List of abbreviation
Abbreviation	Full Name
IPTG	Isoprophyl-β-D-thiogalactoside
HEPES	2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid
HEPPS	4-Hydroxyerhylpiperazine-1-propanesulfonic acid
EDTA	Ethylenediaminetetraacetic acid
kb	Kilo base pairs
DNA	Deoxyribonucleic acid
OD	Optical density
PCR	Polymerase chain reaction
EP tube	Graduated tip bottom centrifuge tube
TY	Tryptone-Yeast extract
rpm	Revolutions per minute
E. coli	Escherichia coli
S. elongatus	Synechococcus elongatus HL7942
A. caulinodans	Azorhizobium caulinodans ORS571
TAE buffer	Buffer solution containing trihydroxymethyl aminomethane, acetic acid, and ethylenediamine tetraacetic acid of certain concentration
CscB	Sucrose permease
Kan	Kanamycin
Cm	Chloramphenicol
Amp	Ampicillin
Ble	Bleomycin

Reagent: BG11 medium powder(hopebio, product number: HB8793), Milli-Q water

Device: Measuring cylinder, Electronic balance, Medicine spoon

Procedure(for a 1L mixture):

1. Accurately weigh 1.7 g of BG11 medium powder and then add 1 L of Milli-Q water.
2. High-pressure steam sterilization at 121℃ for 15 min.
3. If the non-resistant medium is required, use it after cooling. If the resistant medium is required, add antibiotics after cooling.
   Make sure to use it after mixing completely.

P.S. Antibiotic concentration of S. elongatus selection is as follows: Kan 20 ng/μl, Cm 10 ng/μl. Notice that Synechococcus elongatus HL7942 has basic resistance to ampicillin.

Reagent: BG11 medium powder(hopebio, product number: HB8793), Milli-Q water, Agar powder, NaS₂O₃·5H₂O

Device: Measuring cylinder, Electronic balance, Medicine spoon

Procedure(for a 1 L mixture with 2% agar):

1. Accurately weigh 1.7 g of BG11 medium powder, 4.8 g NaS₂O₃·5H₂O, and 20.0 g agar powder. Mix them and then add 1 L of Milli-Q water.
2. High-pressure steam sterilization at 121℃ for 15 min.
3. If the non-resistant medium is required, pour the plate before solidification. If the resistant medium is required, add antibiotics after cooling but before solidification.
4. Make sure to mix well before pouring. Pour the plate until the bottom of the petri dish is completely covered and the thickness reaches 2-3 mm.

P.S. Lack of NaS₂O₃·5H₂O will reduce the hardness of the culture medium.

List of ingredients for BG11 medium powder(hopebio, product number: HB8793)
Component	Content (g/L)
NaNO3	1.5
K2HPO4·3H20	0.04
MgSO4·7H2O	0.075
CaCl2·2H2O	0.036
Citric acid	0.006
Ferric ammonium citrate	0.006
EDTA	0.001
NaCO3	0.02
H3BO3	0.00286
MnCl2·H20	0.00181
ZnSO4·7H2O	0.000222
CuSO4·5H2O	0.000079
Na2MoO4·2H2O	0.00039
Co(NO3)2·6H2O	0.000049

Reagent: LB broth powder(Sangon Biotech, product number: A507002), Milli-Q water

Device: Measuring cylinder, Electronic balance, Medicine spoon

Procedure(for a 1 L mixture):

1. Accurately weigh 25 g of LB broth powder and then add 1 L of Milli-Q water.
2. High-pressure steam sterilization at 121℃ for 15 min.
3. If the non-resistant medium is required, use it after cooling. If the resistant medium is required, add antibiotics after cooling. Make sure to use it after mixing completely.

P.S. Antibiotic concentration of E. coli selection is as follows: Kan 30 ng/μl, Cm 34 ng/μl, and Amp 100 ng/μl.

List of ingredients for LB broth powder(Sangon Biotech, product number: A507002)
Component	Content (g/L)
Tryptone	10
Yeast extract	5
NaCl	10

Reagent: LB broth Agar powder(Sangon Biotech, product number: A507003), Milli-Q water

Device: Measuring cylinder, Electronic balance, Medicine spoon

Procedure(for a 1 L mixture with 1.5% agar):

1. Accurately weigh 40 g of LB broth agar powder and then add 1 L of Milli-Q water.
2. High-pressure steam sterilization at 121℃ for 15 min.
3. If the non-resistant medium is required, pour the plate before solidification. If the resistant medium is required, add antibiotics after cooling but before solidification.
4. Make sure to mix well before pouring. Pour the plate until the bottom of the petri dish is completely covered and the thickness reaches 2-3 mm.

List of ingredients for M9 broth powder(Sangon Biotech, product number: A507024)
Component	Content (g/L)
Na2HPO4	6.8
KH2PO4	3
NaCl	0.5
NH4Cl	1

Reagent: M9 broth powder(Sangon Biotech, product number: A507024), 1 M MgSO₄ solution, 1 M CaCl₂ solution, Milli-Q water, 20% Sucrose solution

Device: Measuring cylinder, Electronic balance, Medicine spoon, BeyoGold Syringe Filter (0.22μm/3mm, PES, Sterile)

Procedure(for a 100 ml mixture):

1. Accurately weigh 1.13 g of M9 broth powder and then add 95 ml of Milli-Q water.
2. Sterilize M9 salts solution, MgSO₄ solution, and CaCl₂ by high-pressure steam sterilization at 121℃ for 15 min respectively. Sterilize 20% sucrose solution by a filter.
3. After cooling, mix 95 ml of M9 salts solution, 200 μl of 1 M MgSO₄ solution, 10 μl of 1 M CaCl₂ solution, and 5 ml of 20% sucrose solution.
4. If the resistant medium is required, add antibiotics after cooling. Make sure to use it after mixing completely.

List of ingredients for LB broth agar powder(Sangon Biotech, product number: A507003)
Component	Content (g/L)
Tryptone	10
Yeast extract	5
NaCl	10
Agar	15

Reagent: M9 broth powder(Sangon Biotech, product number: A507024), 1 M MgSO₄, 1 M CaCl₂, Milli-Q water, glucose

Device: Measuring cylinder, Electronic balance, Medicine spoon
Procedure(for a 1 L mixture):

1. Accurately weigh 11.30 g of M9 broth powder and then add 978 ml of Milli-Q water.
2. Sterilize M9 salts solution, MgSO4 solution, and CaCl2 by high-pressure steam sterilization at 121℃ for 15 min respectively.
3. Prepare glucose solutions of different concentrations and sterilize them by filtration.
4. After cooling, mix 978 ml of M9 salts solution, 20 ml of glucose solution, 2 ml of 1 M MgSO4 solution, 100 μl of 1 M CaCl2 solution.
5. If the resistant medium is required, add antibiotics after cooling. Make sure to use it after mixing completely.

