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</style> <section class="hero-wrap hero-wrap-2" style="background-image: url('/wiki/images/8/8d/T--Tongji_China--NOTEBOOK.jpeg');" data-stellar-background-ratio="0.5">

Experiment protocols

</section> <section id="about-section" class="ftco-section ftco-no-pt">

Fundamental Protocols

Liquid LB medium（Taking 1000 milliliters for example）

Prepare mixture as the following inside a conical flask.

Components	Volume or mass
Tryptone	10 g
NaCl	10 g
Yeast extract	5 g
ddH2O	1000 mL

Then sterilize at 121℃ in an autoclave.

Note: For selective medium, after the temperature drops below 60℃, add the corresponding antibiotic and adjust to its working concentration.

Solid LB medium (Taking 1000 milliliters (nearly 50 dishes) for example)

Prepare mixture as the following inside a conical flask.

Components	Volume or mass
Agar	15 g
Tryptone	10 g
NaCl	10 g
Yeast extract	5 g
ddH2O	1000 mL

Then sterilize at 121℃ in an autoclave.

Note: For selective medium, after the temperature drops below 60℃ and before the medium solidifies, add the corresponding antibiotic and adjust to its working concentration .

Standard PCR (Taking 25 microliters for example)

On the ice, add all components in a PCR tube, making up to 25 µl volume reaction.

Components	Volume or mass
Template	5-50 ng
Forward primer(10μM)	1 μL
Reverse primer(10μM)	1 μL
2 × Phanta® Master Mix (bought from Vazyme, Number: P511-01)	12.5 μL
ddH2O	Up to 25 μL

Gently mix the PCR reactions and centrifuge briefly.
Transfer the PCR tubes to a thermocycler.

PCR program:

Step	Temperature	Time
Initial denaturalization	95℃	30 s
25~35 cycles	95℃	15 s
\	Annealing temperature depending on primer	15 s
\	72℃	Determined by the length of amplified fragments
Final Extension	72℃	5 min
Hold	10℃	∞

1% Agarose Gel Electrophoresis（Taking 30 milliliters for example）

Prepare the bed in which the gel will polymerize and the comb which is suitable.
Weigh 0.3 g of agarose.
Add 30 mL of 1X TAE.
Heat up the solution until the agarose is completely dissolved.
Add 30 µL of Gel-Red SAFE DNA Gel Stain (1000*) to the solution and mix sufficiently.
Pour the solution into the bed and clear all its bubbles with a tip, and then place the comb rightly and tightly.
Wait for its solidification.
Mix the samples with 6X DNA Loading Buffer in a 5:1 ratio. Inject the right amounts of samples into the wells, as well as DNA marker with appropriate molecular weight into the fit well.

DpnI Restriction Enzyme Digestion (Taking 25 microliters for example)

1. Add all the components on ice.

Components	Volume or mass
PCR products	25 μL
DpnI (Bought from NEB, number:#R0176V)	0.5 μL

2. Mix gently and incubate for 1-2 hours at 37°C.

Note: Incubation time varies along the total volume of the reaction.

Gel DNA Extraction and Purification (FastPure® Gel DNA Extraction Mini Kit)

After completion of DNA electrophoresis, excise band containing the target DNA fragments quickly under the ultraviolet lamp with scalpel. Weigh the gel slice in a tube (by measuring the weight difference of an empty centrifuge tube and the centrifuge tube with gel slice).
Add equal volume Buffer GDP. Water bath at 55℃ for 7 to 10 min, adjust the time appropriately according to the size of the gel to ensure it dissolve completely. Mix upside down twice during the water bath to accelerate the dissolution.
Centrifuge briefly to collect droplets on the tube wall. Place the FastPure DNA Mini Columns-G in a Collection Tubes, and transfer the solution (less than 700μL) to the FastPure DNA Mini Columns-G and centrifuge at 12,000 x g for 60 seconds.

Note: Place the FastPure DNA Mini Columns-G in the collection tube if the volume of the solution is more than 700μL, transfer the remaining solution to the adsorption column, and centrifuge again at 12,000 x g for 60 seconds.

Discard the filtrate and place the FastPure DNA Mini Columns-G in the collection tube. Add 300μL Buffer GDP to the FastPure DNA Mini Columns-G, and then keep still for 1 min. Centrifuge at 12,000 x g for 60 seconds.
Discard the filtrate and place the adsorption column in the collection tube. Add 700μL Buffer GW (Confirm absolute ethanol has been added) to the FastPure DNA Mini Columns-G. Centrifuge at 12,000 x g for 60 seconds.

Note:Adding Buffer GW along the FastPure DNA Mini Columns-G wall helps to flush the salt adhering to the tube wall completely.

Repeat step 5.
Discard the filtrate and place the FastPure DNA Mini Columns-G in the collection tube. Centrifuge at 12,000 x g for 2 min.
Place the FastPure DNA Mini Columns-G in a 1.5 mL sterilized centrifuge tube, then add 20 ddH2O to the center of the FastPure DNA Mini Columns-G, place it for 2 min. Centrifuge at 12000xg for 1 min.

Note:Re-added the obtained solution to the FastPure DNA Mini Columns-G so as to attain a higher yield.

Discard the FastPure DNA Mini Columns-G and detect the concentration of DNA solution by Nanodrop spectrophotometer, then store it at -20℃ to prevent DNA degradation.

PCR Clean-up (Vazyme FastPure® Gel DNA Extraction Mini Kit)

Centrifuge PCR products transiently. Measure its volume with a pipette gun and transfer it to a sterilized 1.5 mL centrifuge tube. Supplement the sample volume with ddH2O to 100μL if the sample volume is less than 100μl.
Add 5 times the volume of Buffer GDP, and invert centrifuge tube several times.

Note: If DNA fragments are less than 100 bp, add 1.5 times the volume of absolute ethanol (volume of sample and Buffer GDP).

