iGEM 2019 — Team NU_Kazakhstan

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# Team:NU Kazakhstan/Notebook

NU Kazakhstan

* [Homepage](https://2019.igem.org/Team%3ANU_Kazakhstan)
* Team
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* Project
  + [Description](https://2019.igem.org/Team%3ANU_Kazakhstan/Description)
  + [Design](https://2019.igem.org/Team%3ANU_Kazakhstan/Design)
  + [Experiments](https://2019.igem.org/Team%3ANU_Kazakhstan/Experiments)
  + [Notebook](https://2019.igem.org/Team%3ANU_Kazakhstan/Notebook)
  + [Future Plans](https://2019.igem.org/Team%3ANU_Kazakhstan/Future_Plans)
  + [Modeling](https://2019.igem.org/Team%3ANU_Kazakhstan/Modeling)
  + [Criteria](https://2019.igem.org/Team%3ANU_Kazakhstan/Criteria)
  + [Results](https://2019.igem.org/Team%3ANU_Kazakhstan/Results)
  + [Characterization](https://2019.igem.org/Team%3ANU_Kazakhstan/Characterization)
* * [Parts Overview](https://2019.igem.org/Team%3ANU_Kazakhstan/Parts)
  * [Human Practices](#!)
    + [Human Practices](https://2019.igem.org/Team%3ANU_Kazakhstan/Human_Practices)
    + [Public Engagement and Education](https://2019.igem.org/Team%3ANU_Kazakhstan/Public_Engagement)
  * [Safety](https://2019.igem.org/Team%3ANU_Kazakhstan/Safety)
  * * [Entrepreneurship](https://2019.igem.org/Team%3ANU_Kazakhstan/Entrepreneurship)
  * [Judging Form](https://igem.org/2019_Judging_Form?team=NU_Kazakhstan)

Notebook

# Project Timeline

![](https://static.igem.org/mediawiki/2019/d/d6/T--NU_Kazakhstan--timeline.jpg)