Reagent: Tryptone, Yeast extract, CaCl₂, Milli-Q water

Device: Measuring cylinder, Electronic balance, Medicine spoon

Procedure(for a 1 L mixture):

1. Accurately weigh 5.0 g tryptone, 3.0 g yeast extract, and 0.6 g CaCl₂. Then add 1 L of Milli-Q water.
2. High-pressure steam sterilization at 121℃ for 20 min.
3. If the non-resistant medium is required, use it after cooling. If the resistant medium is required, add antibiotics after cooling. Make sure to use it after mixing completely.

P.S. Antibiotic concentration of A. caulindans selection is as follows: Ble 100 ng/μl, and Amp 100 ng/μl.

Reagent: Tryptone, Yeast extract, CaCl₂, Milli-Q water, Agar powder

Device: Measuring cylinder, Electronic balance, Medicine spoon

Procedure(for a 1 L mixture with 2% agar):

1. Accurately weigh 5.0 g tryptone, 3.0 g yeast extract, 20.0 g agar powder, and 0.6 g CaCl₂. Then add 1L of Milli-Q water.
2. High-pressure steam sterilization at 121℃ for 20 min.
3. If the non-resistant medium is required, pour the plate before solidification. If the resistant medium is required, add antibiotics after cooling but before solidification.
4. Make sure to mix well before pouring. Pour the plate until the bottom of the petri dish is completely covered and the thickness reaches 2-3 mm.

Reagent: BG11 medium powder(hopebio, product number: HB8793), NH₄Cl, NaCl, HEPES, Milli-Q water

Device: Measuring cylinder, Electronic balance, Medicine spoon, BeyoGold Syringe Filter (0.22μm/3mm, PES, Sterile)

Procedure(for a 1 L mixture):

1. Accurately weigh 1.700 g of BG11 medium powder, 6.201 g NaCl, 0.214 g NH₄Cl, and 5.958 g HEPES. Then add 1 L of Milli-Q water. Make the final concentration of NaCl reach 106mM, NH₄Cl reach 4mM, HEPES reach 25mM. Adjust the pH to 7.5.
2. Use the filter to filter the solution or sterilize by high-pressure steam sterilization at 121℃ for 15 min.
3. If the non-resistant medium is required, use it after cooling. If the resistant medium is required, add antibiotics after cooling. Make sure to use it after mixing completely.

Reagent: Glycerol bacteria preserved at -40℃ or -80℃, Ice, Non-resistant liquid medium corresponding to the bacteria strain

Device: Icebox, Ultra clean bench, Culture tubes, Pipette gun, Sterile pipette tips, Refrigerator

Procedure:

1. Take 3-5ml non-resistant liquid medium on the ultra clean bench and put it into the culture tube.
2. Remove the glycerol bacteria from the -40℃ or -80℃ refrigerator and immediately put it into the icebox containing ice.
3. Quickly remove the ice residue of glycerol bacteria on the ultra clean bench by the pipette tip and put it into the culture medium.
4. Quickly put the glycerol bacteria back into the refrigerator before melting.
5. Label the culture tube and put it into the shaker. Culture until the solution turns turbid.

P.S. Avoid repeated freezing and thawing.

Reagent: Glycerol bacteria preserved at -40℃ or -80℃, Ice, Non-resistant TY liquid medium, Resistant TY plate.

Device: Icebox, Ultra clean bench, Culture tubes, Pipette gun, Sterile pipette tips, Refrigerator, NanoDrop One

Procedure:

1. Inoculate 50 μl glycerol bacteria into 5 ml TY liquid medium and culture for 2 ~ 3 days.
2. Perform streak plate method on TY solid medium containing 100 μg/ml ampicillin and culture at 37℃ for 2 days.
3. Inoculate single colonies into 4 ml TY liquid medium containing 100 μg/ml ampicillin and culture until OD₆₀₀ = 0.5 ~ 0.7.

Reagent: Bacterial fluid to be tested, Liquid medium corresponding to the sample, Double distilled water

Device: Ultra clean bench, Pipette gun, Pipette tips, Sterile pipette tips, NanoDrop one ultramicro ultraviolet spectrophotometer(Thermo SCIENTIFIC), Lens papers

Procedure:

1. Open NanoDrop one in advance.
2. Take out the sample of bacterial fluid to be tested on the ultra clean bench.
3. Select the "OD₆₀₀ " tab and click the "OD₆₀₀" option on the home page of NanoDrop one.
4. Fill in "other detection wavelengths" according to the type of bacteria to be tested. For E. coli or *A. caulinodans*, fill in 600 or not. For S. elongatus, fill in 685.
5. Clean the instrument base with double distilled water. Wipe clean with lens papers.
6. Use 1 μl of liquid medium corresponding to the sample as a blank sample. After detecting, clean the instrument base with double distilled water. Wipe clean with lens papers.
7. Add 1 μl bacterial fluid to the base for detection. After detecting, record data and clean the instrument base with double distilled water. Wipe clean with lens papers.
8. Shut down the instrument after use.

P.S. Complete mixing is required before sampling and loading of S. elongatus bacterial fluid.

Reagent: Bacterial fluid to be tested, Liquid medium corresponding to the sample

Device: Ultra clean bench, Pipette gun, Sterile pipette tips, Microplate Reader(Thermo SCIENTIFIC), 96-well plate(or disposable enzyme label strips and enzyme label plate)

Procedure:

1. Open the microplate reader in advance.
2. Add 200μl of bacterial fluid to be tested into 96-well plate(or disposable enzyme label strips and enzyme label plate) on the ultra clean bench. Also add 200 μl of liquid medium corresponding to the sample as a blank sample.
3. Put the 96-well plate(or enzyme label plate) into the microplate reader. Select detection area and wavelength. Then click "start".
4. Record the data after detecting. The OD value of the sample is the sample detection value minus the blank sample detection value.
5. Shut down the microplate reader after use.

Culture

Conditions: 37℃, 220 rpm, no special requirements for lighting.

Inoculation

Reagent: LB liquid medium, Bacterial fluid

Device: Ultra clean bench, Pipette gun, Sterile pipette tips, Culture tubes, Thermostatic shaker

Procedure:

1. Take bacterial fluid in the logarithmic growth period(OD₆₀₀=0.5-0.6).
2. Inoculate the bacterial fluid to the fresh medium at a ratio of 1:100 in the culture tube.
3. Label and put the tube in the thermostatic shaker.

P.S. Steps 1-2 operate on the ultra clean bench

Culture

Conditions: 37℃, no special requirements for lighting.

Inoculation

Reagent: LB plate, Bacterial fluid

Device: Ultra clean bench, Pipette gun, Sterile pipette tips, Thermostatic incubator, Disposable spreaders, Disposable inoculation loops, Parafilm

Procedure:

Spread Plate Method

1. Remove 100 μl bacterial fluid to the LB plate.
2. Use a disposable spreader to evenly coat the bacterial fluid on the LB plate. Keep coating until no flowing liquid on the surface of the plate.
3. Seal the plate with parafilm. Label and put the plate in the thermostatic incubator.

P.S. Steps 1-2 operate on the ultra clean bench

Streak Plate Method

1. Dip the bacterial fluid by a disposable inoculation loop.
2. Streak the LB plate by the inoculation loop with bacterial fluid drop.
3. Seal the plate with parafilm. Label and put the plate in the thermostatic incubator.