Place the FastPure DNA Mini Columns-G in the collection tube. Transfer the solution (less than 700μL) to the FastPure DNA Mini Columns-G and centrifuge at 12000 x g for 60 seconds.If the volume of mixture is more than 700μL, place the adsorption column in the collection tube and then transfer the remaining solution to the FastPure DNA Mini Columns-G, and centrifuge for 60 seconds at 12,000 x g.
Discard the filtrate and place the FastPure DNA Mini Columns-G in the collection tube. Add 700μL Buffer GW (Confirm absolute ethanol has been added) to the FastPure DNA Mini Columns-G. Centrifuge at 12,000 x g for 60 seconds.

Note: Adding Buffer GW along the adsorbent column wall helps to flush the salt adhering to the tube wall completely.

Repeat step 4.
Discard the filtrate and place the adsorption column FastPure DNA Mini Columns-G in the collection tube. Centrifuge at 12,000 x g for 2 min.
Place the FastPure DNA Mini Columns-G in a 1.5 mL sterilized centrifuge tube, then add 20 μL ddH2O to the center of the adsorption column FastPure DNA Mini Columns-G, placing it at room temperature for 2 min. Centrifuge at 12000 x g for 1 min.

Note: Re-added the obtained solution to FastPure DNA Mini Columns-G, and repeat step 7 so as to attain a higher yield.

Discard the adsorption column and detect the concentration of DNA production by Nanodrop spectrophotometer, then store it at -20℃ to prevent DNA degradation.

Two-fragment DNA homologous recombination (Vazyme ClonExpress® II One Step Cloning Kit)

Obtain linear vectors and inserts by PCR.
Purify PCR products, and then detect concentration.
The optimum cloning vector usage and insert fragment usage for ClonExpress® II recombination reaction system is 0.03 pmol and 0.06 pmol, respectively. According to this introduction,the corresponding DNA quality can be roughly calculated by following formulas.

Optimal cloning vectors usage= [0.02* Base pairs number of cloning vectors] ng;

Optimal inserts usage= [0.04* Base pairs number of inserts] ng.

Note:Linear cloning vectors should be used between 50 and 200 ng; insert amplification products should be used between 10 and 200 ng. Select the lowest/highest amount directly when the optimal amount of DNA used exceeds this range by using the above formula.

Arrange the following reaction systems on ice.

Components	Volume
Linear vectors	X μL
Inserts	Y μL
5 X CE Ⅱ Buffer	4 μL
Exnase Ⅱ	2 μL
ddH2O	Up to 20 μL

Note: The values of X and Y are calculated according to the formula of Step 3.

Use a pipette to gently mix (do not shake fiercely), and centrifuge briefly to collect the reaction liquid to the bottom of the tube.
React at 37℃ for 30 min, and then cool on ice.

Multi-fragment DNA homologous recombination (ClonExpress® MultiS One Step Cloning Kit)

Obtain linear vectors and inserts by PCR.
Purify PCR products, then detect concentration.
The optimal DNA usage for the ClonExpress® MultiS recombination reaction system is 0.03 pmol for each fragment (including linearized vector). According to this introduction,the corresponding DNA quality can be roughly calculated by the following formula.

Optimal usage per fragment= [0.02*Base pairs of DNA fragment] ng.

Note: Linear cloning vectors should be used between 50 and 200 ng. Select the lowest/highest amount directly when the optimal amount of DNA used exceeds this range by using the above formula. The amount of each insert should be greater than 10 ng. Use 10 ng directly when using the above formula to calculate the optimum amount of use below this value.

Arrange the following reaction systems on ice.

Components	Volume
Linear vectors	X μL
All inserted fragments	Y1-Yn μL
5* CE MultiS Buffer	4 μL
Exnase MultiS	2 μL
ddH2O	Up to 20 μL

Note: The values of X and Y are calculated according to the formula of Step 3.

Use a pipette to gently suck and mix (do not shake fiercely), and centrifuge briefly to collect the reaction liquid to the bottom of the tube.
React at 37℃ for 30 min, and then cool on ice immediately.

Bacterial Transformation

Add 10 μL of recombination product into 100 μL competent cell, mix gently, and then put tube on the ice for 30 min.
Heat shock for 45 s at 42℃.
Ice bath for 2 min.
Add 900 μL antibiotic-free LB medium into the tube, 220 rpm, 37°C for 1 h, resuscitate the cell.
Preheat the LB solid medium plate with corresponding antibiotics in a 37℃ incubator.
Centrifuge at 5,000 rpm for 5 min and discard 900 μL supernatant.
Suspend the bacteria with the remaining medium, coat the plate containing the correct resistance with a sterile coating rod gently.
Inverted the culture dish in incubator at 37℃ overnight.

Colony PCR (Taking 25 microliters for example)

On ice, add all components in a PCR tube, making up to 25 µl volume reaction.

Components	Volume or mass
F primer (10 μM)	1 μL
R primer (10 μM)	1 μL
Taq PCR Master MIX (2x, blue dye)	12.5 μL
ddH2O	Up to 25 μL

Then pick a colony of the right size and put it in the solution.

Gently mix the PCR reactions and centrifuge briefly.
Transfer the PCR tubes to a thermocycler.

Colony PCR program:

Step	Temperature	Time
Initial denaturalization	95℃	30 s
25~35 cycles	95℃	15 s
\	Annealing temperature depending on primer	15 s
\	72℃	Determined by the length of amplified fragments
Final Extension	72℃	5 min
Hold	10℃	∞

Bacterial Glycerol Stock

In the clean bench, put 500 μL of bacterial overnight culture into the cryovial and add 500 μL of 40% glycerol. Mix it well.
Store at -20 ℃.

