| SUSTechOCEAN - iGEM 2024

Molecular experiment

Plasmid Extraction

FastPure Plasmid Mini Kit-BOX 2 (Vazyme)

1. Collect 1-5 ml of overnight cultured bacterial solution (12-16 hours) into a centrifuge tube and centrifuge at 10,000 rpm (11,500 × g) for 1 minute. Discard the culture medium and invert the tube on absorbent paper to remove residual liquid.
2. Add 250 µl of Buffer P1 (ensure RNase A has been added) to the centrifuge tube containing the cell pellet, and mix gently using a pipette or vortex mixer.
3. Add 250 µl of Buffer P2 to step 2 and invert the tube gently 8-10 times to ensure complete cell lysis.
4. Add 350 µl of Buffer P3 to step 3 and immediately invert gently to neutralize Buffer P2. A white precipitate should form. Centrifuge at 12,000 rpm (13,400 × g) for 10 minutes.
5. Place the FastPure DNA Mini Column into a 2 ml Collection Tube. Carefully transfer the supernatant from step 4 to the column using a pipette, avoiding the precipitate. Centrifuge at 12,000 rpm (13,400 × g) for 30-60 seconds. Discard the waste from the collection tube and return the column to the collection tube.
6. (Optional) Add 500 µl of Buffer PW1 to the column. Centrifuge at 12,000 rpm (13,400 × g) for 30-60 seconds. Discard the waste and return the column to the collection tube.
7. Add 600 µl of Buffer PW2 (ensure it is diluted with absolute ethanol) to the column. Centrifuge at 12,000 rpm (13,400 × g) for 30-60 seconds. Discard the waste and return the column to the collection tube.
8. Repeat step 7.
9. Place the column back into the collection tube and centrifuge at 12,000 rpm (13,400 × g) for 1 minute to dry the column and remove any residual wash buffer.
10. Transfer the column to a new sterile 1.5 ml centrifuge tube. Add 30-100 µl of Elution Buffer to the center of the membrane in the column. Incubate at room temperature for 2 minutes and then centrifuge at 12,000 rpm (13,400 × g) for 1 minute to elute the DNA.
11. Discard the column and store the DNA product at -20°C to prevent degradation.

DNA Extraction Using Chelex-100 Method

1. Label each 1.5 ml microcentrifuge tube. Include one tube for a positive control (E. coli).
2. To each labeled tube, add 200 µL of the sterile Chelex-100 solution. Do not add Chelex to tubes that will be used for sample transfer later.
3. Using a sterile toothpick, pick a single colony from the agar plate and insert it into the tube containing the Chelex solution.
4. Gently shake the tube for 5-10 seconds to ensure the colony is fully mixed with the Chelex solution.
5. Place the tubes in the pre-heated metal bath at 100°C. Incubate for 10 minutes to disrupt the cell membrane and release DNA.
6. After incubation, remove the tubes and place them in an ice box for 2 minutes to cool rapidly.
7. Centrifuge the tubes at 10,000 rpm for 10 minutes. Ensure to prepare equal amount centrifuge tubes for clear labeling.
8. Carefully transfer the supernatant (containing the DNA) to new labeled tubes, avoiding any pellet.
9. Measure the DNA concentration and purity using a Nanodrop spectrophotometer.

Addition:

Chelex-100 chelates divalent metal ions (e.g., Ca²⁺, Mg²⁺) that are necessary for the activity of nucleases, thus preventing DNA degradation.
This method is effective in removing PCR inhibitors, including metal ions and some other substances, without altering the concentration of non-metal ions.

Agarose gel electrophoresis

1) Measure 0.25 g of agarose and place it in a conical flask, then add 25 mL of TAE buffer.
2) Microwave on high for approximately 45 seconds until it boils, shake to disperse the agarose, repeat boiling once more until the solution becomes clear.
3) Add 2.5 μL Gelred dye, mix well, and pour it into a mold fitted with appropriately spaced combs.
4) Allow it to sit until the gel completely solidifies, approximately 30 minutes.
5) Gently and vertically remove the combs, take out the gel, and place it in an electrophoresis chamber.
6) Add TAE buffer until the gel is just covered (with the end containing the sample wells near the negative electrode).
7) Dilute 1 μL 10x loading buffer with 4 μL ultrapure water, mix with 5 μL PCR product, and load it into the sample wells of the gel. Finally, add 5 μL marker into the sample wells.
8) Set the voltage to 120V and run the electrophoresis for 20 minutes.
9) After electrophoresis is complete, remove the gel and capture images using a gel imaging system under ultraviolet conditions.