P.S. Steps 1-2 operate on the ultra clean bench

Culture

Conditions: 30℃, 130 rpm, 10 W lamp tube.

Inoculation

Reagent: BG11 liquid medium, Bacterial fluid

Device: Ultra clean bench, Pipette gun, Sterile pipette tips, Culture tubes, Thermostatic shaker with external lamp tube

Procedure:

1. Take bacterial fluid in the logarithmic growth period(OD₆₈₅≈0.5).
2. Inoculate the bacterial fluid to the fresh medium at a ratio of 1:100 in the culture tube.
3. Label and put the tube in the Thermostatic shaker with an external lamp tube.

P.S. Steps 1-2 operate on the ultra clean bench

Culture

Conditions: 30℃, 8000 lux.

Inoculation

Reagent: BG11 plate, Bacterial fluid

Device: Ultra clean bench, Pipette gun, Sterile pipette tips, Thermostatic light incubator, Disposable spreaders, Parafilm

Procedure:

1. Remove 100 μl bacterial fluid to the BG11 plate.
2. Use a disposable spreader to evenly coat the bacterial fluid on the BG11 plate. Keep coating until no flowing liquid on the surface of the plate.
3. Seal the plate with parafilm. Label and put the plate in the thermostatic light incubator.

P.S. Steps 1-2 operate on the ultra clean bench

Culture

Conditions: 37℃, 180 rpm, no special requirements for lighting.

Inoculation

Reagent: TY liquid medium, Bacterial fluid

Device: Ultra clean bench, Pipette gun, Sterile pipette tips, Culture tubes, Thermostatic shaker

Procedure:

1. Take bacterial fluid in the logarithmic growth period(OD₆₀₀=0.5-0.6).
2. Inoculate the bacterial fluid to the fresh medium at a ratio of 1:100 in the culture tube.
3. Label and put the tube in the thermostatic shaker.

P.S. Steps 1-2 operate on the ultra clean bench

Culture

Conditions: 37℃, no special requirements for lighting.

Inoculation

Reagent: TY plate, Bacterial fluid

Device: Ultra clean bench, Pipette gun, Sterile pipette tips, Thermostatic incubator, Disposable spreaders, Disposable inoculation loops, Parafilm

Procedure:

Spread Plate Method

1. Remove 100 μl bacterial fluid to the TY plate.
2. Use a disposable spreader to evenly coat the bacterial fluid on the TY plate. Keep coating until no flowing liquid on the surface of the plate.
3. Seal the plate with parafilm. Label and put the plate in the thermostatic incubator.

P.S. Steps 1-2 operate on the ultra clean bench

Streak Plate Method

1. Dip the bacterial fluid by a disposable inoculation loop.
2. Streak the TY plate by the inoculation loop with bacterial fluid drop.
3. Seal the plate with parafilm. Label and put the plate in the thermostatic incubator.

P.S. Steps 1-2 operate on the ultra clean bench

Reagent: bacterial fluid to be frozen, Sterile 50% glycerol, -80℃ Refrigerator

Device: Ultra clean bench, Pipette gun, Sterile pipette tips, 1.5 ml EP tube

Procedure(for 500 μl bacterial fluid):

1. Take 500 μl of bacterial fluid whose OD₆₀₀ reaches 0.5.
2. Mix 500 μl bacterial fluid with 500 μl sterile 50% glycerol together in a 1.5 ml EP tube. Make the final concentration of glycerol reach 25%.
3. Label and put the EP tube into the -80℃ refrigerator.

P.S. Operate on the ultra clean bench

Reagent: bacterial fluid to be frozen, Sterile 50% glycerol, Non-resistant BG11 liquid medium, -80℃ Refrigerator

Device: Ultra clean bench, Pipette gun, Sterile pipette tips, Centrifuge, Centrifuge tube, 1.5 ml EP tube

Procedure(for 4 ml bacterial fluid):

1. Take 4 ml of bacterial fluid whose OD₆₈₅ reaches 0.5 in the suitable centrifuge tube.
2. Centrifuge to collect bacteria at 6000 g at 4℃ for 10 min.
3. Discard the supernatant and use 2 ml of non-resistant BG11 liquid medium for resuspension. Centrifuge to collect bacteria at 6000 g at 4℃ for 10 min.
4. Repeat Step 3.
5. Discard the supernatant and use 800 μl of non-resistant BG11 liquid medium for resuspension.
6. Mix 680 μl concentrated liquid with 320 μl sterile 50% glycerol together in a 1.5 ml EP tube. Make the final concentration of glycerol reach 16%.
7. Label and put the EP tube into the -80℃ refrigerator.

P.S. Steps 1, 3-6 should be operated on the ultra clean bench.

Reagent: bacterial fluid to be frozen, Sterile 50% glycerol, Sterile double distilled water, -80℃ Refrigerator

Device: Ultra clean bench, Pipette gun, Sterile pipette tips, Centrifuge, Centrifuge tube, 1.5 ml EP tube

Procedure(for 20 ml bacterial fluid):

1. Take 20 ml of bacterial fluid whose OD₆₀₀ reaches 0.6 in the suitable centrifuge tube.
2. Centrifuge to collect bacteria at 8000 g at 4℃ for 5 min.
3. Discard the supernatant.
4. Use 400 μl sterile 50% glycerol and 600 μl sterile double distilled water to resuspend in the 1.5 ml EP tube. Make the final concentration of glycerol reach 20%.
5. Label and put the EP tube into the -80℃ refrigerator.

P.S. Steps 1, 3-4 should be operated on the ultra clean bench.

Reagent: Double distilled water, 2xPhanta^® Max Master Mix (Dye Plus) P525 or Golden Mix(Green)(TSINGKE, production: TSE101), Template DNA(plasmid DNA), Forward primer(10μM), Reverse primer(10 μM), Ice

Device: Pipette gun, Pipette tips, PCR tube, Icebox, PCR-Instrument, Centrifuge

Procedure:

• 1. Open the PCR-Instrument in advance.
• 2. Mix reagents on ice according to the table below in the PCR tube:

Components	Volume
2 × Phanta® Max Master Mix (Dye Plus) P525 or Golden Mix(Green)	25 μl
Forward primer(10 μM)	2 μl
Reverse primer(10 μM)	2 μl
Template DNA(plasmid DNA)	xμl(10 pg-30 ng)
Double distilled water	up to 50 μl

• 3. Mix the solution system evenly. Carry out rapid centrifugation if the droplets hang on the tube wall.
• 4. Put the PCR tube in the PCR-Instrument. Set the PCR program according to the table below.

Stage/Number of Cycles	Temperature	Time
Stage 1 / 1	95℃	30 s
Stage 2 / 35	95℃	15 s
	(Tm of primer-5)℃	15 s
	72℃	60 s/kb for Phanta® Max Master Mix
		15 s/kb for Golden Mix
Stage 3 / 1	72℃	5 min
Stage 4 / 1	10℃	forever

• 5. Take the tube out and put it in the icebox containing ice.