Plasmid Extraction (Vazyme FastPure® Plasmid Mini Kit)

Take 1 to 5 mL of overnight bacteria culture (12 to 16 h), add it into centrifuge tube (self-prepared), and centrifuge at 10,000 x g for 1 min. Discard the supernatant and pour it upside down on the absorbent paper to absorb the residual culture.
Add 250μL Buffer P1 (confirm RNase A has been added) to the centrifuge tube with the bacterial sediment left, and suspend bacterial cells by vortex.

Note: Complete resuspension of bacteria is critical to yield, and no bacterial clumps should be seen after resuspension.

Add 250μL Buffer P2 and mix gently by inverting the tube 10 times.

Note: Mix gently upside down. Vortex can cause genomic DNA breakage, resulting in the mixing of genomic DNA fragments in the extracted plasmid. At this time the solution became viscous and transparent, indicating that the bacteria had fully lysed. Do not take longer than 5 minutes to avoid plasmid damage.

Add 350μL Buffer P3 and mix gently by inverting the tube 10 times to neutralize Buffer P2 completely. White flocculent precipitation should occur at this time. Centrifuge at 13,000 x g for 10 min.

Note: Invert and blend immediately after adding Buffer P3, to prevent local precipitation from affecting the neutralization effect. Centrifuge again if there is still white precipitate in the supernatant.

Place the FastPure® DNA Mini Column in a Collection Tube 2 ml collection tube. Transfer the supernatant in step 4 to the FastPure® DNA Mini Column carefully, taking care not to suck the precipitate. Then centrifuge at 13,000 x g for 60 seconds. Remove the waste from the collection tube and put the FastPure® DNA Mini Column back into the collection tube.
Add 500 μL Buffer PW1 to the FastPure® DNA Mini Column. Centrifuge at 13,000 x g for 60 seconds. Discard the waste liquid and put the FastPure® DNA Mini Column back into the collection tube.

Note: This step is optional. Take this step if the host bacteria are end A+ and omit it if the host bacteria are end A-.

Add 600 μL Buffer PW2 (confirm it has been diluted with absolute ethanol) to the FastPure® DNA Mini Column. Centrifuge at 13,000 x g for 60 seconds. Discard the waste.
Repeat step 7.
Dry the FastPure® DNA Mini Column by centrifugation at 13,000 x g for 1 min, in order to remove the residual rinse from the FastPure® DNA Mini Column completely.
Placed the FastPure® DNA Mini Column in a new sterilized 1.5 mL centrifuge tube. Add 30 to 100 μL Elution Buffer to the center of the membrane of the FastPure® DNA Mini Column. Place at room temperature for 2 min and centrifuge at 13,000x g for 1 min to elute DNA.

Note: Preheating Elution Buffer to 55℃ can improve elution efficiency, in addition, re-add the obtained solution to the FastPure® DNA Mini Column and centrifuge again as step 10 can also improve elution efficiency.

Discard the FastPure® DNA Mini Column and detect the concentration of DNA solution by Nanodrop spectrophotometer, then store it at -20℃ to prevent DNA degradation.

Preparation of chemically competent cells (CaCl2 method)

Inoculate cryopreserved strain in LB liquid medium at 1:100 and culture overnight in a 37℃ incubator at 200 rpm.
Filter 0.1M calcium chloride solution with 0.45 μm filter to achieve aseptic. Precool the aseptic calcium chloride solution.
After resuscitation, add the bacterial liquid to the LB liquid medium on a scale of one to one hundred, then culture in a 37℃ incubator at 200 rpm until OD600 reach 0.4-0.5.
Place the liquid on ice for 20 minutes.
Take a sterile 1.5 ml EP tube, collect 1 mL of bacterial liquid, centrifuge in a refrigerated centrifuge at 4℃ for 4 minutes, 4000 rpm, then discard the supernatant. The collection should be repeated three times.
Resuspend the bacteria sediment with 1 mL of 0.1 M calcium chloride solution precooled.
Put the EP tube in ice for 30 minutes.
Centrifuge in a refrigerated centrifuge at 4℃ for 4 minutes, 4000r/min.
Discard the supernatant, suspend the bacterial sediment with 350 μL precooled 0.1 M calcium chloride to obtain fresh competent cells.
Split charging or frozen storage according to the needs.

Plasmid Construction of Heme Biosensor System

The process of plasmid construction of heme biosensor system mainly includes experiments as follows:

Standard PCR to obtain target fragments

On the ice, add all components in a PCR tube, making up to 25 µL volume reaction.

Components	Volume or mass
Template	5-50 ng
Forward primer(10μM)	1 μL
Reverse primer(10μM)	1 μL
2 × Phanta® Master Mix (bought from Vazyme, Number: P511-01)	12.5 μL
ddH2O	Up to 25 μL

Gently mix the PCR reactions and centrifuge briefly.
Transfer the PCR tubes to a thermocycler.

PCR program:

Step	Temperature	Time
Initial denaturalization	95℃	30 s
25~35 cycles	95℃	15 s
\	Annealing temperature depending on primer	15 s
\	72℃	Determined by the length of amplified fragments
Final Extension	72℃	5 min
Hold	10℃	∞

1% Agarose Gel Electrophoresis to identify PCR products

Prepare the bed in which the gel will polymerize and the comb which is suitable.
Weigh 0.3 g of agarose.
Add 30 mL of 1X TAE.
Heat up the solution until the agarose is completely dissolved.
Add 30 µL of Gel-Red SAFE DNA Gel Stain (1000*) to the solution and mix sufficiently.
Pour the solution into the bed and clear all its bubbles with a tip, and then place the comb rightly and tightly.
Wait for its solidification.
Mix the samples with 6X DNA Loading Buffer in a 5:1 ratio. Inject the right amounts of samples into the wells, as well as DNA marker with appropriate molecular weight into the fit well.

DpnI Restriction Enzyme Digestion to eliminate templates

1. On the ice, add all the components.

Components	Volume or mass
PCR products	25 μL
DpnI (Bought from NEB, number:#R0176V)	0.5 μL

2. Mix gently and incubate for 1-2 hours at 37°C.

Note: Incubation time varies along the total volume of the reaction.