| Week | Dates | Work completed |
| --- | --- | --- |
| 1 | May 1-5 | - Lab tools and reagents availability check-up  -Measurement of Gibco BG-11’s pH |
| 2 | May 6-12 | - Laboratory introduction & safety training for new team members  - Agar plates preparation according to LB agar protocol  - E.coli (DH5ɑ) streaking on LB agar and incubation  - LB broth media preparation according to general E.coli protocol  - Grown E.coli transfer to LB broth and incubation  - Competent cells preparation according to preparing competent cells protocol  - SOC media (pH=7.18) preparation according to the protocol  - Chemical transformation of competent DH5ɑ cells and streaking on LB agar media |
| 3 | May 13-19 | - Miraprep of cultures according to miraprep protocol  - Cyanobacteria subculturing  - Agarose gel preparation with 10l/100 ml Sybr Safe addition   - Gel electrophoresis of miraprep products  - Measuring the concentration according to Nanodrop protocol  - Dilution of primers  - PCR of liquid chlamy cultures according to liquid PCR protocol  - DNA extraction from SQR- cyanobacteria cultures according to the DNA extraction protocol   - Amplification of the extracted DNA (PCR)   - HydA amplification from the PCR products  - Gel electrophoresis of PCR products  - Nanodrop of the products  - Hydrogenase construct (joined Hyd genes) creation: PCR amplification of Hyd1, Hyd2 and Hyd3 separately  - Check of PCR products via gel electrophoresis |
| 4 | May 20-26 | - Circular polymerase extension cloning (CPEC) (pSyn6+hydrogenase) assembly  - PCR and gel electrophoresis  - Cyanobacteria subculturing  - NS site amplification via PCR  - Gel electrophoresis of the PCR product  - Nanodrop of the PCR products  - Chemical transformation of NS site into E.coli |
| 5 | May 27-June 2 | - DNA extraction from old cyanobacteria cultures  - PCR of hydrogenase construct parts: Hyd1, Hyd2, Hyd3  - Check of SQR gene presence in cyanobacteria via PCR  - Gel electrophoresis of the PCR products  - 4 LB agar plates: 2 with and 2 without spectinomycin preparation  - DH5ɑ streaking on the plates and incubation  - LB broth tubes preparation and transfer of E.coli from agar plates and incubation  - OD measurement of the DH5ɑ cultures via Spectrophotometry  - Competent cells preparation  - Transformation of Hydrogenase construct into the E.coli with higher OD readings   - Plating transformed cultures on spectinomycin plates  - OD measurement of old cyanobacteria cultures  - Cyanobacteria subculturing  - Transformation of cyanobacteria according to Invitrogen protocol  - CPEC (pSyn6+hydrogenase) assembly  - Addition of Ascorbic Acid to C, 58B + Cr, WT + Cr Chlamy cultures  - Preparation of chromium and Ascorbic Acid solutions in TAP S-  - Preparation of the solution with ascorbic acid + chromium in absolute ethanol |
| 6 | June 3-9 | - NaHCO3stock solution preparation  - pH calibration of sodium thiosulfate, NaHCO3and BG-11 via 37% HCl  - BG-11 agar plates with NaHCO3and spectinomycin preparation according to Preparation BG-11 plates protocol  - Gel extraction according BioRad protocol for obtaining pSyn6 and CPEC pure DNA samples  - Transformation of DNA into E.coli  - OD measurement of the samples  - Miraprep of the samples  - Inoculation of cyanobacteria  - Transformation of cyanobacteria according to Stony Brook team protocol  - Preparation of BG11 agar plates  - Liquid PCR  - Nanodrop  - Gel electrophoresis  - DNA purification according to Monarch purification kit protocol  - Nanodrop of the purified samples |
| 7 | June 10-16 | - Re-dilution of stock Hyd samples  - Amplification of Hyd1, Hyd2 and Hyd3 via new PCR protocols  - Samples purification according to Monarch purification kit protocol  - Nanodrop and gel electrophoresis of the samples  - CPEC samples amplification  - Transformation into DH5ɑ strain  - Transfer of cultures from agar plates into LB broth media  - Measurement of absorbance and collection of obtained UV graphs of carbon quantum dots  - Liquid PCR  - Gel electrophoresis |
| 8 | June 17-23 | - Miraprep of transformed culture  - Purification of miraprep samples  - Miraprep samples DNA linearization according to Restriction digestion protocol  - Plasmid to insert ratio recalculation for CPEC  - CPEC (pSyn6+hydrogenase) assembly  - Liquid PCR  - Gel electrophoresis  - Dilution of primers  - HydA amplification via PCR  - Check via nanodrop and gel electrophoresis |