Synechococcus sp.PCC7942
Cyanobacterial Medium Preparation

BG11 Medium

BG11 Blue-Green Medium (Coolaber AML 201-100L)

Store in 4℃

Before use, filter the stock solutions A, B, and C through a 0.22 μm membrane to sterilize them.

Each liter of BG11 medium contains:

988 ml sterile water
10 ml component A
1 ml component B
1 ml component C

After preparation, the pH of the medium is 7.12.

For more detailed components:

Components	Concentration (mg/L)
NaNO₃	1500
K₂HPO₄	40
MgSO₄	36.6
CaCl₂	27.2
Citric acid	6
Ammonium ferric citrate	6
EDTANa₂	1
Na₂CO₃	20
H₃BO₃	2.86
MnCl₂·4H₂O	1.81
ZnSO₄	0.22
Na₂MoO₄·2H₂O	0.39
CuSO₄·5H₂O	0.08
CoCl₂·6H₂O	0.0409

The Culturing condition is:

light intensity	1800lx
Temperature	28℃
Humidity	56%
Light duration	24h

20mg/L NaHCO₃ can be added to help the Cyanobacteria grow faster^[1]

1. Refer to literature to obtain the CscB sequence and submit it to a gene synthesis company for plasmid construction.
2. Plasmid Introduction: Introduce the synthesized plasmid into Synechococcus elongatus PCC7942.
- 2.1. Take 1.5 mL of culture with an OD750 value of 0.5 and transfer it to a 1.5 mL EP tube.
- 2.2. Centrifuge at 6000 g for 10 minutes.
- 2.3. Discard the supernatant and resuspend the pellet in 750 µL of antibiotic-free BG11 liquid medium. Centrifuge at 6000 g for 10 minutes.
- 2.4. Repeat step 2.3.
- 2.5. Discard the supernatant and resuspend in 250 µL of antibiotic-free BG11 liquid medium.
- 2.6. Add the plasmid solution to the tube, ensuring the DNA concentration reaches at least 1 ng/µL.
- 2.7. Wrap the 1.5 mL EP tube in aluminum foil and place it in a dark incubator at 30 °C with shaking at 130 rpm for 24 hours.
- 2.8. After 24 hours, use a sterile spreader to evenly distribute 100 µL of the culture on an antibiotic-containing BG11 plate, spreading until the surface is dry.
- 2.9. Seal the plate with film, label it, and incubate it in a light incubator for 5 days or until single colonies appear. The resulting single colonies indicate successful transformation.

Plasmid Transformation and Resistance Verification

1. Refer to literature to obtain the CscB sequence and submit it to a gene synthesis company for plasmid construction.

2. Plasmid Introduction: Introduce the synthesized plasmid into Synechococcus elongatus PCC7942.

2.1. Take 1.5mL of culture with an OD750 value of 0.5 and transfer it to a 1.5mL EP tube.
2.2. Centrifuge at 6000g for 10 minutes.
2.3. Discard the supernatant and resuspend the pellet in 750µL of antibiotic-free BG11 liquid medium. Centrifuge at 6000g for 10 minutes.
2.4. Repeat step 2.3.
2.5. Discard the supernatant and resuspend in 250µL of antibiotic-free BG11 liquid medium.
2.6. Add the plasmid solution to the tube, ensuring the DNA concentration reaches at least 1 ng/µL.
2.7. Wrap the 1.5mL EP tube in aluminum foil and place it in a dark incubator at 30°C with shaking at 130rpm for 24 hours.
2.8. After 24 hours, use a sterile spreader to evenly distribute 100µL of the culture on an antibiotic-containing BG11 plate, spreading until the surface is dry.
2.9. Seal the plate with film, label it, and incubate it in a light incubator for 5 days or until single colonies appear. The resulting single colonies indicate successful transformation.

light intensity	6000lx
Temperature	30℃
Humidity	56%
Light duration	24h

Plasmid Expression Validation

1.Inoculate single colonies grown on resistance plates into BG11 liquid medium supplemented with 50 µg/mL Amp.

2.After normal culture conditions for 7 days, measure OD685 to reach 0.5. Collect 50 mL of both wild-type PCC7942 and engineered strain PCC7942 by centrifuging at 6000 g for 10 minutes.

3.Discard the supernatant and use the non-resistant BG11 liquid medium for resuspension of both groups. Centrifuge to collect bacteria at 6000 g for 10 min

4.Discard the supernatant. Use 60 ml of non-resistant BG11 liquid medium for resuspension of wild-type S. elongatus. Use 60 ml of resistant BG11 liquid medium for resuspension of S. elongatus transformed with CscB gene.