Gel DNA Extraction and Purification or PCR clean-up to purify PCR products (Vazyme FastPure® Gel DNA Extraction Mini Kit)

1. After completion of DNA electrophoresis, excise band containing the target DNA fragments quickly under the ultraviolet lamp with scalpel. Weigh the gel slice in a tube (by measuring the weight difference of an empty centrifuge tube and the centrifuge tube with gel slice).

2. Add equal volume Buffer GDP. Water bath at 55℃ for 7 to 10 min, adjust the time appropriately according to the size of the gel to ensure it dissolve completely. Mix upside down twice during the water bath to accelerate the dissolution.

3. Centrifuge briefly to collect droplets on the tube wall. Place the FastPure DNA Mini Columns-G in a Collection Tubes, and transfer the solution (less than 700 μL) to the FastPure DNA Mini Columns-G and centrifuge at 12,000 x g for 60 seconds.

Note: Place the FastPure DNA Mini Columns-G in the collection tube if the volume of the solution is more than 700 μL, transfer the remaining solution to the adsorption column, and centrifuge again at 12,000 x g for 60 seconds.

4. Discard the filtrate and place the FastPure DNA Mini Columns-G in the collection tube. Add 300 μL Buffer GDP to the FastPure DNA Mini Columns-G, and then keep still for 1 min. Centrifuge at 12,000 x g for 60 seconds.

5. Discard the filtrate and place the adsorption column in the collection tube. Add 700 μL Buffer GW (Confirm absolute ethanol has been added) to the FastPure DNA Mini Columns-G. Centrifuge at 12,000 x g for 60 seconds.

Note: Adding Buffer GW along the FastPure DNA Mini Columns-G wall helps to flush the salt adhering to the tube wall completely.

6. Repeat step 5.

7. Discard the filtrate and place the FastPure DNA Mini Columns-G in the collection tube. Centrifuge at 12,000 x g for 2 min.

8. Place the FastPure DNA Mini Columns-G in a 1.5 mL sterilized centrifuge tube, then add 20 ddH2O to the center of the FastPure DNA Mini Columns-G, place it for 2 min. Centrifuge at 12,000 x g for 1 min.

Note: Re-added the obtained solution to the FastPure DNA Mini Columns-G so as to attain a higher yield.

9. Discard the FastPure DNA Mini Columns-G and detect the concentration of DNA solution by Nanodrop spectrophotometer, then store it at -20℃ to prevent DNA degradation.

Homologous Recombination (Two-fragment DNA homologous recombination, using Vazyme ClonExpress® II One Step Cloning Kit)

Obtain linear vectors and inserts by PCR.
Purify PCR products, and then detect concentration.
The optimum cloning vector usage and insert fragment usage for ClonExpress® II recombination reaction system is 0.03 pmol and 0.06 pmol, respectively. According to this introduction, the corresponding DNA quality can be roughly calculated by following formulas.

Optimal cloning vectors usage= [0.02* Base pairs number of cloning vectors] ng;

Optimal inserts usage= [0.04* Base pairs number of inserts] ng.

Note: Linear cloning vectors should be used between 50 and 200 ng; insert amplification products should be used between 10 and 200 ng. Select the lowest/highest amount directly when the optimal amount of DNA used exceeds this range by using the above formula.

Arrange the following reaction systems on ice.

Components	Volume
Linear vectors	X μL
Inserts	Y μL
5 X CE Ⅱ Buffer	4 μL
Exnase Ⅱ	2 μL
ddH2O	Up to 20 μL

Note: The values of X and Y are calculated according to the formula of Step 3.

Use a pipette to gently mix (do not shake fiercely), and centrifuge briefly to collect the reaction liquid to the bottom of the tube.
React at 37℃ for 30 min, and then cool on ice.

Bacterial Transformation

Add 10 μL of recombination product into 100 μL competent cell, mix gently, and then put tube on the ice for 30 min.
Heat shock for 45 s at 42℃.
Ice bath for 2 min.
Add 900 μL antibiotic-free LB medium into the tube, 220 rpm, 37°C for 1h, resuscitate the cell.
Preheat the LB solid medium plate with corresponding antibiotics in a 37℃ incubator.
Centrifuge at 5,000 rpm for 5 min and discard 900 μL supernatant.
Suspend the bacteria with the remaining medium, coat the plate containing the correct resistance with a sterile coating rod gently.
Inverted the culture dish in incubator at 37℃ overnight.

Colony PCR

1. On ice, add all components in a PCR tube, making up to 25 µL volume reaction.

Components	Volume or mass
F primer (10 μM)	1 μL
R primer (10 μM)	1 μL
Taq PCR Master MIX (2x, blue dye; Bought from Sangon, number: B639295-0005)	12.5 μL
ddH2O	Up to 25 μL

Then pick a colony of the right size and put it in the solution.

Gently mix the PCR reactions and centrifuge briefly.
Transfer the PCR tubes to a thermocycler.

Colony PCR program:

Step	Temperature	Time
Initial denaturalization	95℃	30 s
25~35 cycles	95℃	15 s
\	Annealing temperature depending on primer	15 s
\	72℃	Determined by the length of amplified fragments
Final Extension	72℃	5 min
Hold	10℃	∞

Bacterial glycerol stock

In the clean bench, put 500 μL of bacterial overnight culture into the cryovial and add 500 μL of 40% glycerol. Mix it well.
Store at -20 ℃.

Plasmid Extraction (Vazyme FastPure® Plasmid Mini Kit)

Take 1 to 5 ml of overnight bacteria culture (12 to 16 h), add it into centrifuge tube (self-prepared), and centrifuge at 10,000 x g for 1 min. Discard the supernatant and pour it upside down on the absorbent paper to absorb the residual culture.
Add 250μL Buffer P1 (confirm RNase A has been added) to the centrifuge tube with the bacterial sediment left, and suspend bacterial cells by vortex.

