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, Pipette tips, PCR tube, Icebox, PCR-Instrument, Centrifuge

Procedure:

1. Turn on the PCR instrument in advance.

2. Mix reagents on ice according to the table below in the PCR tube:

Components	Volume
2 x 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.

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

Procedure:

1. Turn on 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:

4. Mix the solution system evenly. Carry out rapid centrifugation if the droplets hang on the tube wall.

5. Put the PCR tube in the PCR instrument. Set the PCR program according to the table below.

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

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

Device: Pipette, 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, 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.

Cutting Gel Recovery

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, 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, Pipette tips

Procedure:

1. Turn on the 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, 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:

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.

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

Reagent: Recombination product, DH5`\alpha` competent E. coli cells, Ice, Resistant LB plate, Non-resistant LB liquid medium

Device: Pipette, 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 DH5`\alpha` competent E. coli cells and put them on the ice to thaw.

3. After thawing, add 10 μl recombination product into 100μl DH5`\alpha` 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.

Colony Selection and Amplificated Culture

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

Device: Pipette, 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, 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: 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 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 E. coli selection is as follows: Kan 30 ng/μl, and Amp 100 ng/μl.

List of ingredients for LB broth powder (Sangon Biotech, product number: A507002)

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):

4. Accurately weigh 40 g of LB broth agar powder and then add 1 L of Milli-Q water.

5. High-pressure steam sterilization at 121℃ for 15 min.

6. 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.

7. 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)

Reagent: E. coli BL12(DE3) transformants on plate, LB Broth medium with antibiotics, IPTG(1M)

Device: Sterile culture tubes, Pipette and tips, Bunsen flames, 1.5 mL EP tube

Precedure:

1. Work near Bunsen flames.

2. Fill a culture tube with 5 mL of LB medium containing the right antibiotic(s).

3. Using a sterile pipette tip, pick one of the colonies and shoot the pipette tip into the culture tube. Keep the cap of the culture tube loose.

4. Grow the bacteria at 37 °C and 250 rpm overnight.

5. Inoculate the other 5 mL new LB medium with 50 μL overnight culture, culturing it at 37 ºC for 2-3 hours until OD600 reaches 0.6.

6. Extract 1 mL culture a 1.5 mL EP tube for SDS-PAGE sample and add 0.8 μL IPTG(1M) into the culture. Then grow the bacteria at 23 °C and 250 rpm for 16 hours.

7. Extract 1 mL culture for SDS-PAGE sample into a 1.5 mL EP tube and store the rest of culture for other usages.

Reagent: Bacteria culture before and after induction, 5xSDS-PAGE Protein Loading Buffer, Tris-MOPS-SDS Running Buffer, UltraPure sterile water, Deionized water, Protein molecular marker, Coomassie Brilliant Blue Staining Reagent, Coomassie Brilliant Blue Destaining Solution.

Device: ExpressPlus™ PAGE Gel, 10x8, 4-20%, 10 wells, Mini-PROTEAN Tetra Vertical Electrophoresis Cell, Heating block, 1.5 mL EP tube.

Precedure:

Sample-preparation:

1. The sample before induction is the 1mL culture with 0.6 OD600, so there is not any other preparation for it. But the sample after induction should be diluted to 0.6 OD600 and draw 1 mL of the dilution into another EP tube.

2. Centrifuge them in a micro centrifuge at room temperature at the maximum speed for 1 minute and abandon the supernatant.

3. Add 50 μL 5xSDS-PAGE Protein Loading Buffer into each EP tube and mix well. Then incubate them in an 85 ºC heating block for 3 minutes, shaking them intensively during the incubation for several times.

4. Add 100 μL UltraPure sterile water into EP tubes and mix well.

5. UltraPure sterile water into EP tubes and mix well.

6. Centrifuge them in a micro centrifuge at room temperature at the maximum speed for 3 minutes. The supernatant is the sample for further usage.

SDS-PAGE:

1. Place gels into the Vertical Electrophoresis Cell and fill the inner portion between the gel(s) and the gel holder with enough Tris-MOPS-SDS Running Buffer.

2. Load samples and the marker into gel lanes with the appropriate volume depending on gels.

3. Cover the chamber and firmly connect both the anode and the cathode. Set the voltage on the electrophoresis power supply to a constant voltage of 140 V. Turn ON the power supply.

4. Allow the gel to electrophorese for 45-90 minutes. Turn OFF the power immediately after the dye front migrates out from the bottom of the gel.

Coomassie Brilliant Blue Staining:

1. Rinse the gel 3 times for 5 minutes with 100 ml deionized water to remove SDS and buffer salts, which interfere with binding of the dye to the protein. Discard each rinse.

2. Stain the gel with enough Coomassie Brilliant Blue Staining Reagent (20-100 ml) to cover the gel. Stain for 1 hour at room temperature with gentle shaking.

3. Retrieve Coomassie Brilliant Blue Staining Reagent and wash the gel with enough deionized water to wash off the rest of the dye.

4. Incubate the gel with Coomassie Brilliant Blue Destaining Solution to cover the gel, and gently shaking the gel overnight.

5. Observe the band.

Reagent: Protein PAGE Gel after SDS-PAGE, Anhydrous methanol, WB Transmembrane Buffer, WB Rinsing Solution (PBS with 1000× of Tween-20), WB reaction solution (A solution : B solution = 1 : 1).

Device: PVDF membrane, Electrophoresis tank, Mini-PROTEAN Tetra Vertical Electrophoresis Cell, Black Photo Plate, ECL Luminometer, Ice and ice box, tray.

