the adsorption column and store the DNA at $-20^{\circ} \mathrm{C}$.

4. PCR for inserts

4.1 Materials

Template, $2 \times$ Phanta, Primer F, Primer R, ddH₂O


4.2 Protocol

  1. Prepare the reaction solution according to the following table:
    Reagent name Reagent dosage
    Template1 $400 ng$
    $2 \times$ Phanta $25.0 \mu L$
    Primer F $2.0 \mu L$
    Primer R $2.0 \mu L$
    ddH₂O to$50.0 \mu L$
  2. Run the PCR reaction:
    Temperature Duration Number of cycles
    $98^{\circ} \mathrm{C}$ $30 s$ 1 cycle
    $98^{\circ} \mathrm{C}$ $10 s$ 35 cycles
    $55^{\circ} \mathrm{C}$ $5 s$ 35 cycles
    $72^{\circ} \mathrm{C}$ $5 \mathrm{~s} / \mathrm{kb}$ 35 cycles
    $72^{\circ} \mathrm{C}$ $1 min$ 1 cycle

5. Recombination

Vazyme | ClonExpress Multis One Step Cloning Kit (C113)


5.1 Materials

Linearized vector, Insert, $5 \times$ CE MultiS Buffer, Exnase MultiS, ddH₂O


5.2 Protocol

  1. Dilute the vector and insert fragments at an appropriate ratio before preparing the recombination reaction system, and the amount of each component is not less than $1 \mu L$.
  2. Prepare the following reaction on ice:
    Reagent name Reagent dosage
    Linearized Vector $X \mu L$
    Insert $Y_{1}-Y_{n} \mu L$ (Volume of 1-n insertion fragments)
    $5 \times$ CE MultiS Buffer $4 \mu \mathrm{I}$
    Exnase MultiS $2 \mu L$
    ddH₂O to$20 \mu L$
  3. Gently pipette up and down for several times to mix thoroughly (don't vortex!). Centrifuge to collect the reaction solution to the bottom of the tube.
  4. Incubate at $37^{\circ} \mathrm{C}$ for $30 min$ and immediately chill the tube at $4^{\circ} \mathrm{C}$ or on ice.

6. Transformation

  1. Thaw the competent cells on ice (e.g., DH5α Competent Cell, Vazyme #C502).
  2. Pipette $10 \mu L$ of the recombination products to $100 \mu L$ of competent cells, flick the tube wall to mix thoroughly (don't vortex!), and then place the tube on ice for $30 min$ .
  3. Heat shock at $42^{\circ} \mathrm{C}$ water bath for $45 sec$ and then immediately place on ice for $2 - 3 min$.
  4. Add $900 \mu L$ of LB liquid medium (without antibiotics). Then, shake at $37^{\circ} \mathrm{C}$ for $1 h$ at $200-250$ rpm.
  5. Preheat the corresponding resistant LB solid medium plates in a $37^{\circ} \mathrm{C}$ incubator.
  6. Centrifuge the culture at $5,000 \mathrm{rpm}(2,400 \times \mathrm{g})$ for $5 min$ , discard $900 \mu L$ of supernatant. Then, use the remaining medium to suspend the bacteria and use a sterile bent glass rod to gently spread on the plate which contains the appropriate selection antibiotic.
  7. Incubate at $37^{\circ} \mathrm{C}$ for $12-16 \mathrm{~h}$.

7. Colony PCR

Vazyme | $2 \times$ Rapid Taq Master Mix (P222)


7.1 Materials

$2 \times$ Rapid Taq Master Mix, Primer 1, Primer 2, colonies, ddH₂O


7.2 Protocol

  1. Pick a small number of colonies with a pipette tip and shake well in $30\mu L $ of ddH₂O.
  2. Place in $95^{\circ} \mathrm{C}$ and heat for $10 min$, centrifuge at 8000 g for 3 min, and keep the supernatant for next step.
  3. Prepare the reaction system
Reagent name Reagent dosage
ddH₂O to$20 \mu L$
$2 \times$ Rapid Taq Master Mix $10.0 \mu \mathrm{l}$
Primer$1(10 \mu \mathrm{M})$ $1.0 \mu \mathrm{l}$
Primer$2(10 \mu \mathrm{M})$ $1.0 \mu \mathrm{l}$
Template DNA $2.0 \mu \mathrm{l}$

8. Preparation of Competent Shewanella oneidensis MR. 1

8.1 Materials

Shewanella oneidensis MR.1, Sterile pre-chilled water, Sterile pre-chilled $10 \%$ glycerol


8.2 Protocol

  1. Inoculate overnight cultured Shewanella into freshly cultured $5 mL$ LB medium and incubate for $3-3.5 \mathrm{~h}$ to $\mathrm{OD}=0.4-0.6$
  2. Centrifuge at $2,000 \mathrm{rpm}$ for $15 min$ and wash with $200 mM$ sucrose.
  3. Repeat the previous step $3$ times and retain the Bacterial precipitation.
  4. Resuspend the cells with $1 mL$ of sucrose and aliquot $100 \mu L$ for the preparation of competent cells.
  5. Add $400 ng$ of plasmid, electrotransfer into competent cells with $1.5 kV$ , and transform at $30{ }^{\circ} \mathrm{C}$ until the transformant grows.