Note: Complete resuspension of bacteria is critical to yield, and no bacterial clumps should be seen after resuspension.

Add 250μL Buffer P2 and mix gently by inverting the tube 10 times.

Note: Mix gently upside down. Vortex can cause genomic DNA breakage, resulting in the mixing of genomic DNA fragments in the extracted plasmid. At this time the solution became viscous and transparent, indicating that the bacteria had fully lysed. Do not take longer than 5 minutes to avoid plasmid damage.

Add 350μL Buffer P3 and mix gently by inverting the tube 10 times to neutralize Buffer P2 completely. White flocculent precipitation should occur at this time. Centrifuge at 13,000 x g for 10 min.

Note: Invert and blend immediately after adding Buffer P3, to prevent local precipitation from affecting the neutralization effect. Centrifuge again if there is still white precipitate in the supernatant.

Place the FastPure® DNA Mini Column in a Collection Tube 2 ml collection tube. Transfer the supernatant in step 4 to the FastPure® DNA Mini Column carefully, taking care not to suck the precipitate. Then centrifuge at 13,000 x g for 60 seconds. Remove the waste from the collection tube and put the FastPure® DNA Mini Column back into the collection tube.
Add 500μL Buffer PW1 to the FastPure® DNA Mini Column. Centrifuge at 13,000 x g for 60 seconds. Discard the waste liquid and put the FastPure® DNA Mini Column back into the collection tube.

Note: This step is optional. Take this step if the host bacteria are end A+ and omit it if the host bacteria are end A-.

Add 600μL Buffer PW2 (confirm it has been diluted with absolute ethanol) to the FastPure® DNA Mini Column. Centrifuge at 13,000 x g for 60 seconds. Discard the waste.
Repeat step 7.
Dry the FastPure® DNA Mini Column by centrifugation at 13,000 x g for 1 min, in order to remove the residual rinse from the FastPure® DNA Mini Column completely.
Placed the FastPure® DNA Mini Column in a new sterilized 1.5 ml centrifuge tube. Add 30 to 100μL Elution Buffer to the center of the membrane of the FastPure® DNA Mini Column. Place at room temperature for 2 min and centrifuge at 13,000x g for 1 min to elute DNA.

Note: Preheating Elution Buffer to 55℃ can improve elution efficiency, in addition, re-add the obtained solution to the FastPure® DNA Mini Column and centrifuge again as step 10 can also improve elution efficiency.

Discard the FastPure® DNA Mini Column and detect the concentration of DNA solution by Nanodrop spectrophotometer, then store it at -20℃ to prevent DNA degradation.

Plasmid Construction of Bilateral ARG System

The process of plasmid construction of bilateral ARG system mainly includes experiments as follows:

Standard PCR to obtain target fragments

On the ice, add all components in a PCR tube, making up to 25 µL volume reaction.

Components	Volume or mass
Template	5-50 ng
Forward primer(10μM)	1 μL
Reverse primer(10μM)	1 μL
2 × Phanta® Master Mix (bought from Vazyme, Number: P511-01)	12.5 μL
ddH2O	Up to 25 μL

Gently mix the PCR reactions and centrifuge briefly.
Transfer the PCR tubes to a thermocycler.

PCR program:

Step	Temperature	Time
Initial denaturalization	95℃	30 s
25~35 cycles	95℃	15 s
\	Annealing temperature depending on primer	15 s
\	72℃	Determined by the length of amplified fragments
Final Extension	72℃	5 min
Hold	10℃	∞

1% Agarose Gel Electrophoresis to identify PCR products

Prepare the bed in which the gel will polymerize and the comb which is suitable.
Weigh 0.3 g of agarose.
Add 30 mL of 1X TAE.
Heat up the solution until the agarose is completely dissolved.
Add 30 µL of Gel-Red SAFE DNA Gel Stain (1000*) to the solution and mix sufficiently.
Pour the solution into the bed and clear all its bubbles with a tip, and then place the comb rightly and tightly.
Wait for its solidification.
Mix the samples with 6X DNA Loading Buffer in a 5:1 ratio. Inject the right amounts of samples into the wells, as well as DNA marker with appropriate molecular weight into the fit well.

DpnI Restriction Enzyme Digestion to eliminate templates

1. On the ice, add all the components.

Components	Volume or mass
PCR products	25 μL
DpnI (Bought from NEB, number:#R0176V)	0.5 μL

2. Mix gently and incubate for 1-2 hours at 37°C.

Note: Incubation time varies along the total volume of the reaction.

Gel DNA Extraction and Purification or PCR clean-up to purify PCR products (Vazyme FastPure® Gel DNA Extraction Mini Kit)

1. After completion of DNA electrophoresis, excise band containing the target DNA fragments quickly under the ultraviolet lamp with scalpel. Weigh the gel slice in a tube (by measuring the weight difference of an empty centrifuge tube and the centrifuge tube with gel slice).

2. Add equal volume Buffer GDP. Water bath at 55℃ for 7 to 10 min, adjust the time appropriately according to the size of the gel to ensure it dissolve completely. Mix upside down twice during the water bath to accelerate the dissolution.

3. Centrifuge briefly to collect droplets on the tube wall. Place the FastPure DNA Mini Columns-G in a Collection Tubes, and transfer the solution (less than 700 μL) to the FastPure DNA Mini Columns-G and centrifuge at 12,000 x g for 60 seconds.

Note: Place the FastPure DNA Mini Columns-G in the collection tube if the volume of the solution is more than 700 μL, transfer the remaining solution to the adsorption column, and centrifuge again at 12,000 x g for 60 seconds.

4. Discard the filtrate and place the FastPure DNA Mini Columns-G in the collection tube. Add 300 μL Buffer GDP to the FastPure DNA Mini Columns-G, and then keep still for 1 min. Centrifuge at 12,000 x g for 60 seconds.