Reagent: Double distilled water, 2xPhanta^® Max Master Mix (Dye Plus) P525, Forward primer(10 μM), Reverse primer(10 μM), Ice, Bacterial fluid to be used

Device: Pipette gun, Pipette tips, PCR tube, Icebox, PCR-Instrument, Centrifuge, Metal bath heater, 1.5 ml EP tube

Procedure:

• 1. Open the PCR-Instrument in advance. Set the metal bath heater to 95℃.
• 2. Add 100 μl bacterial fluid into a 1.5 ml EP tube. Put the tube on the metal bath heater for 10 minutes.
• 3. Mix reagents on ice according to the table below in the PCR tube:

Components	Volume
2 × Phanta® Max Master Mix (Dye Plus) P525	25 μl
Forward primer(10μM)	2 μl
Reverse primer(10μM)	2 μl
Heated bacterial fluid	1-2 μl (according to the concentration of the bacteria)
Double distilled water	up to 50 μl

• 3. Mix the solution system evenly. Carry out rapid centrifugation if the droplets hang on the tube wall.
• 4. Put the PCR tube in the PCR-Instrument. Set the PCR program according to the table below.

Stage/Number of Cycles	Temperature	Time
Stage 1 / 1	95℃	30 s
Stage 2 / 35	95℃	15 s
	(Tm of primer-5)℃	15 s
	72℃	60 s/kb
Stage 3 / 1	72℃	5 min
Stage 4 / 1	10℃	forever

• 5. Take the tube out and put it in the icebox containing ice.

Reagent: 1xTAE buffer, Agarose, 10000xNucleic acid dye

Device: Pipette gun, Pipette tips, Measuring cylinder, Electronic balance, Medicine spoon, Microwave oven, Gel-making tank, Gel-making plate, Gel-making comb

Procedure(for a 1% nucleic acid electrophoresis gel):

1. Accurately weigh 0.3 g agarose and then add 30 ml of 1xTAE buffer.
2. Heat to boiling in the microwave oven at least 3 times to dissolve agarose completely.
3. Put a gel-making plate on the gel-making tank and put a gel-making comb on it.
4. Add 3 μl 10000xNucleic acid dye in the agarose solution when it's cooled to about 60℃.
5. Pour the agarose solution into the gel-making mold.
6. Wait about 20 minutes until the gel sets. Remove the comb.

Reagent: Nucleic acid electrophoresis gel, DNA marker, PCR product, 1xTAE buffer

Device: Pipette gun, Pipette tips, Electrophoresis apparatus, Electrophoresis tank for nucleic acid

Procedure:

1. Add 1xTAE buffer into the electrophoresis tank for nucleic acid. Put the gel into the tank and make sure it can be submerged by TAE buffer.
2. Make sure the loading pore is close to the negative side.
3. Add 10 μl DNA marker into the loading pore. Add 50 μl PCR product into the loading pore. Record the position of the sample added.
4. Open the electrophoresis apparatus and connect it with the electrophoresis tank for nucleic acid.
5. Set the voltage to 100 V. Run 30 minutes or as appropriate.

P.S. The choice of DNA marker is based on the length of the PCR product.

Reagent: Post-electrophoresis nucleic acid gel

Device: Gel imager(azure biosystems c150), Computer, Disposable gloves

Procedure:

1. Take out post-electrophoresis gel with disposable gloves on the hands.
2. Open the computer and gel imager. Open the software of the gel imager.
3. Put the gel in the imager. Set the exposure mode and capture the image of the gel.
4. Find the target bands and save the image.

Reagent: Post-electrophoresis nucleic acid gel, Gel recovery kit(FastPure^® Gel DNA Extraction Mini Kit DC301 from Vazynme), double distilled water

Device: UV Transilluminator, Scalpel, 1.5ml EP tube, Metal bath heater, Pipette gun, Pipette tips, Electronic balance

Procedure:

1. Set the metal bath heater to 55℃ in advance.
2. Precisely cut the gel containing the target bands with a scalpel under the UV transilluminator.
3. Accurately weigh the pieces of gel. Put them into 1.5 ml EP tubes respectively.
4. According to the instructions in the kit recover the DNA fragment until the last step. Use double distilled water but not the buffer to dissolve the DNA fragment.

Reagent: Gel recovery product, double distilled water

Device: NanoDrop one ultramicro ultraviolet spectrophotometer(Thermo SCIENTIFIC), Lens papers, Pipette gun, Pipette tips

Procedure:

1. Open NanoDrop one in advance.
2. Select the "nucleic acid " tab and click the "double strain DNA" option on the home page of NanoDrop one.
3. Clean the instrument base with double distilled water. Wipe clean with lens papers.
4. Use 1 μl of double distilled water as a blank sample. After detecting, wipe clean with lens papers.
5. Add 1 μl gel recovery product to the base for detection. After detecting, record data and clean the instrument base with double distilled water. Wipe clean with lens papers.
6. Shut down the instrument after use.

Reagent: Gel recovery product, double distilled water, Exnase II, 5xCE II buffer, Ice

Device: Pipette gun, Pipette tips, Icebox, PCR-Instrument, PCR tube

Procedure:

• 1. Open the PCR-instrument in advance.
• 2. Mix reagents on ice according to the table below in the PCR tube:

Components	Volume
Linearization carrier	x μl (up to 0.03 pmol)
Insert Fragment	y μl (up to 0.06 pmol)
5 × CE II buffer	4 μl
Exnase II	2 μl
Double distilled water	up to 20 μl

• 3. Mix the solution system evenly. Carry out rapid centrifugation if the droplets hang on the tube wall.
• 4. Put the PCR tube in the PCR-Instrument. Set the PCR program according to the table below.

Stage/Number of Cycles	Temperature	Time
Stage 1 / 1	37℃	30 min
Stage 2 / 1	10℃	forever

• 5. Take the tube out and put it in the icebox containing ice.

Reagent: Recombination product, DH5α competent E. coli cells, Ice, Resistant LB plate, Non-resistant LB liquid medium

Device: Pipette gun, Sterile Pipette tips, Icebox, Metal bath heater, Ultra clean bench, Disposable spreader, 1.5ml EP tube

Procedure:

1. Set the metal bath heater to 42℃ in advance.
2. Take DH5α competent E. coli cells and put them on the ice to thaw.
3. After thawing, add 10 μl recombination product into 100μl DH5α competent E. coli cells. Put it on ice for 30 minutes.
4. Heat shock the transformation tube for 90 seconds. Cool it on the ice for 3 minutes immediately after heat shock.
5. Add 600 μl non-resistant LB liquid medium in the transformation tube. Culture the transformed bacteria for 45 minutes.
6. Use a disposable spreader to evenly coat 100 μl of the bacterial fluid on the resistant LB plate. Keep coating until no flowing liquid on the surface of the plate.
7. Seal the plate with parafilm. Label and put the plate in the thermostatic incubator.

P.S. The cultural time should not last too long, or the single colony can't be obtained.

