F

Protocols

PREPARING THE SUBCULTURE OF CYANOBACTERIA

1. Resuspend and take 200μL of cyanobacteria from the old culture.
2. Place into fresh 50 mL BG-11 media.
3. Leave it under continuous illumination on the shaker at room temperature in the hood.
4. Check OD at 750nm.

TRANSFORMATIONS OF CYANOBACTERIA (OBTAINED FROM STONY BROOK IGEM TEAM)

1. Measure the OD750 (must be approx. 0.7)
2. 15mL per transformation must be centrifuged at maximum speed for 10 min at room temperature.
3. Remove the supernatant by pipetting.
4. Resuspend pellet in 10 mL of 10 mM NaCl solution and centrifuge at maximum speed for 10 minutes.
5. Resuspend the pellet in 0.3 mL of BG-11 and transfer to a microcentrifuge tube.
6. Add between 50ng and 2ug of DNA.
7. Wrap the microcentrifuge tube in aluminum foil to protect from light and incubate at 30°C for 24 hours.
8. Plate the entire volume (0.3mL) of cells on the big plates containing antibiotics by streaking.

PREPARATION OF NaHCO₃ SOLUTION STOCK

1.For this mix 4.2 g of NaHCO₃, 0.3 g of Hepes and 300 ul of BG-11.
2.Fill out to 50 ml with ddH₂O.

PREPARATION OF BG-11 PLATES

Please, work at sterile conditions.
1. Prepare two autoclaved flasks.
2. To the first flask add 90 ml of BG-11 media, to the second flask add 88.2 ml of it.
3. After that add 2.7 g of bacteriological agar to the first flask and 0.54 g of sodium thiosulfate to the second flask.
4. Autoclave both flasks.
5. Add 1.8 ml of NaHCO₃ stock solution to the second flask.
6. Mix two flasks by pouring the flask 2 into the first flask.
7. Add needed spectinomycin to the final concentration 10ug/ml.
8. Pour into petri dishes, the amount should be enough for 2 big and 2 small plates

GENERAL / E.COLI PROTOCOLS

PREPARATION OF M9 MINIMAL MEDIA

1. Prepare 5x stock by dissolving 30g of Na₂HPO₄, 15g of KH₂PO₄, 5g of NH₄Cl, 2.5g of NaCl and 15g of CaCl₂ in one liter of distilled water.
2. Autoclave the 5x stock.
3. For 1x working solution dilute the 5x solution and add 1ml of 1M MgSO₄∙7H₂O and 10 ml of 20% glycerol solution.

PREPARATION OF LURIA BERTANI BROTH, MILLER

1. Suspend 20 grams of the Luria Bertani broth in 1 liter of distilled water.
2. Heat if necessary to dissolve the medium completely.
3. Sterilize by autoclaving at 15 lbs pressure (121°C) for 15 minutes.
4. Dispense as desired.

PREPARATION OF LURIA BERTANI AGAR PLATES

1. Susptend 37 grams of the LB agar, Miller in 1 litre of distilled water
2. Mix the solution and sterilize by autoclaving
3. Wait until the agar becomes warm
4. Add needed amount of the antibiotic of interest, for the spectinomycin we had used the final concentration of 50 ug/ml
5. Add 20 ml of the mixture into the plate while avoiding bubbles
6. Wait until the plates are cooled down to the room temperature and label with the date, antibiotic name and its concentration

PREPARING CHEMICALLY COMPETENT E.COLI

1. Prepare 0.1M CaCl₂ and 0.1M CaCl₂+ 15% glycerol solutions beforehand.
2. Take 1 colony of E.coli from LB plates and inoculate in 10mL of LB in 50 ml Falcon tube.
3. Place the tube with DH5-alpha E.coli strains in LB into the shaking incubator at 250 rpm at 37 °C until OD600 reaches 0.2-0.3.
4. Once the OD600 reaches 0.2-0.3, place the tube with DH5-alpha strain on ice for 15 min. Keep in ice solutions of 0.1M CaCl₂ and 0.1M CaCl₂ + 15% glycerol, too.
5. Centrifuge cells at 4 ̊C for 10 min.
6. Resuspend pellet with 3 ml of 0.1M CaCl₂ and put on ice for 30 min.
7. Centrifuge cells again and resuspend in 300 µL of 0.1M CaCl₂ + 15% glycerol.
8. Prepare 50 µL aliquots of resulting solution in separate Eppendorf tubes.
9. Place aliquots in a Cold room at - 80 °C.