5. Discard the filtrate and place the adsorption column in the collection tube. Add 700 μL Buffer GW (Confirm absolute ethanol has been added) to the FastPure DNA Mini Columns-G. Centrifuge at 12,000 x g for 60 seconds.

Note: Adding Buffer GW along the FastPure DNA Mini Columns-G wall helps to flush the salt adhering to the tube wall completely.

6. Repeat step 5.

7. Discard the filtrate and place the FastPure DNA Mini Columns-G in the collection tube. Centrifuge at 12,000 x g for 2 min.

8. Place the FastPure DNA Mini Columns-G in a 1.5 mL sterilized centrifuge tube, then add 20 ddH2O to the center of the FastPure DNA Mini Columns-G, place it for 2 min. Centrifuge at 12,000 x g for 1 min.

Note: Re-added the obtained solution to the FastPure DNA Mini Columns-G so as to attain a higher yield.

9. Discard the FastPure DNA Mini Columns-G and detect the concentration of DNA solution by Nanodrop spectrophotometer, then store it at -20℃ to prevent DNA degradation.

Homologous Recombination (Two-fragment DNA homologous recombination, using Vazyme ClonExpress® II One Step Cloning Kit)

Obtain linear vectors and inserts by PCR.
Purify PCR products, and then detect concentration.
The optimum cloning vector usage and insert fragment usage for ClonExpress® II recombination reaction system is 0.03 pmol and 0.06 pmol, respectively. According to this introduction,the corresponding DNA quality can be roughly calculated by following formulas.

Optimal cloning vectors usage= [0.02* Base pairs number of cloning vectors] ng;

Optimal inserts usage= [0.04* Base pairs number of inserts] ng.

Note: Linear cloning vectors should be used between 50 and 200 ng; insert amplification products should be used between 10 and 200 ng. Select the lowest/highest amount directly when the optimal amount of DNA used exceeds this range by using the above formula.

Arrange the following reaction systems on ice.

Components	Volume
Linear vectors	X μL
Inserts	Y μL
5 X CE Ⅱ Buffer	4 μL
Exnase Ⅱ	2 μL
ddH2O	Up to 20 μL

Note: The values of X and Y are calculated according to the formula of Step 3.

Use a pipette to gently mix (do not shake fiercely), and centrifuge briefly to collect the reaction liquid to the bottom of the tube.
React at 37℃ for 30 min, and then cool on ice.

Bacterial Transformation

Add 10 μL of recombination product into 100 μL competent cell, mix gently, and then put tube on the ice for 30 min.
Heat shock for 45 s at 42℃.
Ice bath for 2 min.
Add 900 μL antibiotic-free LB medium into the tube, 220 rpm, 37°C for 1h, resuscitate the cell.
Preheat the LB solid medium plate with corresponding antibiotics in a 37℃ incubator.
Centrifuge at 5,000 rpm for 5 min and discard 900 μL supernatant.
Suspend the bacteria with the remaining medium, coat the plate containing the correct resistance with a sterile coating rod gently.
Inverted the culture dish in incubator at 37℃ overnight.

Colony PCR

1. On ice, add all components in a PCR tube, making up to 25 µl volume reaction.

Components	Volume or mass
F primer (10 μM)	1 μL
R primer (10 μM)	1 μL
Taq PCR Master MIX (2x, blue dye; Bought from Sangon, number: B639295-0005)	12.5 μL
ddH2O	Up to 25 μL

Then pick a colony of the right size and put it in the solution.

2. Gently mix the PCR reactions and centrifuge briefly.

3. Transfer the PCR tubes to a thermocycler.

Colony PCR program:

Step	Temperature	Time
Initial denaturalization	95℃	30 s
25~35 cycles	95℃	15 s
\	Annealing temperature depending on primer	15 s
\	72℃	Determined by the length of amplified fragments
Final Extension	72℃	5 min
Hold	10℃	∞

Bacterial glycerol stock

In the clean bench, put 500 μL of bacterial overnight culture into the cryovial and add 500 μL of 40% glycerol. Mix it well.
Store at -20 ℃.

Plasmid Extraction (Vazyme FastPure® Plasmid Mini Kit)

Take 1 to 5 ml of overnight bacteria culture (12 to 16 h), add it into centrifuge tube (self-prepared), and centrifuge at 10,000 x g for 1 min. Discard the supernatant and pour it upside down on the absorbent paper to absorb the residual culture.
Add 250μL Buffer P1 (confirm RNase A has been added) to the centrifuge tube with the bacterial sediment left, and suspend bacterial cells by vortex.

Note: Complete resuspension of bacteria is critical to yield, and no bacterial clumps should be seen after resuspension.

Add 250μL Buffer P2 and mix gently by inverting the tube 10 times.

Note: Mix gently upside down. Vortex can cause genomic DNA breakage, resulting in the mixing of genomic DNA fragments in the extracted plasmid. At this time the solution became viscous and transparent, indicating that the bacteria had fully lysed. Do not take longer than 5 minutes to avoid plasmid damage.

Add 350μL Buffer P3 and mix gently by inverting the tube 10 times to neutralize Buffer P2 completely. White flocculent precipitation should occur at this time. Centrifuge at 13,000 x g for 10 min.

Note: Invert and blend immediately after adding Buffer P3, to prevent local precipitation from affecting the neutralization effect. Centrifuge again if there is still white precipitate in the supernatant.

Place the FastPure® DNA Mini Column in a Collection Tube 2 ml collection tube. Transfer the supernatant in step 4 to the FastPure® DNA Mini Column carefully, taking care not to suck the precipitate. Then centrifuge at 13,000 x g for 60 seconds. Remove the waste from the collection tube and put the FastPure® DNA Mini Column back into the collection tube.
Add 500μL Buffer PW1 to the FastPure® DNA Mini Column. Centrifuge at 13,000 x g for 60 seconds. Discard the waste liquid and put the FastPure® DNA Mini Column back into the collection tube.