Reagent: Antibiotic screening plate culturing transformed bacteria, Resistant LB liquid medium

Device: Pipette gun, Sterile Pipette tips, Ultra clean bench, Culture tubes

Procedure:

1. Use a sterile pipette tip to pick up the single colony on the plate.
2. Put the pipette tip stained with colony into the culture tube containing resistant LB liquid medium. Culture the bacterial fluid for about 12 hours.

Reagent: TIANprep Mini Plasmid Kit(TIANGEN^® , product number: DP103-02) or NucleoSpin^® Plasmid, Mini kit for plasmid DNA(MACHEREY-NAGEL), double distilled water, Bacterial fluid to be used

Device: 1.5 ml EP tube, Metal bath heater, Pipette gun, Pipette tips, Electronic balance, Centrifuge

Procedure:

1. Set metal bath heater to 55℃ in advance.
2. According to the instructions in the kit recover the DNA fragment until the last step. Use double distilled water but not the buffer to dissolve the plasmids.

Reagent: Plasmid solution, double distilled water

Device: NanoDrop one ultramicro ultraviolet spectrophotometer(Thermo SCIENTIFIC), Lens papers, Pipette gun, Pipette tips

Procedure:

1. Open NanoDrop one in advance.
2. Select the "nucleic acid " tab and click the "double strain DNA" option on the home page of NanoDrop one.
3. Clean the instrument base with double distilled water. Wipe clean with lens papers.
4. Use 1 μl of double distilled water as a blank sample. After detecting, wipe clean with lens papers.
5. Add 1 μl plasmid solution to the base for detection. After detecting, record data and clean the instrument base with double distilled water. Wipe clean with lens papers.
6. Shut down the instrument after use.

Reagent: S. elongatus bacterial fluid, Non-resistant BG11 liquid medium, Double distilled water

Device: Pipette gun, Sterile pipette tips, Ultra clean bench, 1.5 ml EP tube, NanoDrop One

Procedure:

1. 1:100 add the S. elongatus bacterial fluid to 200 ml BG11 liquid medium.
2. Remove 10 μl of bacterial fluid each time for measurement in the ultra clean bench. Measure and record the OD₆₈₅ value.

Reagent: Plasmid to be transformed, S. elongatus bacterial fluid, Non-resistant BG11 liquid medium, Resistant BG11 plate

Device: Pipette gun, Sterile pipette tips, Ultra clean bench, Centrifuge, 1.5 ml EP tube, Thermostatic light incubator, Disposable spreaders, Parafilm, Tinfoil, Thermostatic dark shaker

Procedure:

1. Take 1.5 ml of bacterial fluid whose OD₆₈₅ reaches 0.5 in the 1.5 ml EP tube.
2. Centrifuge to collect bacteria at 6000 g for 10 min.
3. Discard the supernatant and use 750 μl of non-resistant BG11 liquid medium for resuspension. Centrifuge to collect bacteria at 6000 g for 10 min.
4. Repeat Step 3.
5. Discard the supernatant and use 250 μl of non-resistant BG11 liquid medium for resuspension.
6. Add plasmid solution into the tube. Make the concentration of DNA reach at least 1 ng/μl.
7. Wrap the 1.5 ml EP tube with tinfoil. Put it into the thermostatic dark shaker with 30℃ and 130 rpm for 24 hours.
8. After 24 hours, use a disposable spreader to evenly coat 100 μl of the bacterial fluid on the resistant BG11 plate. Keep coating until no flowing liquid on the surface of the plate.
9. Seal the plate with parafilm. Label and put the plate in the thermostatic light incubator. Culture for 5 days or until the single colony occurs. These single colonies are transformants.

P.S. Steps 1, 3-6, 8 should be operated on the ultra clean bench.

Reagent: S. elongatus bacterial fluid to be induced, 1M IPTG solution, Resistant BG11 liquid medium

Device: Pipette gun, Sterile pipette tips, Ultra clean bench, Centrifuge, Centrifuge tube, Thermostatic shaker with external lamp tube

Procedure(for 5 ml bacterial fluid):

1. Take 4.995 ml of bacterial fluid whose OD₆₈₅ reaches 0.5 in the centrifuge tube.
2. Centrifuge to collect bacteria at 6000 g for 10 min.
3. Discard the supernatant and use 1ml of non-resistant BG11 liquid medium for resuspension. Centrifuge to collect bacteria at 6000 g for 10 min.
4. Discard the supernatant and use 4.995 ml of resistant BG11 liquid medium for resuspension.
5. Add 5 μl of 1 M IPTG solution into the bacterial fluid. Mix them evenly.
6. Label and put it into the thermostatic shaker with an external lamp tube with 30℃ and 130 rpm.

P.S. Steps 1, 3-5 should be operated on the ultra clean bench.

Reagent: Wild-type S. elongatus bacterial fluid, S. elongatus bacterial fluid transformed with CscB gene, Non-resistant BG11 liquid medium, Resistant BG11 liquid medium, 1 M IPTG solution, 1 M NaCl solution, Plant Sucrose Content Assay Kit(boxbio, product number: AKPL006M)

Device: Pipette gun, Sterile pipette tips, Ultra clean bench, Culture tube, Thermostatic shaker with external lamp tube, Centrifuge, Centrifuge tube

Procedure:

• 1. Take 60 ml of wild-type S. elongatus bacterial fluid whose OD₆₈₅ reaches 0.5 in the centrifuge tube. Take 60 ml of S. elongatus bacterial fluid transformed with CscB gene whose OD₆₈₅ reaches 0.5 in the centrifuge tube.
• 2. Centrifuge each group to collect bacteria at 6000 g for 10 min.
• 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.
• 5. Mix reagents according to the table below in the culture tube: (C represents the control groups; E represents the experiment groups)

	Group C-1	Group C-2	Group C-3	Group C-4	Group E-1	Group E-2	Group E-3	Group E-4
Wild-type S. elongatus bacterial fluid (μl)	5000	4995	4500	4495	0	0	0	0
S. elongatus bacterial fluid transformed with cscB gene (μl)	0	0	0	0	5000	4995	4500	4495
1M IPTG solution (μl)	0	5	0	5	0	5	0	5
1M NaCl solution (μl)	0	0	500	500	0	0	500	500
Final volume of fluid (ml)	5	5	5	5	5	5	5	5
Number of parallel groups	3	3	3	3	3	3	3	3

• 6. Label and put them into the thermostatic shaker with an external lamp tube with 30℃ and 130 rpm. Start timing at the same time.
• 7. Measure and record OD₆₈₅ value at 0 hours, 24 hours, 48 hours, and 72 hours.
• 8. Take 100 μl bacterial fluid from each tube at 24 hours, 48 hours, and 72 hours. Centrifuge to obtain supernatant at 6000 g for 10 min.
• 9. Measure and record the concentration of sucrose in the supernatant with the assay kit.

P.S. Steps 1, 3-5, 8, and all sampling operation should be operated on the ultra clean bench.