PREPARATION OF SOC MEDIA (100ml):

1.Add 80ml of of dH₂O to a sterile media bottle.
2.Add 2g of Tryptone, 0.5g of Yeast extract and 0.05g of NaCl to the media bottle.
3.Mix until all of the components are dissolved.
4. Adjust pH to 7 with NaOH
5.Transfer the solution into the sterile volumetric flask and add the remaining 20 ml of water
6.Transfer the content of the flask into media bottle and autoclave the content.
7.After autoclaving, store at 4°C.

CHEMICAL TRANSFORMATION was carried out according to IGEM Protocols

1. Resuspend DNA in selected wells in the Distribution Kit with 10µl dH₂O. Pipet up and down several times, let sit for a few minutes. Resuspension will be red due to the cresol red dye.
2. Label 1.5ml tubes with a part name or well location. Fill lab ice bucket with ice, and pre-chill 1.5ml tubes (one tube for each transformation, including your control) in a floating foam tube rack.
3. Pipette 50µl of competent cells into the 1.5ml tube: 50µl in a 1.5ml tube per transformation. Tubes should be labeled, pre-chilled, and be in a floating tube rack for support. Keep all tubes on ice. Don’t forget a 1.5ml tube for your control.
4. Pipette 1µl of resuspended DNA into the 1.5ml tube: Pipette from well into the appropriately labeled tube. Gently pipette up and down a few times. Keep all tubes on ice.
5. Pipette 1µl of control DNA into the 2ml tube: Pipette 1µl of 10pg/µl control into your control transformation. Gently pipette up and down a few times. Keep all tubes on ice.
6. Close 1.5ml tubes, incubate on ice for 30 min: Tubes may be gently agitated/flicked to mix solution but return to ice immediately.
7. Heat shock tubes at 42°C for 45 sec: 1.5ml tubes should be in a floating foam tube rack. Place in a water bath to ensure the bottoms of the tubes are submerged. Timing is critical.
8. Incubate on ice for 5 min: Return transformation tubes to an ice bucket.
9. Pipette 950µl SOC media to each transformation
10. Incubate at 37°C for 1 hour, shaking at 200-300 rpm.
11. Spin down cells at 6800g for 3 mins and discard 850µL of the supernatant.
12. Resuspend the cells in the remaining 150µL, and pipette each transformation onto Petri plates
13. Spread with a sterilized spreader or glass beads immediately. This increases the chance of getting colonies from lower concentration DNA samples.
14. Incubate transformations overnight (14-18 hr) at 37°C: Incubate the plates upside down (agar side up).

MIRAPREP

1. Inoculate 50 ml of bacterial culture in appropriate selective media and incubate on a shaker at 250 rpm at 37 °C overnight.
2. Next day, transfer the bacterial culture into a 50 ml tube and spin at 4000xg at 4 °C for 10 minutes.
3. Discard the supernatant and resuspend the pellet in 2 ml of resuspension buffer with 50 ul/ml RNase (ThermoFisher #EN053) freshly added.
4. Add 2 ml of lysis buffer to the bacterial suspension and invert the tube 3-4 times.
5. Incubate it at room temperature for 3 minutes.
6. Add 2 ml of neutralization buffer and invert the tube 3-4 times.
7. Quickly distribute the bacterial lysate into 1.5ml centrifuge tubes (approx. 4 tubes) by pouring, not pipetting.
8. Centrifuge at room temperature at 13,200xg for 10 minutes.
9. Collect supernatants in 15 ml tube and discard pellets.
10. Add 1x volume of 96% ethanol (approx. 5 ml) into the supernatant and mix it thoroughly for 5 seconds.
11. The Centrifuge DNA purification method is used

CENTRIFUGE DNA PURIFICATION:

1. Load the sample spin-columns (approx. 700 ul)
2. Spin the column for 1 minute at 13,200xg after the addition of each aliquot.
3. After each spin discard the flow through and repeat steps 1 and 2 if needed.
4. Add 500ul of wash buffer into column.
5. Spin them at 13,200xg at room temperature for 1 minute.
6. Discard the flow through.
7. Repeat the steps 4-6
8. Centrifuge the empty columns one more time for 1.5 minutes to remove any residue buffer.
9. After this, discard old collection tube and put the column into a new tube.
10. Add 5-35 ul of 60°C nuclease free water (depending on the expected amount of DNA in column) to the column and incubate for 1-2 minutes.
11. Spin at 13,200xg at room temperature for 2 minutes.
12. Combine the DNA from all columns in one tube if needed.
13. Store the samples at -20°C.