	Wild-type Non-Induced Group A	Wild-type Induced Group A	Wild-type Non-Induced Group B	Wild-type Induced Group B	Transformed Non-Induced Group A	Transformed Induced Group A	Transformed Non-Induced Group B	Transformed Induced Group B
Wild-type PCC7942 fluid (ml)	5	5	2	2	0	0	0	0
S. elongatus bacterial fluid transformed with cscB gene (μl)	0	0	0	0	5	5	2	2
1M IPTG solution (μl)	0	5	0	5	0	5	0	5
ddH2O(μl)	5	0	5	0	5	0	5	0
1M NaCl solution(μl)	0	0	0	0	0	0	0	0
BG11 medium (ml)	50	50	50	50	50	50	50	50
Number of parallel groups	2	2	2	2	2	2	2	2

5. Use a sucrose content detection kit (Boxbio AKPL006M) to measure sucrose concentration in the supernatant of 72-hour cultures every 24 hours.

5.1. Take 1mL of mixed culture medium, centrifuge at 10,000rpm for 2 minutes, and collect the supernatant.
5.2. Add 2 mg of Reagent IV and decolorize at 80 °C for 30 minutes.
5.3. After decolorization, centrifuge at 5000 g at room temperature for 10 minutes and collect the supernatant.
5.4. Measure the absorbance at 480 nm using a microplate reader.
5.5. Subtract the concentration of the blank tube from the measurement to obtain ΔA, and use the standard curve to determine sucrose concentration.

6. Use a sucrose content detection kit (Boxbio AKPL006M) to measure sucrose concentration in the cyanobacterial cells every 24 hours for 72 hours.

6.1. Take approximately 50 mg of the pellet, freeze at -20 °C for 10 minutes, then thaw at 40 °C. Repeat this twice.
6.2. Disrupt the pellet in an ice bath using an ultrasonic power of 400 W, with a cycle of 3 seconds of disruption followed by 3 seconds of rest, for a total of 12 minutes.
6.3. Add 1 mL of extraction solution, mix well, and extract at 80 °C for 10 minutes, shaking 5 times. After cooling to room temperature, centrifuge at 5000 g for 10 minutes and collect the supernatant.
6.4. Add 2 mg of Reagent IV and decolorize at 80 °C for 30 minutes.
6.5. After decolorization, centrifuge at 5000 g at room temperature for 10 minutes and collect the supernatant.
6.6. Measure the absorbance at 480 nm using a microplate reader.

Reagent	Sample	Standard Solution	Blank
Sample	40
Standard Solution Diluent		40
Distilled Water			40
Reagent1	20	20	20
Mix and heat at 95℃ for 5 minutes, then cool to room temperature.
Reagent2	280	280	280
Reagent3	80	80	80
Mix and incubate at 95℃ for 30 minutes. After cooling to room temperature, measure the absorbance

[1] Wang, K., Wei, W., Zheng, C., & Xu, H. (2005). Optimization of culture conditions for transgenic Synechococcus PCC7942 and its wild type. *Journal of Zhejiang University of Technology*, 33(3), 5.

S.oneidensis MR-1

Experiment 1: Determination of Formate Concentration Using the Thiobarbituric Acid Colorimetric Method

Reagent/Preparation:

R1-a/Thiobarbituric Acid Aqueous Solution [50mL]:

Dissolve 0.8g of thiobarbituric acid in 40mL of ddH₂O. Stir gently for 2-5 minutes. Adjust the pH to 9-10 using 6M NaOH solution (if pH exceeds 10, adjust using 1M diluted sulfuric acid). Heat the solution to 60°C and stir until fully dissolved (about 5-15 minutes). Bring the volume to 50mL. Store in a 50mL centrifuge tube in a refrigerator at 4°C. Multiple tubes can be prepared.

R1-b/1:3 Diluted Sulfuric Acid Solution [100mL]:

Carefully and slowly add 25mL of 98% concentrated sulfuric acid to 75mL ddH₂O while continuously stirring to mix and dissipate heat. Once cooled to room temperature, transfer to a 250mL blue-capped bottle and store. The final H₂SO₄ concentration should be 4.6M.

R1-c/1M Sodium Formate Standard Solution [50mL]:

Weigh 3.4g of sodium formate (MW = 68.01) into a beaker. Add 40mL ddH₂O and stir to fully dissolve. Bring the volume to 50mL. Store at room temperature in a 50mL centrifuge tube.

R1-d-(1-5)/Series of Gradient Sodium Formate Standard Solutions [1mL]:

R1-d-1: 10µL of R1-c + 990µL ddH2O
R1-d-2: 50µL of R1-c + 950µL ddH2O
R1-d-3: 125µL of R1-c + 875µL ddH2O
R1-d-4: 375µL of R1-c + 625µL ddH2O
R1-d-5: 1000µL of R1-c

R1-e/Charcoal Active:

Located on the shelf above the sterilization area in Laboratory 447.