| 9 | June 24-30 | - Mixing with ZnCl2 and burning in furnaces of two cyanobacteria samples (sulfide and control)  - Washing with HNO3 and HCl, filtering and drying two cyanobacteria samples (sulfide and control one)   - PAM stock dilution  - HydA amplification from PAM  - Liquid PCR  - Gel electrophoresis  - Preparation of TAP media  - Analysis of the prepared carbon dots |
| 10 | July 1-7 | - Check of reagents presence for BG-11 media preparation  - Linearization of PAM HydA and p233 Maturation factor (MF) DNA by BAM-HI  - Gel electrophoresis check  - Psba1 amplification   - CPEC (pSyn6+hydrogenase) assembly  - Verification of insert integration into the vector through amplification of region  - Linearization of CPEC DNA  - Nanodrop and gel electrophoresis   - Liquid PCR  - Gel electrophoresis |
| 11 | July 8-14 | - Polymerase cycling assembly (PCA) creation  - Miraprep  - Nanodrop and gel electrophoresis  - Preparation of the TAP media  - Liquid PCR   - Gel electrophoresis |
| 12 | July 15-21 | - Gel electrophoresis   - Hydrogenase construct assembly  - CPEC (pSyn6+hydrogenase) assembly  - Linearization of CPEC sample  - Nanodrop and gel electrophoresis of CPEC samples  - Purification of psba1 gene  - HydA amplification from PAM  - Transformation of CPEC  - Liquid PCR  - Subculturing |
| 13 | July 22-28 | - Subculturing of cultures from agar plates into LB broth  - Check for the contamination in the reactants  - Nanodrop  - Gel electrophoresis  - PCR  - Enzyme digestion of CPEC miraprep samples  - HydA and PsbA1 assembly via Polymerase Cycling Assembly (PCA) |
| 14 | July 29- August 4 | - MF plasmid check via nanodrop  - Linearization of MF plasmid  - Gel electrophoresis  - HydA and PsbA1 assembly via Polymerase Cycling Assembly (PCA) |
| 15 | August 5-11 | - Primers redesigning  - Cleaning lab  - Subculturing of cultures |
| 16 | August 12-18 | - Preparation of ingredients for the reduction assay |
| 17 | August 19-25 | - Preparation of chromium reduction assay  - Liquid PCR for the presence of SQR  - Filter paper preparation  - Latex’s pH measurement |
| 18 | August 26 - September 1 | - Dilution of primers  - Amplification of Hyd 1, Hyd2 and Hyd3  - Purification of Hyd DNAs according to Monarch protocol  - Nanodrop  - Hydrogenase assembly via Polymerase Cycling Assembly (PCA) from Hyd1, Hyd2 and Hyd3  - Amplification of obtained PCA samples  - Subculturing from liquid culture of cyanobacteria to latex on filter paper  - Rhodopsin amplification   - Gel electrophoresis  - Overlaps (to Hyd1and pSyn6) addition to SQR gene  -pSyn6 digestion with Kpn and BgL-II restriction enzymes |
| 19 | September 2-8 | - Hyd1 and Hyd3 amplification  - Gel electrophoresis  - Hydrogenase assembly via Polymerase Cycling Assembly (PCA) from Hyd1, Hyd2 and Hyd3  - Amplification of obtained PCA samples  - Insert (Hydrogenase construct) integration into pSyn6 vector  - Survivability of cyanobacteria assay on latex covered filter papers  - Purification of Hyd1, Hyd2, Hyd3 according to Monarch purification kit protocol  - Assembly (PCA) from Hyd1, Hyd2 and Hyd3 with a new approach  - Amplification of obtained PCA samples  - Gel electrophoresis check of PCA samples  - Hyd2 amplification with extended cycles of PCR |
| 20 | September 9-15 | - Purification of Hyd1, Hyd2, Hyd3 with nuclease free water  - Amplification of Hyd1, Hyd2 and Hyd3 with a new Master Mix Phusion for the high GC content  - Gel extraction according to Invitrogen Gel Extraction kit protocol   - Nanodrop |
| 21 | September 16-22 | - Insertion of psba1+HydA into MF vector  - HydA and psba1 amplification  - Purification of the amplified samples  - Insert and MF digestion with BamHI-HF  - Purification of the digested samples  - Ligation of insert and MF  - Amplification of construct using E.coli  - Transformation of construct into cyanobacteria |
| 22 | September 23-29 | - Ligation of insert and MF  - H2S Survivability assay of cyanobacteria  - Transformation of construct into cyanobacteria |
| 23 | September 30- October 6 | - Preparation of liquid cultures with/without chloramphenicol antibiotic  - Work on characterization part: mRFP generator (Part:BBa\_K861173)  - Transformation of mRFP generator into E.coli  - Nanodrop  - Gel electrophoresis of the samples  - Transformation into BL21 (DE3) according to the BioBrick part protocol  - H2S Survivability assay of cyanobacteria  - Preparation of the TAP media |
| 24 | October 7-13 | - HydA amplification from the miraprep samples  - H2S Survivability assay of cyanobacteria |
| 25 October 14-20 | - H2S Survivability assay of cyanobacteria  - HydA amplification from the miraprep samples | |
| 26 | October 21-22 | - Transformation of construct into cyanobacteria  - Hydrogen production assay |