Reagent: E. coli bacterial fluid transformed with recombinant plasmid, E. coli bacterial fluid transformed with empty vector, Resistant LB liquid medium, M9 minimal liquid medium with sucrose

Device: Ultra clean bench, Pipette gun, Sterile pipette tips, NanoDrop One

Procedure:

1. Culture engineering E. coli transformed with recombinant plasmid and engineering E. coli transformed with empty vector in 3 ml resistant LB liquid medium until OD₆₀₀ reaches 0.6.
2. Store them at 4℃ as seeds for the following steps.

Reagent: E. coli seeds with recombinant plasmid, E. coli seeds with empty vector, Resistant LB liquid medium, M9 minimal liquid medium with sucrose

Device: Ultra clean bench, Pipette gun, Sterile pipette tips, NanoDrop One

Procedure:

1. 1:100 add the seeds required for the certain experiment to 100 ml M9 minimal liquid medium with sucrose respectively. Add antibiotics corresponding to the resistance they have.
2. Culture these groups of E. coli. Measure and record their OD₆₀₀ every 2 hours. Take out 80 μl of liquid on the ultra clean bench for the test each time. If OD₆₀₀ is higher than 1.5, measure again after dilution until its OD₆₀₀ is lower than 1.5.

P.S. The plasmids required for our experiments contain pUC-Empty, pUC-J23101-SacC, pUC-J23107-SacC, and pUC-J23109-SacC.

Reagent: E. coli seeds with recombinant plasmid, E. coli seeds with empty vector, Resistant LB liquid medium, M9 minimal liquid medium with different glucose concentration

Device: Ultra clean bench, Pipette gun, Sterile pipette tips, NanoDrop 100, Black 96-well plate, Transparent 96-well plate

Procedure:

1. Preparation of M9 minimal medium: In 1L M9 minimal medium 33.9g Na2HPO4, 15g KH2PO4, 2.5g NaCl, 5g NH4Cl, 0.36g MgSO4, glucose with a concentration gradient is supplied as the sole carbon source.
2. Dilute the E. coli cultures that are stored at 4 degrees 1:100 into 3ml LB medium and culture overnight.
3. Then the cultures were diluted 1:100 into 3mL LB medium and cultured for about 3 hours. Use these cultures(OD600 0.6~0.8) as seeds.
4. Dilute the seeds 1:100 to 600ul M9 minimal medium and culture for 6 hours. Add 200ul into each well of 96-well plate. Measure OD600 and fluorescence intensity of each sample with a multimode plate reader.

Reagent: E. coli seeds transformed with BBa_K4115039, E. coli seeds transformed with BBa_K4115040, Resistant LB liquid medium, Autoinducer(3O C6 HSL) solution

Device: Ultra clean bench, Pipette gun, Sterile pipette tips, NanoDrop one, Transparent 96-well plate, 1.5 ml EP tube, Black bottom 96-well plate, Centrifuge, Centrifuge tube

Procedure:

Signal Sender

• 1. Dilute the E. coli seeds transformed with BBa_K4115039 1:100 into 3ml LB liquid medium and culture overnight.
• 2. Dilute the overnight cultures 1:100 into 3ml LB liquid medium and culture for 3h. Measure OD600 with Nanodrop One.
• 3. Dilute the culture with LB liquid medium until OD₆₀₀ is about 0.1. Add 600 μl diluted cultures to each 1.5ml EP tube. Add 0.6 μl autoinducer solution to each tube. Make the final concentrations of the autoinducer reach the range from 10^-4 to 10^-14 M. Then culture them.
• 4. After culturing for 3 hours, add 200 μl to each well of the black bottom 96-well plate for fluorescence intensity testing. Use an excitation wavelength of 485nm and an emission wavelength of 535nm. Record the data. Record the data.
• 5. Add 200 μl to each well of the transparent 96-well plate for OD600 measurement.

Signal Receiver

• 1. Dilute the E. coli seeds transformed with BBa_K4115040 1:100 into LB liquid medium and culture overnight. Dilute the E. coli seeds transformed with BBa_K4115039 1:100 into 3ml LB liquid medium and culture overnight.
• 2. Centrifuge the overnight E. coli bacterial fluid transformed with BBa_K4115040 (express LuxI constitutively) at 12000 rpm for 1 min. Take out the supernatants for use.
• 3. Dilute the overnight E. coli bacterial fluid transformed with BBa_K4115039 until OD₆₀₀ is about 0.1.
• 4. Set 3 groups as follows:

Group name	Blank	Autoinducer	Signal sender
Medium	LB medium	LB medium with 10-7 autoinducer	Supernatant from Step 2

1:1000 add the 10^-3 M autoinducer into the diluted cultures in order to set the autoinducer group. Centrifuge the diluted cultures at 3500 rpm for 10 min and then resuspend the cells with the supernatant in Step 2 in order to set the signal sender group.

• 5. Culture all these three groups for 3h.
• 6. After culturing, add 200 μl of bacterial fluid of each group to each well of the black bottom 96-well plate for fluorescence intensity testing from each group. Use an excitation wavelength of 485nm and an emission wavelength of 535nm.
• 7. Add 200 μl of bacterial fluid of each group to each well of the transparent 96-well plate for OD₆₀₀ measurement.

Reagent: Initial bacterial fluid, Ice, Resistant TY liquid medium

Device: Ultra clean bench, Culture tubes, Pipette gun, Sterile pipette tips, NanoDrop One

Procedure:

1. Seed initial cultures at a ratio of 1:100 into 100 ml of TY liquid medium containing 100 μg/ml ampicillin and culture.
2. Measure and record the OD₆₀₀ value of the culture medium every 4 hours.

Reagent: Initial bacterial fluid, BG11 medium powder(hopebio, product number: HB8793), NH₄Cl, NaCl, HEPPS, Milli-Q water, Acetate, Sucrose

Device: Ultra clean bench, Measuring cylinder, Electronic balance, Medicine spoon, BeyoGold Syringe Filter (0.22μm/3mm, PES, Sterile)

Procedure:

1. Dissolve 11.7 g BG11 powder, 106 mM sodium chloride, 4 mM ammonium chloride, 25 mM HEPPS and 10 mM acetate into 1 L Milli-Q water. Adjust the pH to 7.5. Mark this solution as CoBG11-acetate.
2. Dissolve 11.7 g BG11 powder, 106 mM sodium chloride, 4 mM ammonium chloride, 25 mM HEPPS and 20 g sucrose into 1 L Milli-Q water. Adjust the pH to 7.5. Mark this solution as CoBG11-sucrose.
3. Use the filter to filter the both solution.
4. Seed initial cultures at a ratio of 1:100 into 100 ml CoBG11-acetate or CoBG11-sucrose liquid medium in a shaker at 37℃, 180 rpm.
5. Measure and record the OD₆₀₀ value of the culture medium every day.