Experimental Procedure:

Preparation of Supernatant from Bacterial Culture and Blank Media:

In the biosafety cabinet, for each type of medium, transfer 1mL of bacterial culture into a 1.5mL centrifuge tube and label as 1-n. Transfer 4mL of the corresponding blank media into separate centrifuge tubes.

Centrifuge the 1mL bacterial culture tubes at 12,000rpm for 3 minutes to obtain the supernatant.

Reaction with Thiobarbituric Acid Reagent:

Prepare six 2mL centrifuge tubes and label them as A1 to A6, which will be used for standard sample testing. Additionally, prepare the same number of 2mL centrifuge tubes as the number of samples, labeling them as S1 to Sn. For example, if there are two samples, label them as S1 and S2.

Follow the corresponding steps. Steps marked with an asterisk (*) are decolorization steps, which can be used to remove interference from other pigments and aldehydes, as needed.

Step 1:
- Add 400µL of blank media to tubes A1 to A6.
- Add 476µL of centrifuged supernatant 1 to tube S1.
- Add 476µL of centrifuged supernatant 2 to tube S2.
*Step 1 (Decolorization):
- Add 600µL of blank media to tubes A1 to A6.
- Add 714µL of centrifuged supernatant 1 to tube S1.
- Add 714µL of centrifuged supernatant 2 to tube S2.
Step 2:
- Add 76µL of R1-d-1 to tube A1.
- Add 76µL of R1-d-2 to tube A2.
- Add 76µL of R1-d-3 to tube A3.
- Add 76µL of R1-d-4 to tube A4.
- Add 76µL of R1-d-5 to tube A5.
- Add 76µL of ddH₂O to tube A6.
*Step 2 (Decolorization):
- Add 114µL of R1-d-1 to tube A1.
- Add 114µL of R1-d-2 to tube A2.
- Add 114µL of R1-d-3 to tube A3.
- Add 114µL of R1-d-4 to tube A4.
- Add 114µL of R1-d-5 to tube A5.
- Add 114µL of ddH₂O to tube A6.
*Step 3-1 (Decolorization):
- Add an appropriate amount of R1-e activated charcoal powder to each tube using a yellow pipette tip.
*Step 3-2 (Decolorization):
- Vortex each tube for 5-10 seconds to ensure thorough mixing.
*Step 3-3 (Decolorization):
- Heat the tubes in a metal bath at 80°C for 30 minutes.
*Step 3-4 (Decolorization):
- Leave the tubes uncovered for 5 minutes to cool to room temperature.
- Centrifuge at 12,000rpm for 5 minutes.
- Transfer 400µL of the supernatant to a new set of 2mL centrifuge tubes.
Step 4:
- Add 50µL of R1-b to each of the tubes (A1 to A6 and S1 to S2).
Step 5:
- Add 1000µL of R1-a to each of the tubes (A1 to A6 and S1 to S2).

Invert the tubes to mix thoroughly, then heat in a metal bath at 100°C for 20 minutes. A yellow coloration of varying degrees will be visible.

Measure the absorbance using a 96-well plate and a microplate reader:

Add 200µL of the heated reaction mixture to each well of the 96-well plate. Then, measure the absorbance at 450nm using the microplate reader in Lab 450.

No.	Known Formate Concentration (mM)	Absorbance (450nm)	Calculated Formate Concentration (mM) (450nm)
A1	1.6
A2	8
A3	20
A4	60
A5	160
A6	0

Experiment 2: Construction of The Device for Electrosynthesis of Formate by S.oneidensis MR-1

No./Equipment:

M2-a/[Maisheng]MS152D (0-15V 0-2A 30W) adjustable regulated DC power supply:

M2-b/[Sanaisi] MFC microbial fuel electrolyzer glass bottle set (double chamber frosted flange 250mL):

M2-c/[DuPont] Nafion N117 proton exchange membrane (3cm*3cm):

Before the first use or after each use, heat it with 50mL of 3% hydrogen peroxide solution at 80oC for 60min, then heat it with 50mL of 1M dilute sulfuric acid at 80oC for 60min, and finally store it with ddH2O for later use.

M2-d-(1-3)/electrode:

M2-d-1:
[Luton] Graphite rod electrode, 6*90mm
M2-d-2:
[Keshenghe] Carbon Energy Carbon Cloth, 10*10cm, W0S1011
M2-d-3:
Silver electrode glass

Before the first use or after each use, heat it with 50mL of 3% hydrogen peroxide solution at 80oC for 60min, then heat it with 50mL of 1M dilute sulfuric acid at 80oC for 60min, and finally store it with ddH2O for later use.