MEASURING THE CONCENTRATION (NANODROP)

1.Launch ND-8000 V2.2.1.
2.Clean Nanodrop with an ethanol and Kimtech paper towels
3. Make sure that pedestals are clean.
4. Blank with 2ul of Nuclease-Free water.
5. Blank with 2ul of Elution buffer or water, depending on the solvent of the DNA
6. Load 1ul of resuspended DNA sample on the pedestal.
7. Choose/label blanked wells and run Nanodrop.
8. Clean with ethanol and Kimtech paper towels after each use.
9. Place the cover lid back.

DNA GEL ELECTROPHORESIS

1. Prepare a 1% agarose gel by adding 1g of agarose powder into 100ml of 1X TAE buffer.
2. Heat it until agarose is completely dissolved.
3. Add ethidium bromide to the final concentration 0.5ug/ml of solution.
4. Pour the solution into the tray avoiding any bubbles.
5. Allow the solution to solidify (approx. 15-20 minutes).
7. Remove comb, pour 1X TAE buffer with ethidium bromide to cover the gel and load ladder and samples (5ul of DNA and 1 ul of Loading Dye for 6x dye).
8. Run the gel at 100V for 35 minutes.
9.Visualize bands under UV light.

GEL EXTRACTION

1.Cut bands of interest from the agarose gel into the tubes with the known mass.
2.Weight the gel on the scale sensitive to1.00×10-10g.
3.Add Gel solubilization buffer to the tube based on its mass. The proportion is 3 ml of buffer to the 1 mg of excised gel portion.
4.Place the tube into 50 °C water bath for 10 minutes. Invert the tubes every 3 minutes.
5.After the gel is dissolved, add 1 gel volume of isopropanol (to 1mg of sample approximately 1ml).
6.Extract the DNA with the Centrifuge DNA purification method.

RESTRICTION DIGESTION

1. Add nuclease-free water.
2. Add buffer 5µl.
3. Add DNA up to 1 ug.
4. Add 10 units of restriction enzyme.
5. Incubate for 5-60 minutes at 37°C.
6. Inactivate enzyme at 80°C or 65°C (depending on the enzyme) for 20 minutes if heat-inactivation is needed.

LIGATION

1. Add nuclease-free water
2.Combine 100 ng of vector and 3 times molar excess of insert.
3. Add 5 µl ligase buffer and 1 µl ligation enzyme.

POLYMERASE CHAIN REACTION (PCR)

General procedure followed NEB Protocol Phusion® High-Fidelity PCR Master Mix with HF Buffer
1. Calculate the needed amount of nuclease-free water, primers, template DNA and master mix.
2. Add the named compounds in the mentioned order.
3. Add master mix keeping tubes on ice.
4. Prepare the protocol for the amplification on the PCR machine. Please note that temperatures for annealing to be chosen according to the primers used.

DNA extraction

1.Take 250 ul of culture, centrifuge at max speed for 10 min at 4 °C.
2. Quickly remove supernatant and make sure it does not contain parts of the cells’ pellet.
3. Resuspend cell pellet in 20 ul of 0.2% Triton. Heat the tubes with cells at 98°C for 10 min.
4. Centrifuge at 14,000 g and take up the supernatant.
5. Extract the supernatant with 150 ul of hexane and remove the lower aqueous layer into separate tubes. This layer contains DNA. Make sure you do not take up hexane.
6. Set up PCR reaction using 3ul of DNA. Use standard reaction protocol for Phusion Master Mix with primers for SQR.

PCR & DNA purification: Please note Monarch DNA Purification Kit was used for this procedure.

1. Add Binding buffer to the sample depending on the size of the desired DNA. The proportion for the dsDNA >2kb is 2 μl of buffer to 1 μl sample, for the dsDNA <2kb is 5 μl of buffer to 1 μl of sample and for the ssDNA 7 μl of buffer to 1 μl of sample.
2. Extract the DNA with the Centrifuge DNA purification method.