Reagent: Recovered bacterial fluid, Ice, Resistant TY liquid medium, Sterile double distilled water, Sterile 10% glycerol

Device: Ultra clean bench, Icebox, Culture tubes, Pipette gun, Sterile pipette tips, Centrifuge, Centrifuge tube, Refrigerator, 1.5 ml EP tube

Procedure:

1. Seed initial cultures at a ratio of 1:100 into 100 ml of TY liquid medium containing 100 μg/ml ampicillin and culture until OD₆₀₀ = 0.4 ~ 0.6.
2. Pre-cool sterile double distilled water and sterile 10% glycerol.
3. Place the cultures on ice for 30 min, then centrifuge the culture medium at 6000 rpm for 10 min to remove the supernatant and resuspend in 35 ml sterile pre-cold water.
4. Centrifuge the culture medium again at 6000 rpm for 10 min to remove the supernatant and resuspend in 20 ml sterile pre-cold water.
5. Repeat the last step but resuspend the bacteria with 2.5 ml sterile pre-cold 10% glycerol instead of 20 ml water.
6. Divide the glycerol bacteria into sterile 1.5 ml EP tubes (150 μl each), and store at -80℃.

Reagent: Prepared glycerol bacterial, Ice, Resistant TY liquid medium, Sterile double distilled water, Absolute ethanol, Plasmid solution to be transformed

Device: Ultra clean bench, Icebox, Culture tubes, Pipette gun, Sterile pipette tips, Centrifuge, Centrifuge tube, Refrigerator, 1.5 ml EP tube, electroporation cuvettes, BIO-RAD MicroPulser

Procedure:

1. re-cool sterile double distilled water and absolute ethanol.
2. Wash the 2 mm electroporation cuvettes with pre-cold sterile water and pre-cold absolute ethanol successively.
3. Melt the electrocompetent cells on ice. Add 10 μl plasmid solution into cells, and leave it on ice for 30 min.
4. Transfer cells into a cuvette and avoid bubbles in the liquid.
5. Use BIO-RAD MicroPulser to electroporate at 2500V for 5ms.
6. Transfer cells back to 1.5 ml EP tube, add 800 μl TY liquid medium and culture for 7~8 hours.
7. Centrifuge the culture medium at 4000 rpm for 4 min, remove 700 μl supernatant and resuspend the cell with the remaining liquid.
8. Perform the spread plate method for the concentrated cell liquid on TY solid medium containing the correct antibiotics and culture for 1 day.

Reagent: Bacterial plate, TIANamp Bacteria DNA Kit(TIANGEN)

Device: Pipette gun, Pipette tips, Centrifuge, Centrifuge tube, 1.5 ml EP tube

Procedure:

1. Inoculate single colonies into 4 ml of TY liquid medium containing 100 μg/ml ampicillin and 100 μg/ml bleomycin and culture for 1 day.
2. Extract the genomic DNA of A. caulinodans by the TIANamp Bacteria DNA Kit.

Reagent: S. elongatus bacterial fluid transformed with CscB gene, 1 M IPTG solution, 1 M NaCl solution, Plant Sucrose Content Assay Kit(boxbio, product number: AKPL006M), E. coli bacterial fluid transformed with SacC gene, Resistant LB liquid medium, Non-resistant CoBG11 liquid medium, NaCl-free non-resistant CoBG11 liquid medium, 20% Sucrose solution

Device: Pipette gun, Sterile pipette tips, Refrigerator, Ultra clean bench, Culture tube, Thermostatic shaker with external lamp tube, Centrifuge, Centrifuge tube, Thermostatic shakerProcedure:

• 1. Take two portions of 50 ml of S. elongatus bacterial fluid transformed with CscB gene, whose OD₆₈₅ reaches 0.5 in the centrifuge tube. Mark these two portions as group 1 and group 2 respectively.
• 2. Centrifuge each group to collect bacteria at 6000 g for 10 min.
• 3. Discard the supernatant. Use the non-resistant CoBG11 liquid medium for resuspension of group 1. Use the NaCl-free non-resistant CoBG11 liquid medium for resuspension of group 2. Centrifuge each group to collect bacteria at 6000 g for 10 min.
• 4. Discard the supernatant. Use 50 ml of non-resistant CoBG11 liquid medium that is already added 50 μl of 1 M IPTG solution for resuspension of group 1. Use 50 ml of NaCl-free non-resistant CoBG11 liquid medium for resuspension of group2.
• 5. Culture both two groups for 84 hours. Measure and record OD₆₈₅ value. The data of group 1 is recorded as OD1 and OD2 for group 2.
• 6. Centrifuge each group to collect supernatant at 6000 g for 10 min. Mark the supernatant of group 1 as the supernatant⁺ and that of group 2 as the supernatant^-.
• 7. Dilute the supernatant of the group with large OD value in a ratio of their OD value(higher than 1).
• 8. Add 1 M NaCl solution into supernatant^- and make the final concentration of NaCl reach 106 mM(for instance, add 4.76 ml of 1 M NaCl solution into 40 ml supernatant^-).
• 9. Measure and record the concentration of sucrose in the both two groups of supernatant with the assay kit.
• 10. 1:100 add E. coli bacterial fluid transformed with SacC gene into 50 ml resistant LB liquid medium. Culture until the OD₆₀₀ reaches 0.5.
• 11. Add 20% sucrose solution into the non-resistant CoBG11 liquid medium and make the concentrate of sucrose reach the higher value measured in Step 9.
• 12. Prepare four solutions for the following steps according to the table below.

Label	Solution 1	Solution 2	Solution 3	Solution 4
Component	Non-resistant CoBG11	Non-resistant CoBG11 with sucrose from Step11	Supernatant-	Supernatant+
Volume (ml)	40

• 13. The volume of E. coli bacterial fluid to be used mainly depends on its OD₆₀₀. The following steps take OD₆₀₀=0.5 as an example.
• 14. Take four portions of 10.5 ml of E.coli bacterial fluid in centrifuge tube. Centrifuge all portions to collect bacteria at 4000 g for 10 min.
• 15. Discard the supernatant and use the non-resistant CoBG11 liquid medium for resuspension of all portions. Centrifuge all portions to collect bacteria at 4000 g for 10 min. Mark four portions as group 1 - 4.
• 16. Suspend group 1 - 4 of E. coli with 21 ml of solution 1 - 4 respectively and obtain 100% bacterial fluid. Set the control groups and experiment groups according to the following table.

100% bacterial fluid
Group	1	2	3	4
Component	Solution 1	Solution 2	Solution 3	Solution 4
Theoretical OD600 value	0.25
Volume per tube (ml)	4
Number of tubes	3

• 17. Dilute the remaining 9 ml of 100% bacterial fluid with 9 ml of the corresponding solution and obtain 50% bacterial fluid. Set the control groups and experiment groups according to the following table.

50% bacterial fluid
Group	1	2	3	4
Component	Solution 1	Solution 2	Solution 3	Solution 4
Theoretical OD600 value	0.13
Volume per tube (ml)	4
Number of tubes	3

• 18. Dilute the remaining 6 ml of 50% bacterial fluid with 6 ml of the corresponding solution and obtain 25% bacterial fluid. Set the control groups and experiment groups according to the following table.