No./Reagent:

R2-a/4×Shewanella BasalBuffer Solution (SBBS)【500mL】:

0.92g NH₄Cl
0.450g K₂HPO₄: Reagent at 450
0.450g KH₂PO₄
0.234g MgSO₄·7H₂O
0.450g (NH₄)₂SO₄
47.662g HEPES: reagent at 450, MW=238.31

After dissolution, store in a 500 mL blue-cap bottle.

R2-b/1M sodium lactate solution [200mL]:

Add 20.016g of 90% D-lactic acid (MW=90.08) to 120mL of ddH₂O. After brief stirring (1-2min), adjust the pH to 7-8 with 6M NaOH solution, then make the volume to 200mL and store in a 250mL blue-cap bottle.

R2-c/1M sodium nitrate solution [50mL]:

Weigh 4.25g NaNO₃(MW=84.99), dissolved toAdd ddH₂O and adjust the volume to 50mL. Store in a 50mL centrifuge tube.

R2-d-(1-2)/cathode culture solution [200mL]:

R2-d-1: SBBS-o cathode culture medium (for Shewanella oneidensis> MR-1):
- 0.25g LB Broth powder
- 47.5mL R2-a
- 4mL R2-b
Dissolve in 180mL, adjust pH to 7.2-7.5, make up to 200mL so that the final volume contains 95%SBBS, 5%LB, 20mM sodium lactate, and sterilize.
R2-d-2: SBBS-f cathode culture medium (for Shewanella fidelis):
- 0.374g 2216E powder
- 47.5mL R2-a
- 4mL R2-b
- 0.793g glucose
- 4g NaCl
Dissolve in 180mL, adjust pH to 7.2-7.5, make up to 200mL so that the final volume contains 95%SBBS, 5%2216E, 2%NaCl, 20mM sodium lactate, 20mM glucose, and sterilize.

R2-e/anolyte【200mL】:

1.491g KCl
200uL 1M H₂SO₄

Use ddH2O was added to make the volume 200mL, so that the final KCl was 100mM, H₂SO₄ 1mM.

R2-f/Tail gas absorption liquid [400mL]:

96g NaOH

Slowly dissolves in ddH₂O, and the final volume was 400mL.NaOH is 6M.
The 400mL tail gas absorption liquid can absorb 2.4mol of CO₂, a total of 53.76L CO₂ (25oC, 1atm); if calculated at a CO₂ flow rate of 60mL/min, it can absorb CO₂ for 14 hours.

R2-g-(1-2)/Electron Mediator:

R2-g-1: Riboflavin(RF), MW=376.36, 53.14uM = 4mg/200mL.
R2-g-2: 2-Hydroxy-1,4-naphoquinone(2-HNQ), MW=174.15, 53.14uM=1.85mg/200mL.

R2-h/reduced graphene oxide (rGO):

The working concentration is 0.2mg/mL.

Experimental steps:

Inoculate 2mL of Shewanella broth into Cathode culture solution 200mL middle, and cultured in a 35 oC incubator 4h.
Inoculate 2mL of Shewanella liquid culture medium into the cathode culture medium again, add 4mL R2-b after filtration and sterilization, add an electrode M2-d, add electron mediator R2-g, and make the final concentration of electron mediator 53.14uM, and continue to culture in a 35oC incubator for 8-12 hours.
Assemble electrodes, double-chamber electrolytic cell glass bottles, proton exchange membranes, gas inlets and outlets, tail gas absorbers, and check for leaks.
Add 200 mL of R2-e anolyte to the anode chamber.
In the clean benchTransfer200mL cathode culture solution containing bacteriato the cathode chamber and replenishAfter filtration sterilization4mL R2-b and 200uL R2-c, so thatSodium nitrateThe concentration is 1mM.
Turn on the adjustable regulated DC power supply, adjust the voltage to 1V, and check the positive and negative connections, as well as the waterproof measures.
Open the CO₂ valve, control the air flow rate to 60mL/min, strictly control the ventilation time, and connect the gas outlet to the tail gas absorption bottle to ensure that no excess CO₂ leaks into the laboratory.
Electrosynthesis every 4Hour, From the small air inlet on the upper right side of the cathode chamber of the double-chamber electrolytic cell glass bottle Pick 1000uL + 800uL Tube Samples, Determination OD600 Bacterial density and formate content.