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# Team:NU Kazakhstan/Experiments

NU Kazakhstan

* [Homepage](https://2019.igem.org/Team%3ANU_Kazakhstan)
* Team
  + [Team Members](https://2019.igem.org/Team%3ANU_Kazakhstan/Team)
  + [Collaborations](https://2019.igem.org/Team%3ANU_Kazakhstan/Collaborations)
  + [Attributions](https://2019.igem.org/Team%3ANU_Kazakhstan/Attributions)
* Project
  + [Description](https://2019.igem.org/Team%3ANU_Kazakhstan/Description)
  + [Design](https://2019.igem.org/Team%3ANU_Kazakhstan/Design)
  + [Experiments](https://2019.igem.org/Team%3ANU_Kazakhstan/Experiments)
  + [Notebook](https://2019.igem.org/Team%3ANU_Kazakhstan/Notebook)
  + [Future Plans](https://2019.igem.org/Team%3ANU_Kazakhstan/Future_Plans)
  + [Modeling](https://2019.igem.org/Team%3ANU_Kazakhstan/Modeling)
  + [Criteria](https://2019.igem.org/Team%3ANU_Kazakhstan/Criteria)
  + [Results](https://2019.igem.org/Team%3ANU_Kazakhstan/Results)
  + [Characterization](https://2019.igem.org/Team%3ANU_Kazakhstan/Characterization)
* * [Parts Overview](https://2019.igem.org/Team%3ANU_Kazakhstan/Parts)
  * [Human Practices](#!)
    + [Human Practices](https://2019.igem.org/Team%3ANU_Kazakhstan/Human_Practices)
    + [Public Engagement and Education](https://2019.igem.org/Team%3ANU_Kazakhstan/Public_Engagement)
  * [Safety](https://2019.igem.org/Team%3ANU_Kazakhstan/Safety)
  * * [Entrepreneurship](https://2019.igem.org/Team%3ANU_Kazakhstan/Entrepreneurship)
  * [Judging Form](https://igem.org/2019_Judging_Form?team=NU_Kazakhstan)

F

Experiments

![](https://static.igem.org/mediawiki/2019/6/61/T--NU_Kazakhstan--Notebook.jpg)

# 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](https://international.neb.com/protocols/2012/09/06/protocol-phusion-high-fidelity-pcr-master-mix-with-hf-buffer-m0531)
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

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# Team:NU Kazakhstan/Results