CYANOBACTERIA ON FILTER PAPER TEST

1. Spin down the falcon tube with cyanobacteria of interest for 10 minutes at 4000 rpm
2. Remove the excess supernatant, leaving 200 ul in the tube.
3. Transfer 200 ul of latex into 0.5 ml Eppendorf tube.
4. Prepare the filter paper by placing it into small petri dish.
5. Mix the cyanobacteria with the latex and shake vigorously.
6. Before the two layers separate, transfer the mixture to the filter paper, drop by drop, and giving some time for the drops to dry out.
7. Fill the Petri dish with the BG-11, so that the filter paper is barely below the surface.

PREPARATION OF CARBON DOTS

1. Spin down all cultures
2. Resuspend in TAP S- media (TAP media that has no sulfate)
3. Leave cultures for overnight
4. If you have 4 tubes, for 2 of them add Cr (VI) at a final concentration of 10 mM
5. The other two leave overnight without any treatment
6. Leave all of them under the light
7. Next day, add ascorbic acid (AA) at a final concentration of 0.2 M to one of the algae cultures that contain Cr (VI) and one that does not contain Cr (VI)
8. There should be 1 culture with AA+Cr (VI), 1 culture with AA, 1 culture with Cr (VI) and 1 culture without any treatment (pure control)
9. After shaking vigorously leave all cultures for four hours under the light
10. Spin down all cultures
11. Resuspend them in 9-11 ml of 96% Ethanol
12. Shake vigorously and leave in the fridge
13. Place all cultures to the oven at 150°C for 6 hours
14. Filter

SUBCULTURING OF ALGAE (C. reinhardtii)

1. Resuspend and take 1 ml of algae from the old solution of TAP+arginine
2. Place into fresh 40 mL TAP+arginine
3. Check OD at 750nm

PREPARATION OF TAP MEDIA

1. Prepare the following components:

Component	Volume (1 L total volume)
1M Tris Base	20 ml
Phosphate Buffer II	1 ml
Solution A	10 ml
Hutner’s reagent	1 ml
Glacial acetic acid	1 ml

a. Phosphate buffer II preparation

Component	Volume (100 ml total volume)
K₂HPO₄	10.8 g
KH₂PO₄	5.6 g

b. Solution A preparation

Component	Volume (500 ml total volume)
NH₄Cl	20 g
MgSO₄ x 7H₂O	5 g
CaCl₂ x 2H₂O	2.5 g

2. 400 mL of TAP medium need to be prepared for autoclaving with amount of salts identical for 500 mL.
3. Autoclave. Let it cool down
4. Filter
5. Prepare arginine
6. Filter arginine
7. Add arginine (final concentration in TAP medium should be 100 mg/L)
8. Adjust pH of medium to 7.0-7.3
9. After pH adjustment, finalize the volume up to 500 mL

SULFIDE SURVIVAL ASSAY

1. Transfer 10 ml of subculture into the glass tube. Prepare 1 tube per 1 culture.
2. Add 10 uL of 0.1M H₂S into the tube.
3. To provide anaerobic conditions close the glass tubes with the stopper and aluminum seal using crimper.
4. Place the needle designated for air to flow-out and ensure that it penetrates the rubber stopper
5. Place another needle connected to nitrogen source into the tube ensuring that it penetrates the rubber stopper and dip into the liquid culture.
6. Flush the glass tubes for min at medium speed.
7. When the flushing is over, first remove the needle for air flow out, immediately after removing the needle with nitrogen source.
8. Prevent leakage of nitrogen by wrapping the stopper with parafilm.

GAS PRESSURE AND TEMPERATURE MEASUREMENT

1. Attach tubing to the end of the ideal gas apparatus (pasco, scientific) and wrap it with parafilm to prevent gas leakage.
2. Attach needle to the another end of the tubing, and wrap it with parafilm to prevent gas leakage.
3. Penetrate needle as fast as possible into the anaerobic tube with cyanobacterial sample.
4. Pull out syringe to take out air from the cyanobacterial sample tube.
5. Record volume of the collected gas.
6. Take out needle from the tube and quickly transfer end of the ideal gas apparatus to the end of pressure detector.
7. Join two ends.
8. Open Pasco Capstone and start measuring.
9. Wait till the graph will have stable line. After push plunge, and you will notice change in the graph.
10. Wait till the stabilization of the graph, and release plunge. Wait another time to stabilize graph.
11. Save the data.
12. Calculate pressure and temperature of sample gas.

HYDROGEN TEST

1. Place the lit match near the rubber end of the anaerobic tube.
2. Pierce the rubber on the tube into the flame by the needle

Team:NU Kazakhstan/Experiments