Safety measures:

Complete the device for leak testing and use a tray to carry the device to prevent water from spilling onto the laboratory bench.
The power supply unit is higher than the glass bottle of the dual chamber electrolytic cell and all wires are off the countertop to prevent contact with water.
The CO2 flow rate is set to the lowest setting, i.e. 60 mL/min.Strictly control the ventilation time, each time is 4 hours,The gas outlet is connected to the tail gas absorption bottle to ensure that no excess CO₂ leaks into the laboratory.
The required carbon dioxide cylinders are in the gas cylinder cabinet next to the glove box and connected to the N3 channel.

The gas cylinder must be unscrewed before use and must be closed after use, see Figure 1 for the gas cylinder valve.

The D valve at N3 must be opened (vertically) before use and closed (horizontally) after use.The A table at N3 indicates the gas outflow rate, and the B table indicates the pressure of the gas in the gas cylinder. The B number will decrease as the gas in the gas cylinder decreases. The E valve can adjust the gas outflow rate. The E valve must be adjusted during the use of the gas cylinder. The tables and valves at N3 are shown in Figure 2.

The gas in the gas cylinder flows out from N3 and reaches N3-1 through the pipeline. The gas valve at N3-1 must be opened (vertically) before use and closed (horizontally) after use., the table and valve at N3-1 are shown in Figure 3.

Figure 1:Gas cylinder valve

Figure 2. Meters and valves at N3

Figure 3. Meters and valves at N3-1

Experiment 3: Preparation and Transformation of S.oneidensis MR-1 Competent Cells

No./Equipment:

M3-a/[Bio-Rad] 1mm shock cup:

After use, wash with ddH₂O and anhydrous ethanol alternately for 10 times, starting with ddH₂O. Then irradiate with UV light for 30 minutes in a clean bench, and finally dry for later use.

No./Reagent:

R3-a/0.5M KCl solution [50mL]:

1.86 g KCl was added50ml de-ionized water to dissolve, loaded into a 50mL centrifuge tube.

R3-b/1M glucose solution [50mL]:

9.01g Glucose (M = 180.18) was dissolved in 50mL of deionized water, filtered and sterilized, and then placed in a 50 mL centrifuge tube.

R3-c/1M magnesium sulfate solution [50mL]:

Dissolve 12.32g of MgSO₄·7H₂O (M=246.4) in 50 ml of deionized water, filter and sterilize, and place in a 50mL centrifuge tube.

R3-d/SOB (Super Optimal Broth) culture medium [100mL]:

R3-a, 0.5mL
NaCl, 0.05g
Tryptone, 2g
Yeast extract, 0.5g

pH = 6.8-7.2, dilute to 98mL and sterilize, store at room temperature

Before use, add 2 mL of R3-c (1 M magnesium sulfate solution).

R3-e-(1-2)/SOC (Super Optimal Catabolite Repression Broth) culture medium [10mL]:

R3-e-1:SOC culture medium:
After sterilization(After adding R3-c)R3-d, 9.8mL
R3-b (1M glucose solution), 200uL after filtration and sterilization
Store in refrigerator at 4 degrees.
R3-e-2:SOC-s (SOC for SOMR-1) culture medium:
R3-d after sterilization (after adding R3-c), 9.8mL
R2-b (1M sodium lactate solution), 200uL after filtration and sterilization
4 degrees refrigeratorsave.

R3-f/TB (Terrific Broth) culture medium:

Yeast extract, 24g/L
Tryptone, 12g/L
K2HPO4 (M=174.18), 12.54g/L
KH2PO4 (M=136.09), 2.313g/L
Glycerol (M=92.09), 4mL/L (0.4% v/v)

R3-g/1M sorbitol buffer [50mL]:

Dissolve 9.109g of sorbitol solid (the reagent is in the lower layer of the 450 ZengLab reagent cabinet) in 50mL of deionized water, put it into a 50mL centrifuge tube, and store it in a refrigerator at 4 degrees.

R3-h-(1-2)/antibiotic solution [1mL]:

R3-h-1: Ampicillin, working concentration 50-100 µg/mL
R3-h-2: Kanamycin, working concentration 50 µg/mL

R3-I/IPTG (Isopropyl β-D-1-thiogalactopyranoside) solution [1mL]:

Working concentration is 1 mM

R3-J/TSA (Tryptic Soy Agar) culture medium:

Tryptone: 15g/L
Soytone: 5g/L
Sodium chloride (NaCl):5g/L
Agar: 15g/L

Experimental steps:

Extract plasmid

Shewanella competent cell preparation

Take 100uL of seed solution and inoculate it into 100mL SOB liquid medium. Cultivate at 30℃ and 250r/min until OD600 is about 0.4.
Transfer the bacterial solution into a pre-cooled 50 mL centrifuge tube and place on ice for 5 minutes.
Take 1mL of bacterial solution in a 1.5mL centrifuge tube and centrifuge at 12000g for 1 min. Discard the supernatant, resuspend the bacterial pellet in 330uL of pre-cooled 1M sorbitol buffer, centrifuge again at 12000g for 1 min, and discard the supernatant.
Gently resuspend the cells in 40uL of pre-cooled 1M sorbitol buffer. Place on ice and perform transformation within 15 minutes. If not used immediately, store in a -80℃ refrigerator.