25% bacterial fluid
Group	1	2	3	4
Component	Solution 1	Solution 2	Solution 3	Solution 4
Theoretical OD600 value	0.06
Volume per tube (ml)	4
Number of tubes	3

• 19. Measure and record OD₆₀₀ as the value of 0 hours. Label and put them into the thermostatic shaker to culture. Start timing at the same time.
• 20. Measure and record OD_{600 every 3 hours until culturing them for 48 hours.}

Reagent: Initial E .coli bacterial fluid, Initial S .elongatus bacterial fluid, Initial A .caulinodans bacterial fluid, CoBG11 liquid medium, Sucrose

Device: Pipette gun, Sterile Pipette tips, Centrifuge, Centrifuge tube, 1.5 ml EP tube, NanaDrop One, BeyoGold Syringe Filter (0.22μm/3mm, PES, Sterile), Thermostatic shakerProcedure:

1. Add 20 g of sucrose into the CoBG11 liquid medium to obtain CoBG11-sucrose liquid medium. Sterilize by the filter.
2. Seed initial cultures at a ratio of 1:100 for E. coli and A. caulinodans, and at a ratio of 1:20 for S. elongatus into 100 ml CoBG11-sucrose liquid medium in a thermostatic shaker(for E. coli, at 37℃, 220 rpm; for A. caulinodans, at 37℃, 180 rpm; for S. elongatus, at 30℃, 130 rpm with lamp tube).
3. Measure and record the OD value of each culture medium every 4h (E. coli), 6h(A. caulinodans), or 12h (S. elongatus).

Reagent: Initial E .coli bacterial fluid, Initial S .elongatus bacterial fluid, Sodium alginate powder, Calcium chloride, CoBG11-sucrose liquid medium and CoBG11-acetate liquid medium(see 'Experiments on A. caulinodans'-'Measurement of Growth Curves'), Physiological saline, Milli-Q water

Device: Pipette gun, Sterile Pipette tips, Centrifuge, Centrifuge tube, 1.5 ml EP tube, NanaDrop One, BeyoGold Syringe Filter (0.22μm/3mm, PES, Sterile), Thermostatic shaker, Microscope, Coverslip, Fluorescence microscope

Procedure:

1. Add 2 g sodium alginate powder to 100 ml hot water, heat and stir until completely dissolved.
2. Leave sealed for several hours until the bubbles all disappear.

Procedure:

1. Add 2.22 g calcium chloride to 100 ml Milli-Q water. Make the concentration of the calcium chloride reach 0.2 M.
2. Use the filter to filter the solution.

Procedure:

1. Centrifuge the microorganism fluid at 4000 rpm for 15 min. Remove the supernatant and weigh the wet weight.
2. For every 1 g cell, resuspend in 1 ml physiological saline, add 20 ml sodium alginate solution and stir until well combined.
3. Transfer the alginate-cell mixture into a small syringe (needle removed) and drips naturally into 20 ml 0.2 M calcium chloride solution. Meanwhile, stir with an electromagnetic stirrer.
4. Soak for 5 hours, then transfer the immobilized cells into CoBG11-sucrose medium (for E. coli and S. elongatus) or CoBG11-acetate medium (for A. caulinodans), culture in a shaker (for E. coli, at 37℃, 220 rpm; for A. caulinodans, at 37℃, 180 rpm; for S. elongatus, at 30℃, 130 rpm).

Procedure:

1. Transfer the immobilized cells into 1.5 ml EP tubes and weigh wet weight.
2. For every 1 g immobilized cell, add 1 ml 0.2 M phosphate buffer (pH=7.5).
3. Shake violently for 15 min until all solids dissolve.
4. Measure the OD₆₀₀ (for E. coli and A. caulinodans) or OD₆₈₅ (for S. elongatus) value of the lysate.
5. Perform the above operations once a day for about a week.

Procedure:

1. Drop 10 μl lysate onto the microslide and cover the coverslip.
2. Place the sample under a fluorescence microscope and obtain the image.
3. Perform image analysis to get the average fluorescence intensity.

Reagent: Initial E .coli bacterial fluid, Initial S .elongatus bacterial fluid, Initial A .caulinodans bacterial fluid, Sodium alginate powder, Calcium chloride, CoBG11-sucrose liquid medium and CoBG11-acetate liquid medium(see Experiments on A. caulinodans-Measurement of Growth Curves), Physiological saline, Milli-Q water

Device: Pipette gun, Sterile Pipette tips, Centrifuge, Centrifuge tube, 1.5 ml EP tube, NanaDrop One, BeyoGold Syringe Filter (0.22μm/3mm, PES, Sterile), Thermostatic shaker, Microscope, Coverslip, Fluorescence microscope

Procedure:

1. Dissolve 11.7g BG11 powder, 106mM sodium chloride, 4mM ammonium chloride, 25mM HEPPS, 20g sucrose, and 10mM acetate into 1 l Milli-Q water. Adjust the pH to 7.5.
2. Use the filter to filter the solution.

Procedure:

1. Centrifuge the microorganism fluid at 4000 rpm for 15 min. Remove the supernatant and weigh the wet weight.
2. For every 1 g cell, resuspend in 1 ml physiological saline, add 20 ml sodium alginate solution and stir until well combined.
3. Transfer the alginate-cell mixture into a small syringe (needle removed) and drips naturally into 20 ml 0.2 M calcium chloride solution. Meanwhile, stir with an electromagnetic stirrer.
4. Soak for 5 hours, then transfer the immobilized cells into CoBG11-PLUS medium and culture in a light shaker at 30℃, 130 rpm.

Procedure:

1. Transfer the immobilized cells into 1.5 ml EP tubes and weigh wet weight.
2. For every 1 g immobilized cell, add 1 ml 0.2 M phosphate buffer (pH=7.5).
3. Shake violently for 15 min until all solids dissolve.
4. Measure the OD₆₀₀ (for E. coli and A. caulinodans) or OD₆₈₅ (for S. elongatus) value of the lysate.
5. Perform the above operations once a day for about a week.

Reagent: E .coli bacterial fluid carries LuxI gene, E .coli bacterial fluid carries LuxR-Plux-sfGFP sequence, CoBG11-sucrose liquid medium(see 'Experiments on A. caulinodans'-'Measurement of Growth Curves'), Physiological saline, Sodium alginate solution

Device: Pipette gun, Sterile Pipette tips, Centrifuge, Centrifuge tube, 1.5 ml EP tube, NanaDrop One, BeyoGold Syringe Filter (0.22μm/3mm, PES, Sterile), Thermostatic shakerProcedure:

1. Centrifuge the microorganisms culture at 4000 rpm for 15 min, remove the supernatant and weigh wet weight.
2. For every 1 g cell, resuspend in 1 ml physiological saline, add 20 ml sodium alginate solution and stir until well combined.
3. Transfer the alginate-cell mixture into a small syringe (needle removed) and drips naturally into 20 ml 0.2 M calcium chloride solution. Meanwhile, stir with an electromagnetic stirrer.
4. Soak for 5 hours, then transfer the immobilized cells into CoBG11-sucrose medium and culture in a shaker at 37℃, 100 rpm.