9. Measurement of the Shewanella oneidensis MR.1

9.1 Materials

Bacterial culture medium, Cellulase


9.2 Protocol

  1. Take 3 sterilized ep tube, labeled 1, 2, and 3. Add $2.5 mL$ of LB culture medium and $500 \mu L$ bacteria respectively.
  2. Set the initial OD value of the bacterial solution to $0.02$ . The volume of bacterial culture medium to be added is calculated.
  3. Put it into a shaker for culture, and measure the OD value every $3 \ hours$ .

10. Agarose Gel Electrophoresis

10.1 Materials

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


10.2 Protocol

  1. Add $1.2g$ of agarose and $80 mL$ of $1 \times$ 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 $5 min$, stopping the microwave oven every $30 sec$ 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 $60^{\circ} \mathrm{C}$ before adding $2 \mu L$ GelRed nucleic acid dyes.
  5. Place the comb into the $15 \times 7 \mathrm{~cm}$ slot of the tray and pour the molten agarose into the gel tray.
  6. Allow $30-40 min$ 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 $1 \times$ TAE buffer.
  9. Load the samples and the marker.
  10. Close the lid of gel tank and apply a voltage of $75 V$.
  11. 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.

11. Polyacrylamide gel electrophoresis

11.1 Materials

Stacker A, Stacker B, Resolver A (12%), Resolver B, APS, Protein.


11.2 Protocol

  1. Mix each component upside down 6-8 times before use.
  2. Preparation of the lower gel: take the same volume of Resolver A 2.7 $mL$ and Resolver B 2.7 $mL$ each, and mix well.
  3. Preparation of upper gel: take the same volume of Stacker A 0.75 $mL$ and Stacker B 0.75 $mL$, each, and mix well.
  4. Add $60.0 \mu L$ of APS to the mixing solution in step 2 and mix well. immediately. Inject into the gelatinizing glass plate so that the liquid level is about 1.5 cm from the upper edge of the short glass plate.
  5. Add $15.0 \mu L$ of APS to the mixing solution in step 3 and mix well immediately. Without waiting for the lower gel to solidify, gently inject the mixed solution into the gel-making glass plate and gently insert the comb teeth.
  6. After the gel has solidified (about 15min at room temperature), the comb teeth can be removed and used for electrophoresis. The recommended electrophoresis voltage is 150V, and the electrophoresis can be stopped when the bromophenol blue indicator reaches the bottom edge.
  7. Then when the sample is loaded, add 5 μL of 10-180 kDa protein label.
  8. 5ug protein loading buffer is loaded in the protein sample we detect.
  9. Heated at 95 degrees for 10min, and $10.0 \mu L$ protein is loaded after 150kv electrophoresis for 1h, and then decolorized.

12. Pi content detection

12.1 Materials

Molybdate, Ascorbic acid, ddH2O


12.2 Protocol

  1. Prepare molybdate solution at a concentration of $130 \mathrm{~g} / \mathrm{L}$ and ascorbic acid solution at a concentration of $10 \%(w / v)$.
  2. the supernatant to be measured was mixed with molybdate solution, ascorbic acid solution and water at a volume ratio of $1: 2: 1: 49$
  3. Detect the absorbance value at 710 nm was determined after standing for 10 min .

13. PolyP content detection

13.1 Materials

Bacteria, Lysate, Hepes buffer, DAPI


13.2 Protocol

  1. $10^{8}$ bacteria cultured overnight $(O D=1.0)$ were washed once with $20 mM$ Hepes buffer solution.
  2. Centrifuged at $12,000 rpm$ for $5 min$ , then resuspended with $1 mL$ of Hepes buffer.
  3. It is heated at $95{ }^{\circ} \mathrm{C}$ for $10 min$ , and then placed on ice to cool.
  4. Take $10 uL$ of lysate and add it to the mixture of $900 uL$ of Hepes buffer and $100 uL$ of $100 mM$ DAPI solution to mix well. Mix well every $5 min$.
  5. Repeat the operation twice.
  6. Take $200uL$ of the mixture to detect the fluorescence density. Conditions are set to excite at $415 nm$ and emission at $550 nm$.

14. ATP content detection

Enhanced ATP Assay Kit


14.1 Materials

ATP detection working solution, ATP detection reagent diluent


14.2 Protocol

  1. $10^{8}$ bacteria $(O D=1.0$ ) cultured overnight are taken and centrifuged at $12,000 rpm$ for $5 min$.
  2. Add $200\mu L$ of lysate for follow-up operations. After lysis, centrifuge at $12,000 \mathrm{~g}$ at $4^{\circ} \mathrm{C}$ for 5 min, take the supernatant.
  3. Prepare an appropriate amount of ATP testing solution according to the ratio of $100 \mu L$ of ATP detection solution required for each sample or standard.
  4. Add $100 \mu L$ of ATP detection solution to the test hole or test tube. Leave at room temperature for $3-5 min$.
  5. Add $20 \mu L$ of sample or standard to the test well or test tube, quickly mix well with a micropipette, at least $2 sec$ apart, and measure the RLU value with a chemiluminescence instrument.