Transformation of Shewanella

Add 10uL of plasmid solution (concentration of plasmid solution is about 50ng/uL) to 40uL of sorbitol buffer containing competent bacteria, mix in cold for 60-90s, and transfer to a cold electroporation cup.
The conversion voltage was 0.55 KV, and the cells were electroporated in a 1 mm electroporation cup (Bio-Rad, MicroPulserTM) (200 Ω resistor, 25 uF capacitor).
Immediately after transformation, transfer the bacterial solution to 1 ml of pre-cooled SOC culture medium and resuscitate at 30℃ 225r/min for 1-1.5h.
Centrifuge at 12000 g for 1 min, discard the supernatant and resuspend in 200 uL LB.
Spread the bacterial solution on LB solid culture medium containing 100 ng/uL antibiotics and culture at 30°C.

Target gene verification

If the strain can grow normally on the selective medium containing antibiotics, it means that the resistance gene in the plasmid has been successfully expressed, which proves that the plasmid has been successfully introduced.
In order to maximize the expression of the target gene, 1 mM IPTG was added and cultured for about 32 hours.

Vibrio Natriegens

Medium Preparation

1. LB (Luria-Bertani) Medium (g/L):

Peptone: 10g
Yeast extract: 5g
NaCl: 10g

2. LBv2 Medium:

Add v2 salts to LB medium:

NaCl: 204mmol/L
KCl: 4.2mmol/L
MgCl₂: 23.14 mmol/L

3. LB3 Medium (g/L):

Peptone: 10g
Yeast extract: 5g
NaCl: 30g

4. BHIv2 Medium (g/L):

BHI: 37g
NaCl: 204mM
KCl: 4.2mM
MgCl₂: 23.14mM

5. Electroporation Buffer:

680mM Sucrose + 7mM Potassium bicarbonate

6. Improved M9 Medium Formula (per 1L):

Solution A: M9 Stock Solution 5x (for 100ml calculation):: 6g

Na₂HPO₄·12H₂O	27g
KH₂PO₄	5.1g
NaCl	1.75g
NH₄Cl	0.75g

Adjust pH to 7.4 using 1M NaOH and sterilize by autoclaving.

Solution B: MEM Vitamins 1000X

Filter-sterilize.

Solution C: Trace Element Solution 500X (100ml)

FeSO₄·7H₂O	0.3g
CaCl₂·2H₂O	0.3g
MnCl₂·4H₂O	0.06g
CoCl₂·6H₂O	0.04g
CuCl₂·2H₂O	0.015g
ZnSO₂·7H₂O	0.035g
H₃BO₄	0.001g
(NH₄)₆Mo₇O₂₄·4H₂O	0.0125g
EDTA	0.25g

Filter-sterilize.

Solution D: MgSO₄ Stock Solution 1mol/L (100ml)

MgSO₄·7H₂O	24.6g

Sterilize by autoclaving.

Solution E: CaCl2 Stock Solution 1mol/L (50 ml)

CaCl₂·2H₂O	7.35g

Sterilize by autoclaving.

Solution F: Sodium Formate/Sucrose/Sodium Lactate Solution 5X (200 ml)

Formate/Sucrose/Sodium Lactate Solution	10g

Sterilize by autoclaving.

Preparation of 1L M9 Medium (prepare fresh before use):

Combine 200ml of Solution A with 500ml of water. Then add 5ml of Solution D, followed by 0.2ml of Solution E. Add 200ml of Solution F (optional), and adjust the volume with water to 1L.

Cultivation, Preservation, and Competent Cell Preparation of Vibrio Natriegens

Protocol 1:

1.Cultivation and Preparation of Competent Vibrio Natriegens using LB3 Medium
2. Inoculate 100 µL of seed culture into 100mL of LB3 liquid medium and incubate at 37°C, 250 rpm until OD600 reaches approximately 0.4.
3. Transfer the culture to a pre-chilled 50mL centrifuge tube and ice bath for 5 minutes.
4. Centrifuge at 4°C, 2000 × g for 5 minutes and discard the supernatant. Wash the cell pellet twice with 1mL of pre-chilled 1mol/L sorbitol buffer (total of three washes).
5. Gently resuspend the cell pellet in 250µL of pre-chilled 1mol/L sorbitol solution. Dispense 50 µL aliquots into tubes and store at -80°C if not used immediately.