Note: This step is optional. Take this step if the host bacteria are end A+ and omit it if the host bacteria are end A-.

Add 600μL Buffer PW2 (confirm it has been diluted with absolute ethanol) to the FastPure® DNA Mini Column. Centrifuge at 13,000 x g for 60 seconds. Discard the waste.
Repeat step 7.
Dry the FastPure® DNA Mini Column by centrifugation at 13,000 x g for 1 min, in order to remove the residual rinse from the FastPure® DNA Mini Column completely.
Placed the FastPure® DNA Mini Column in a new sterilized 1.5 ml centrifuge tube. Add 30 to 100μL Elution Buffer to the center of the membrane of the FastPure® DNA Mini Column. Place at room temperature for 2 min and centrifuge at 13,000x g for 1 min to elute DNA.

Note: Preheating Elution Buffer to 55℃ can improve elution efficiency, in addition, re-add the obtained solution to the FastPure® DNA Mini Column and centrifuge again as step 10 can also improve elution efficiency.

Discard the FastPure® DNA Mini Column and detect the concentration of DNA solution by Nanodrop spectrophotometer, then store it at -20℃ to prevent DNA degradation.

Site-directed Mutagenesis PCR

When constructing bilateral ARG system, we met a lot of problems. For example, there might be some mutations shown in sequencing results, and then we might adopt site-directed mutagenesis PCR to repair it.

1. Gene splicing by overlap extension PCR (SOE PCR)

a) Complete complementary primers were designed to repair the plasmid which had some point mutations.

b) DpnI restriction enzyme digestion.

c) Bacterial transformation.

2. Terminal repair PCR

a) Back-to-back primer design: add missing bases in the end of the primer.

b) Ligation:

On the ice, add all the components.

Components	Volume or mass
PCR products	140 ng
T4 ligase	1 μL
T4 Buffer	2 μL
ddH2O	Up to 20 μL

Then, mix gently and incubate overnight at 16°C.

c) DpnI restriction enzyme digestion.

d) Bacterial transformation.

3. Repeat fragments repair PCR

a) Design primers on both sides of repeat fragments to get rid of redundant repeat fragments.

b) Ligation:

On the ice, add all the components.

Components	Volume or mass
PCR products	140 ng
T4 ligase	1 μL
T4 Buffer	2 μL
ddH2O	Up to 20 μL

Then, mix gently and incubate overnight at 16°C.

c) DpnI restriction enzyme digestion.

d) Bacterial transformation.

4. Two fragments homologous recombination repair PCR

a) Design primers to clone fragments required from the plasmid with point mutation in promoter region and design primers to clone promoter required from the other plasmid (pSB1A2-R0051).

b) The rest experimental process of it was the same as normal process of plasmid construction of bilateral ARG system.

5. Enzymatic ligation repair PCR

a) Design primers to clone fragments required from the plasmid with point mutation in promoter region and design primers to clone promoter required from the other plasmid (pSB1A2-R0051).

b) Double digestion:

On the ice, add all the components.

Components	Volume or mass
PCR products	140 ng
KpnI (Takara QuickCut)	1 μL
NdeI (Takara QuickCut)	1 μL
10X QuickCut Buffer	5 μL
ddH2O	Up to 50 μL

Mix gently and incubate for 1-2 hours for double digestion at 37°C.

Note: Incubation time varies along the total volume of the reaction.

c) 1% Agarose Gel Electrophoresis to identify PCR products:

1. Prepare the bed in which the gel will polymerize and the comb which is suitable.

2. Weigh 0.3 g of agarose.

3. Add 30 mL of 1X TAE.

4. Heat up the solution until the agarose is completely dissolved.

5. Add 30 µL of Gel-Red SAFE DNA Gel Stain (1000*) to the solution and mix sufficiently.

6. Pour the solution into the bed and clear all its bubbles with a tip, and then place the comb rightly and tightly.

7. Wait for its solidification.

8. Mix the samples with 6X DNA Loading Buffer in a 5:1 ratio. Inject the right amounts of samples into the wells, as well as DNA marker with appropriate molecular weight into the fit well.

d) Ligation:

On the ice, add all the components.

Components	Volume or mass
PCR products	140 ng
T4 ligase	1 μL
T4 Buffer	2 μL
ddH2O	Up to 20 μL

Then, mix gently and incubate overnight at 16°C.

e) DpnI restriction enzyme digestion.

f) Bacterial transformation.

Heme Biosensor System

Characterization of HrtO

The purpose of this step is to measure the expression strength of HrtO without HrtR. E.coli-wildtype is the negative control. E.coli-pSB1C3-K608010 is the positive control.

Culture E.coli-pUC-GW-HrtO-GFP, E.coli-pSB1C3-K608010 and E.coli-wildtype to logarithmic phase. Measure the fluorescence.

Inoculate E.coli-pUC-GW-HrtO-GFP, E.coli-pSB1C3-K608010 and E.coli-wildtype in LB medium. Resuscitate it in incubator at 37℃ and 200rpm, overnight.
Inoculate the bacterial solution into a 96-well plate in the manner of 100 μL/well, with 3 groups of each concentration in parallel.LB medium was used as the control group.
Measure the OD600 and fluorescence intensity (excitation: 475nm.Emission: 515nm) of the plates by the Microplate spectrophotometer.
The following formula was used to calculate the relative fluorescence intensity of bacteria at different Hemin concentrations, using LB medium as blank.

Characterization of HBS

The purpose of this step is to measure the sensitivity of HBS to heme.

Culture E.coli-pSB1K3-ChuA-HrtR-HrtO-GFP with introduction of different concentration of hemin for several hours, measure the fluorescence.