15. NADH content detection

NAD+ /NADH Assay Kit with WST-8


15.1 Materials

NADH preparation liquid, NAD+s>/NADH extract, Reaction buffer, Chromogenic solution


15.2 Protocol

  1. $10^{8}$ bacteria $(O D=1.0$ ) cultured overnight were also taken and centrifuged at $12,000 rpm$ for $5 min$ .
  2. Add $200 uL$ of lysate for follow-up operations. After lysis, centrifuge at $12,000 \times \mathrm{~g}$ at $4^{\circ} \mathrm{C}$ for $5 min$ and take the supernatant,
  3. Pipette $50-100 \mu L$ of the sample to be tested in a centrifuge tube and heat for $30 min$ on $60^{\circ}$ C water bath or PCR instrument to break down NAD⁺. If insoluble matter is produced after heating, centrifuge at $10,000 \mathrm{~g}$ for $5 min$ at room temperature or $4^{\circ} \mathrm{C}$ and pipette $20 \mu L$ of the supernatant as the sample to be tested into a 96-well plate.
  4. Configure the following systems:
    Regant name Blank Standard Sample
    Samples to be tested - $20 \mu \mathrm{l}$ $20 \mu \mathrm{l}$
    NAD+/NADH extract $20 \mu \mathrm{l}$ - -
    Alcohol dehydrogenase working solution $90 \mu \mathrm{l}$ $90 \mu \mathrm{l}$ $90 \mu \mathrm{l}$
  5. Incubate at $37^{\circ} \mathrm{C}$ for $10 min$ in the dark.
  6. Mix the chromogenic solution appropriately, then add $10 \mu L$ of chromogenic solution to each well, mix well, and incubate at $37^{\circ} \mathrm{C}$ for $30 min$ in the dark, at this time orange-yellow formazan will be formed.

Measure the absorbance at $450 nm$ .


16. Scanning electron microscopy and transmission electron microscope characterization

16.1 Materials

Glutaraldehyde, Ethanol


16.2 Protocol

  1. Cells were collected by centrifugation for $5 min$ at $12,000 \times \mathrm{rpm}$ and then fixed with $5 \%$ glutaraldehyde at $4^{\circ} \mathrm{C}$ for $12 h$ .
  2. Samples were dehydrated in increasing concentrations of ethanol $(25 \%, 50 \%, 75 \%, 95 \%$, and $100 \%$ ) for scanning electron microscopy measurements (SU8020, Japan) or transmission electron microscope (FEI Tecnai G2 F30, USA).

17. Half-cell experiment

17.1 Materials

Amperotentiostat (CHI1040C, Zhenhua, China), $3 \mathrm{M}\ \mathrm{KCl} \ \mathrm{Ag} / \mathrm{AgCl}$ electrode, Carbon cloth, M9 buffer, Sodium lactate and Luria-Bertani culture


17.2 Protocol

  1. A single-chamber electrochemical reactor ( $28 mL$ working volume) MEC system consisting of three electrodes in the Half-Cell Laboratory.
  2. Prepare the electrode solution. The working electrode is carbon cloth, the reference electrode is a $3 \mathrm{M}\ \mathrm{KCl} \ \mathrm{Ag} / \mathrm{AgCl}$ electrode and the counter electrode is a graphite rod. The electrolytes were M9 buffer, $20 mM$ sodium lactate and $5 \% \mathrm{LB}$, and different concentrations of AgNO₃.
  3. The maximum output current was recorded with an amperotentiostat (CHI1040C, Zhenhua, China) by current-time (I-t) measurement ( $200 mV$ on the reference electrode).

18. Full-cell experiment

18.1 Materials

Two-chamber electrochemical reactor,sodium lactate, KHPO₄, KH₂PO₄, M9 minimal medium, proton exchange membrane, Carbon cloth


18.2 Protocol

  1. The MFC system of a two-chamber electrochemical reactor ( $140 mL$ working volume) with electrodes connected via a $2 \mathrm{k} \Omega$ external resistor is used to record the output voltage. Carbon cloth of $1 \times 2 \mathrm{~cm}^{2}, 2.5 \times 3.5 \mathrm{~cm}^{2}$ and proton exchange membrane (Nafion 117, United States DuPont) were used as electrodes and separators, respectively.
  2. Prepare the electrode solution. The cathode solution is $50 mM$ potassium ferricyanide, $50 mM$ K₂HPO₄, and 50 mM KH₂PO₄ solutions. The anode solution is M9 minimal medium, $20 mM$ sodium lactate, and $5 \%$ LB (Luria-Bertani) medium.
  3. Turnover cyclic voltammetry at $1 \mathrm{mV} / \mathrm{s}$ scan rate. Test polarization discharge curves and output power density curves at $0.1 \mathrm{mV} / \mathrm{s}$.