Protocol 2: Cultivation of Vibrio Natriegens in BHIv2

1. Inoculate Vibrio Natriegens into BHIv2 and incubate overnight at 30°C, 200rpm.
2. Dilute 1:100 into fresh BHIv2 medium and incubate at 37°C, 200 rpm until OD600 reaches 0.5.
3. Transfer the culture into five 50mL centrifuge tubes and incubate on ice for 15 minutes.
4. Centrifuge at 4°C, 6000 rpm for 10 minutes and discard the supernatant.
5. Wash with 10mL of cold electroporation buffer and centrifuge again at 4°C, 6000 rpm for 10 minutes. Discard the supernatant and repeat 3-4 times.
6. Resuspend in 150µL of buffer (gently pipetting up and down) and dispense into three tubes. Store at -80°C if not used immediately.

Preservation of Vibrio Natriegens:

1. Rinse the bacteria on the plate with 0.5mL of sterile water and transfer to a 2mL tube.
2. Add 0.5mL of glycerol, seal with parafilm, and store at -20°C.

Transformation of Vibrio Natriegens

Protocol 1:

1. Take 50µL of competent V. N. and add 100 ng of plasmid. Gently mix and transfer to a pre-chilled 1 mm electroporation cuvette. Let it sit for 3 minutes before electroporation at a voltage of 0.7-0.9kV (Bio-Rad, MicroPulser™).
2. Immediately after electroporation, add 1mL of pre-chilled LB3 medium and transfer the mixture to a 2mL centrifuge tube.
3. Incubate at 37°C, 250rpm for 45 minutes.
4. Plate the culture on LB3 solid medium containing 100ng/µL ampicillin and incubate at 30°C overnight.

Protocol 2:

1. Add plasmid (at least 25ng/µL, with a concentration above 70ng/µL) to each 100µL of prepared competent cells. Gently mix and transfer to a pre-chilled 1mm electroporation cuvette.
2. Apply a pulse of 1200/1300V, 25µF, and 200Ω. Immediately add 500µL of pre-warmed (42°C) BHIv2 medium and gently shake in the cuvette for 3-5 minutes.
3. Transfer to a 2mL centrifuge tube and incubate at 37°C, 200rpm for 2 hours.
4. Centrifuge at 8000rpm for 2 minutes, discard the supernatant, and resuspend the pellet in 100µL of BHIv2.
5. Plate 20µL on an antibiotic-free plate and 80µL on an ampicillin-resistant plate.

Verification of Target Gene Expression

1. Testing Plasmid Uptake:

Inoculate the transformed strains onto LB3 solid medium containing ampicillin. If the strains grow normally on this selective medium, it indicates successful expression of the ampicillin resistance gene, confirming successful plasmid uptake.

2. Testing Plasmid Expression:

Preparation and Inoculation of Medium:
- Prepare 200 mL of LB3 medium in six flasks, with flasks A, B, and C inoculated with Vibrio Natriegens transformed with the target plasmid (engineered strains), and flasks D, E, and F with wild-type strains. Add 10 sterilized glass beads to each flask and, once the strains reach the log phase, add 0.8 g of chitin powder. Add IPTG to each flask to a final concentration of 1mM, followed by inoculation with the respective strains.
- Additionally, prepare one flask with transformed Vibrio Natriegens and one with wild-type, adding glass beads without chitin powder, labeled G and H.
Fluorescence Detection:
- After inoculation, cultivate to log phase. Every 10 minutes, take 200µL samples from each flask and transfer them to a black 96-well plate. Measure mCherry fluorescence intensity with an excitation wavelength of 580 nm and emission wavelength of 620nm. Similarly, measure EGFP fluorescence with excitation at 488 nm and emission at 510nm. Record fluorescence changes over 240 minutes to assess target gene expression.
Fusion Protein Extraction and Detection:
- Process the strains that have been cultured overnight and supplemented with chitin. First, centrifuge the cell suspension at 8000g for 10 minutes (4 °C), resuspend the pellet in PBS. Then, use an ultrasonic processor (VCX-130, Sonics & Materials Inc.) to disrupt the cells at 30% power for 2 minutes. Centrifuge the lysate at 12000 g for 20 minutes (4°C) and detect the presence of the target protein using SDS-PAGE.
Chitin Quality Assessment:
- After 24 hours of cultivation, add kanamycin to a final concentration of 100µg/mL. Filter the cultures (ABCDEFG) through a 0.45µm membrane, followed by a 0.22µm membrane. After drying at 60°C in an oven, measure the weight of the membranes to calculate the calcification rate.