Preparation of hemin reserve solution: Dissolve Hemin powder by 1 M NaOH, diluted with ddH2O to a final concentration of 500 μM, and sterilized by 0.45 μM needle filter.
Diluted Hemin reserve solution with LB medium to LB medium containing Hemin, with Heme concentration of 0, 1, 2, 3 and 4 μM.
Resuscitation: Inoculate Glycerin bacteria transferred with pSB1K3-ChuA-HrtR-HrtO-GFP in LB medium. Resuscitate it in incubator at 37℃, 200 rpm, overnight.
Induction: Inoculate the resuscitated bacterial solution into the above medium with different Hemin concentration at 1:100. Culture it in a incubator at 37℃, 200 rpm for 6 h.
Inoculate the induced bacterial solution into a 96-well plate in the manner of 100 μL/well, with 3 groups of each concentration in parallel. The LB of corresponding hemin concentration was used as the control group, add the same amount with to do the blank.
Measure the OD600 and fluorescence intensity (excitation: 475 nm.Emission: 515 nm) of the plates by the Microplate spectrophotometer.
The following formula was used to calculate the relative fluorescence intensity of bacteria at different Hemin concentrations, use LB medium with the same concentration of Hemin in the sample bacteria as blank.

Bilateral ARG System

Ultrasonic imaging of gas vesicles

The separate imaging results of ARG1 and ARG2 in mice intestine had been verified by SJTU-BioX-Shanghai in 2018. So we would put emphasis on the distinction between ARG1 and ARG2 by putting the critical collapse pressure of ARG2. Meanwhile, verifying if ARG1 intensity induced by heme was strong enough to be detected was necessary.

Induce E.coli-pHBS-cI-pBAS by various concentration of hemin. The concentration gradient and induction time would be based on the characterizing results of HBS.
Dissolve agar of 8% in 95℃ and mix with induced bacterical before solidification.
After solidification, implement the first time of ultrasonic imaging. Put the critical collapse pressure of ARG2, and then implement the second time of ultrasonic imaging.

Characterization of HBS-BAS

The HBS-BAS may be remade by other teams to express other reporter proteins in the future, while there is no particular characterization data of R0051 and I12007. So we planned to characterize the HBS-BAS, using the combination of pHBS-cI and pA1c-R0051-mRFP-I12007-GFP.

Under ideal conditions, meaning no leakage of HrtO or heme insufficiency, only the mRFP expresses with no heme and only the GFP expressed with the heme. But practically, the result may be a blend of these two. So we want to confirm the ratio of expression of mRFP and GFP under various heme concentrations.

Meantime, the functional verification of the heme signal amplifier would also be based on this characterization.

Inoculate E.coli-pHBS-cI-pA1C-R0051-RFP-I12007-GFP, E.coli-HrtO-cI-pA1C-R0051-RFP-I12007-GFP, E.coli-pSB1C3-K608010, E.coli-pSB1K3-J04450 and E.coli-wildtype in LB medium. Resuscitate it in incubator at 37℃ and 200 rpm, overnight.
Inoculate the bacterial solution into a 96-well plate in the manner of 100 μL/well, with 3 groups in parallel. LB medium was used as the control group.
Measure the OD600 and fluorescence intensity (GFP: ex: 475 nm, em: 515 nm; RFP: ex:588 nm, em: 635 nm) of the plates by the Microplate spectrophotometer.
The following formula was used to calculate the relative fluorescence intensity of bacteria, using LB medium as blank.

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Team:Tongji China/Experiments Protocols/content

Contents

Experiment protocols

Fundamental Protocols

Liquid LB medium（Taking 1000 milliliters for example）

Solid LB medium (Taking 1000 milliliters (nearly 50 dishes) for example)

Standard PCR (Taking 25 microliters for example)

1% Agarose Gel Electrophoresis（Taking 30 milliliters for example）

DpnI Restriction Enzyme Digestion (Taking 25 microliters for example)

Gel DNA Extraction and Purification (FastPure® Gel DNA Extraction Mini Kit)

PCR Clean-up (Vazyme FastPure® Gel DNA Extraction Mini Kit)

Two-fragment DNA homologous recombination (Vazyme ClonExpress® II One Step Cloning Kit)

Multi-fragment DNA homologous recombination (ClonExpress® MultiS One Step Cloning Kit)

Bacterial Transformation

Colony PCR (Taking 25 microliters for example)

Bacterial Glycerol Stock

Plasmid Extraction (Vazyme FastPure® Plasmid Mini Kit)

Preparation of chemically competent cells (CaCl2 method)

Plasmid Construction of Heme Biosensor System

Standard PCR to obtain target fragments

1% Agarose Gel Electrophoresis to identify PCR products

DpnI Restriction Enzyme Digestion to eliminate templates

Gel DNA Extraction and Purification or PCR clean-up to purify PCR products (Vazyme FastPure® Gel DNA Extraction Mini Kit)

Homologous Recombination (Two-fragment DNA homologous recombination, using Vazyme ClonExpress® II One Step Cloning Kit)

Bacterial Transformation

Colony PCR

Bacterial glycerol stock

Plasmid Extraction (Vazyme FastPure® Plasmid Mini Kit)

Plasmid Construction of Bilateral ARG System

Standard PCR to obtain target fragments

1% Agarose Gel Electrophoresis to identify PCR products

DpnI Restriction Enzyme Digestion to eliminate templates

Gel DNA Extraction and Purification or PCR clean-up to purify PCR products (Vazyme FastPure® Gel DNA Extraction Mini Kit)

Homologous Recombination (Two-fragment DNA homologous recombination, using Vazyme ClonExpress® II One Step Cloning Kit)

Bacterial Transformation

Colony PCR

Bacterial glycerol stock

Plasmid Extraction (Vazyme FastPure® Plasmid Mini Kit)

Site-directed Mutagenesis PCR

Heme Biosensor System

Characterization of HrtO

Characterization of HBS

Bilateral ARG System

Ultrasonic imaging of gas vesicles

Characterization of HBS-BAS