NU Kazakhstan

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Results

# Results Due to complications with amplification and assembly of synthetic sequences of genes that we ordered like complex stable secondary structures and a lot of long repeats we switched to using plasmids that we received from as a kind gesture from Daniel C. Ducat. Plasmid p233 that contains hydrogenase maturation factors HydEF and HydG under psbA1 promoter and HydA under psbA1 promoter in 0015 plasmids. To assemble psbA1 and HydA genes, firstly, amplification of psbA1 (503 bp) from p233(11830 bp) and HydA(1998 bp) from plasmid PAM (5004 bp) were done. As it can be seen from Figure 1 and Figure 2 successful amplification of genes of interest was verified via gel electrophoresis. ![](https://static.igem.org/mediawiki/2019/3/39/T--NU_Kazakhstan--resultspsbA.jpg) Figure1: PsbA1 amplification gel results. ![](https://static.igem.org/mediawiki/2019/e/ef/T--NU_Kazakhstan--resultshydAband.jpg) Figure2: HydA amplification gel results Amplified HydA and psbA1 genes were assembled via Polymerase Cycling Assembly (PCA) and also verified via gel electrophoresis as shown on Figure 3. ![](https://static.igem.org/mediawiki/2019/8/8a/T--NU_Kazakhstan--resultsinsert.jpg) Figure 3. HydA and PsbA1 assembly gel results After transformation of the assembled construct into the DH5alpha cloning strain, DNA extraction results illustrated bands higher than empty vector p233 (also known as MF (Maturation Factor)). All samples were linearized with BamHI-HF beforehand. ![](https://static.igem.org/mediawiki/2019/d/d7/T--NU_Kazakhstan--resultsconstruct.jpg) Figure4. Linear assembled construct digested by BamH gel results To ensure the presence of HydA gene in the construct, the samples were linearized by EcoRV and used as template for HydA amplification (see Figure 5). Overall, four samples were assessed for the HydA and three were positive for the gene as bands corresponding to ~1.7-1.8 kb were visualized on the gel. As a negative control p233 (also known as MF) without insert was used which did not show any band for HydA as expected. ![](https://static.igem.org/mediawiki/2019/6/6e/T--NU_Kazakhstan--resultshydAampaftermiraprep.jpg) Figure5. HydA verification in the construct To verify the integration of construct into cyanobacteria psbA1+hydA(~3.1 kb) amplification was performed using forward psbA1 and reverse NSb primers. NSb primer was used due to complications during amplification process with HydA primers. Out of five samples, the 5th band without ladder had an expected band for ~3.1 kb. ![](https://static.igem.org/mediawiki/2019/0/0a/T--NU_Kazakhstan--resultscyanohydA.jpg) Figure6. HydA+PsbA1 amplification from the construct integrated into cyanobacteria The effect of genetic modification on survivability of cyanobacteria in a hydrogen sulfide (H2S) rich and anaerobic environment was assessed qualitatively. The samples of nearly same OD were constantly shaken in the shaking incubator. From Figure 7, it can be seen that transformed HydA+ cells differ significantly from the wild-type by its color illustrating the higher survival rate compared to the wild-type strains. Blotted paper was also assessed using survival assay with the same condition only few adjustments for the paper. It was soaked in the medium with sulfide and w/o sulfide. Tubes were flushed with nitrogen until air is replaced. 5 days later tubes were assessed on paper colour to confirm survival. As Ducat stated limited growth is supported in Cyanobacteria as they use Hydrogen as reductant in alternative metabolic pathways. This is supported by this survival assay and thus hydrogen presence by HydA activity. ![](https://static.igem.org/mediawiki/2019/1/1e/T--NU_Kazakhstan--resultssurvivalassay.jpg) Figure 7. Sulfide survival assay Pop test was performed as a classical way to detect hydrogen in the air one of our tubes showed exact same result to a lesser extent as the control reaction of Zinc + Hydrochloric Acid. Zinc + HCl reaction was done in the same sealed anaerobic tube. Stopper was punctured using need and excess gas was let out straight to the flame of match instead of usual “Pop” sound we heard whistle in both cases and same reaction of flame to the gas that went out in control and our culture tube. This leads us to be sure that hydrogen is being produced: Control reaction video: Transformed cells video: Wild type video: Hydrogen gas production measurement There were two cultures cultured in two different times (04.10.19 and 16.10.19). They were flushed by nitrogen gas in order to make anaerobic conditions. The pressure and temperature of samples were measured on 22.10.19. The Ideal Gas Apparatus, PASCO sensor PASCO Capstone software were used to measure pressure and temperature of the system. Below, you can see the photo of our measurement setup: ![](https://static.igem.org/mediawiki/2019/4/43/T--NU_Kazakhstan--resultFig8.jpg) Figure 8. Gas measurement apparatus We collected gas sample from our cultures grown in anaerobic conditions into the special measuring cylinder. We measured the volume of the collected gas, then we applied pressure by pushing down the plunge. This pressure was measured by special Pasco detectors. From the Pasco Capstone software, we obtained the values of pressure