Procedure:

Preparing protein gel after SDS-PAGE

Trarsmembrane:

1. Soak the PVDF membrane in anhydrous ethanol for 1-2 minutes.

2. Pour the membrane transfer solution in the tray, put a splint and open it, put a sponge on both sides of the splint, then put a white cushion, then put the membrane transfer filter paper. Place the protein gel on the black side of the plate.

3. Clean the PVDF membrane with membrane transfer solution, then put it on the white deck side, close the plywood so that the protein gel and the PVDF membrane align, and try to exhaust the air in it, and then fix the plywood.

4. Put the assembled wet transfer module into the electrotransfer apparatus.

5. Put ice in the white ice box on the black side.

6. Close the lid and set the current to 400mA for 30 minutes.

Rinsing and antibody incubation:

1. First rinse the PVDF membrane with a small amount of rinsing solution

2. Then incubate with corresponding antibody for 30 minutes.

3. Rinse with rinsing solution for three times with shaking, each time 5 minutes.

Reaction and observation:

1. Add a small amount of reaction solution onto a black photographic plate.

2. Lay the PVDF membrane on it carefully, taking care that one side of the PVDF membrane touches the reaction solution first, and then place the other side slowly, taking care that there are no air bubbles.

3. Drop the remaining reaction solution and observe the result with ECL Luminometer and record the result.

Reagent: Bacterial culture, LB medium.

Device: 96-well plate, Multimode plate reader (SpectraMax i3).

Procedure:

1. Dilute the bacteria culture with LB medium to 1.0 OD600.

2. Add 200 μL dilution into the well of the 96-well plate.

3. Place the plate into the multimode plate reader.

4. Setting the reading area, the luminescence-testing mode, the range of wavelength, and the step.

5. Close the plate reader and start the reading.

6. Store and analyze data.

Reagent: BG11 medium powder(product number: HB8793), NaHCO3,Milli-Q water

Device: Measuring cylinder, Electronic balance, Medicine spoon, Syringe, Filter membrane

Procedure(for a 1L mixture with 0.1M NaHCO3):

Mix the two sterilized solutions. 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: Kan 10 ng/μl. Notice that Synechococcus elongatus HL7942 has basic resistance to ampicillin.

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

Device: Measuring cylinder, Electronic balance, Medicine spoon, Syringe, Filter membrane

Procedure(for a 1 L mixture with 0.1M NaHCO3 and 1.5% agar):

1. Accurately weigh 1.7 g of BG11 medium powde and 15.0g agar powder. Mix them and then add 900mL of Milli-Q water.

2. High-pressure steam sterilization at 121℃ for 15 min.

3. Weigh 8.4g NaHCO3, dissolve it in 100mL Milli-Q water, inhale it into the syringe in the ultra-clean workbench, and then push the solution through the filter membrane for sterilization.

4. Add the NaHCO3 solution into sterilized BG-11 agar solution and mix well before cooling.

5. 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.

6. 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: LB liquid medium, Bacterial culture

Device: clean bench, Pipette, Pipette tips, Culture tubes, Thermostatic shaker

Procedure:

1. Take fresh BG11 medium (containing NaHCO3) and place it in a shake flask or other culture container.

2. Take logarithmic-phase bacterial culture (OD750=0.2-0.8) and add it to the medium at a 1:100 ratio, then vigorously mix by shaking or stirring.

3. Label and put the tube in the shaker.

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

Liquid Culture: 12 mL culture tubes containing 5 mL of cyanobacterial culture were placed in a constant temperature shaker at 25°C, shaken at 150 rpm, and illuminated with an external LED light source (approximately 6000 lux).

Solid Culture: Plates with cyanobacterial culture were placed in a constant temperature light incubator at 30°C, with a light intensity of approximately 6000 lux.

Reagent: cyanobacterial culture, BG11 liquid medium

Device: culture tube(12 mL), Pipette, Pipette tips, Thermostatic shaker, Clean bench

1. Take 1.5 mL of cyanobacterial culture in the logarithmic growth phase (od750 > 0.2) and transfer it to a sterile centrifuge tube. Centrifuge at 5000 rpm for 10 minutes.

2. Discard the supernatant in a laminar flow hood and resuspend the pellet in 750 μL of BG11 medium. Centrifuge at 5000 rpm for 10 minutes.

3. Discard the supernatant in a laminar flow hood. Resuspend the bacterial pellet in 250 μL of BG11 liquid medium.

4. Add 500 ng of plasmid DNA/linearized fragment to the centrifuge tube.

5. Cover the centrifuge tube with aluminum foil and incubate in the dark on a shaker at 30°C with a shaking speed of 150 rpm for 24 hours.

6. Preheat the solid culture medium, and after incubation, plate the bacterial suspension onto plates containing BG-11 solid medium with kanamycin resistance at a concentration of 10 ng/μL. Plate 125 μL on each plate. Incubate at 30°C under constant light for 5 days (or until single colonies appear). Single colonies represent transformants.

Reagent: cyanobacterial culture, Double distilled water, 2xPhanta(®) Max Master Mix (Dye Plus) P525, Forward primer(10 μM), Reverse primer(10 μM), Ice

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

1. Take 1 mL of the algal culture, or pick a bit of cyanobacteria from the solid culture medium and dissolve it in 10 μL of BG-11 liquid medium.

2. Freeze at -80°C for 20 minutes, then thaw in a 60°C water bath for 10 minutes.

3. Repeat step 2.

4. Take 1 μL of the algal culture as the PCR template.

5. Conduct PCR steps according to "Colony PCR"