and temperature. Using the Ideal Gas equation, we measured the number of moles: PV = nRT Volume contains the parasitic volume of apparatus. In order to compensate for the difference of OD, we obtained a ratio of number of moles of gas produced per OD of culture measured. For the Wild type cultures we obtained the following graph: ![](https://static.igem.org/mediawiki/2019/c/cc/T--NU_Kazakhstan--resultFigure9.png) Figure 9. The hydrogen production measurements for the Wild Type For the Transformed cultures: ![](https://static.igem.org/mediawiki/2019/b/b5/T--NU_Kazakhstan--resultFigure1.png) Figure 10. The hydrogen production of the transformed cyanobacteria (SBA21) As it can be seen, there is an increase in gas production by transformed cyanobacterial cultures, and clear decrease of gas produced by the wild type. In our measurement, we constructed the experiment by variating the parameter of H2S presence. H2S is known to be the common PSII inhibitor, which we are aiming to accomplish. This fact should increase the production of Hydrogen gas. We did the measurement for two types of cultures, SBA21 and HydA1, that were transformed in different days. We also performed the same measurement with Wild type. ![](https://static.igem.org/mediawiki/2019/a/aa/T--NU_Kazakhstan--graphResult.png) Figure 11. Hydrogen production by various cell cultures ![](https://static.igem.org/mediawiki/2019/f/fb/T--NU_Kazakhstan--errors.png) Error Propagation As it can be seen, Wild type does not deal with the survival in the presence of H2S, and that is why both proliferation and hydrogen gas are greatly inhibited. On the other hand, there is a clear difference in the production of gas for the transformed cultures. H2S increases yield for the gas production for both HydA1 and SBA21, and works as we expected. Beside of this, there is clear increased hydrogen production comparing to the Wild type in overall. The increased hydrogen production in the presence of H2S proves that our part works as expected. Survival test of bacteria on filter paper Bacteria were fixed on the filter paper by mixing equal amount of latex and culture, and placing dropwise on the paper, and solution of BG11 was poured until it topped the paper. Initially, the pH of latex was measured by a pH-meter, but the value was quite fluctuating. Therefore, it was decided to use pH-indicator strips. The resultant pH was 6. This was quite acceptable for the culture. Starting from the first day, the BG11 solution was being changed everyday. This was done due to the high mass convection inside hood. However, this caused high consumption of BG11. Therefore, it was decided to put larger plate on top of the plate with filter paper, to prevent evaporation of BG11. On average, in 3 weeks, the color of bacteria changed from light green to pale yellow, meaning the life expectancy was about 2 weeks considering equal amounts of culture and latex, exposure to surroundings, and slow evaporation rate of BG11. ![](https://static.igem.org/mediawiki/2019/6/69/T--NU_Kazakhstan--resultspapers.jpg) Figure 12. The survival test of bacteria on the filter paper. Carbon Quantum Dots Microalgae C. reinhardtii containing chromate reductase gene Six cultures named 62, 53, 58A, 58B, C and wild type were checked for the presence of chromate reductase (ChrR) gene by using PCR on liquid cultures. ![](https://static.igem.org/mediawiki/2019/7/75/T--NU_Kazakhstan--resultsreductasegene.jpg) Figure 13. ChrR gene extraction from cultures. From this, we could identify the presence of the gene in several strains and chose the corresponding biomass for preparing Carbon Quantum Dots. The CQDs were prepared from the strains containing Chromate reductase gene. We performed excitation wavelength-dependent fluorescence measurements to detect the optimum wavelength. We excited the solutions of CQDs with the range of 350-450 nm and obtained the results presented on figure 14. ![](https://static.igem.org/mediawiki/2019/6/6f/T--NU_Kazakhstan--grahChang2.jpg) Figure 14. 3D data for the fluorescence of CQDs As it can be observed, the fluorescence properties of our CQDs can be tuned upon different excitation wavelengths. Based on this study, we identified that most pleasurable fluorescence is obtained upon the excitement with 425 nm. The graph is presented on figure 15. ![](https://static.igem.org/mediawiki/2019/9/94/T--NU_Kazakhstan--changgrah1.jpg) Figure 15. Fluorescence of CQDs excited with 425 nm The maximum peaks are observed on 482, 611, 661 nm. The first peak overlaps with the absorption of Gloebacter Rhodopsin which is in 500 nm range, and the third one is close to the absorbance of the PSI which is in the range of 700 nm. We also coated cyanobacteria strains with Carbon Quantum Dots and performed the analysis with the Confocal Microscopy, using the 409 nm excitation wavelength. ![](https://static.igem.org/mediawiki/2019/7/70/T--NU_Kazakhstan--carbdot.png) Figure 16. The picture of coated cyanobacteria from the confocal microscopy As it can be seen, half of the cyanos are coated with fluorescent CQDs. We also performed time-resolved fluorescence measurement, and using the Decay Fit software we modeled the major fluorescent events occurring with CQDs. The modeling description can be seen in detail on the Modeling web page.