# Protocols Here we will describe the methods that we used in the lab to perform routine molecular cloning operations. This will be divided into 9 sections, to which you can proceed through the contents menu on the left of the page. # Microbiology ### E. coli cultivation E. coli cultures were cultivated on LB (Lysogeny Broth) medium and its solid version, LB-agar or LA. SOB or SOC medium was used in transformation protocols to rehabilitate cells after heat shock. LB medium composition: 10 g/l tryptone, 5 g/l yeast extract, 10 g/l NaCl, pH = 7.5, autoclaved at 22 psi. LA composition: 10 g/l tryptone, 5 g/l yeast extract, 10 g/l NaCl, 1.5% w/v agar, pH = 7.5, autoclaved at 22 psi. SOC composition: 20 g/l tryptone, 5g/l yeast extract, 0.584 g/l NaCl, 0.186 g/l KCl, 2.03 g/l MgCl2, 2.46 g.l MgSO4*7H2O, pH = 7.5, autoclaved at 22 psi. Liquid cultures with V≤15 ml were grown in loosely closed 50 ml Eppendorf tubes at 37°С overnight (16-20 h) with 200 rpm shaking. LIquid cultures with V≤5 ml were grown in 5 ml Eppendorf tubes at 37°С for 6-12 h with 200 rpm shaking. Colonies on solid medium were grown overnight in a static incubator at 37°С. ### Plasmid extraction Extraction is performed with eurogene "Plasmid MiniPrep" without any digressions ### Chemical transformation of E. coli 1. Take out bacteria from the -80°C freezer and immediately place on ice for 10 minutes or until just thawed. 2. Turn on the UV in the laminar hood for about 15 minutes to sterilize the working environment before opening the test tube with the bacteria to avoid any risk of contamination. 3. Pour fresh agar-LB (Luria Broth) medium in Petri dishes with the required antibiotic. 4. Mix 33-85 μl of competent cells with 2-78 μl of the ligation reaction mixture in the sterile box, incubate on ice for 15-40 minutes*. 5. Perform a heat shock for 45 sec at 42°C. 6. Immediately transfer cells in ice and incubate for 2 minutes. 7. Resuspend the bacteria with 500-1000 μl of SOB/SOC medium (Super Optimal Broth). 8. Incubate at 37°C constantly shaking at 180 rpm for 30-60 minutes. 9. Centrifuge the cells for 4 minutes at 3500 g. 10. Discard the supernatant, leaving around 50 µl of the medium, resuspend the bacteria in the remaining LB medium and plate out on a prepared Petri dish. Incubate the plates overnight at 37°C. > * — For proper results it is essential to perform as many transformation controls as possible: transform the bacteria without DNA (contamination control), only the linearized digested vector (to check if the DNA is properly digested), positive control with purified non-digested DNA (to check whether everything is alright with competent cells and the transformation). # Molecular cloning ## PCR This protocol is provided by the supplier — Evrogen — for Tersus® polymerase mix. Combine in the following order: | Component|20 µl reaction|50 µl reaction|Final concentration| |Sterile water|to 20 µl|to 50 µl|-| |10X Tersus Plus buffer*|2 µl|5 µl|1X| |50X dNTP mix|0.4 µl|1 µl|1X (0.2 mM each)| |Upstream primer|variable|variable|0.5 µM| |Downstream primer|variable|variable|0.5 µM| |DNAtemplate|variable|variable|1-250 ng| |50X Tersus polymerase|0.4 µl|1 µl|1X| > * — Optionally 5X Tersus Red Buffer can be used (then add 4(10) µl in 20(50) µl sample and adjust water volume accordingly). Cycling instructions: ||Stage|Cycle number|Temperature|Time| |1|Initial denaturation|1|92-95°C|3min| |2|Denaturation||92-95°C|30 sec| |3|Annealing||Ta (55-68°C)|30 sec| |4|Elongation|Repeat steps 2–4 20–35 times |72°C|1 min per 1.5 kb| |5|Final extension|1|72°C|3 min| > Ta – primer annealing temperature, depending on the specific primer structure, may deviate ±5°C from the primer Tm. The Tm was calculated using the kindly supplied SnapGene software. ### Colony PCR 1. Prepare a clean dish with the required medium for clone selection, mark and number regions for future clones. 2. Using a sterile yellow tip or another appropriate instrument gently touch a single colony, transfer some of biomass onto the pre-marked dish in a form of a small streak, resuspend the rest of biomass in the aliquot of sterile mQ, following the numeration. 3. Repeat two previous stages with 5–8 colonies in total 4. Place the new dish at 37°C and incubate o/n. * 5. Heat sampled mQ aliquots at 95°C up to 5 min 6. Prepare a PCR Master Mix with 5X Red Buffer** and pipet into the PCR tubes 16 µl each (of 20 µl total volume of the sample). 7. Add 6 µl of lysate onto Master Mix samples. 8. Run the PCR program, reckon to perform 35 cycles of the PCR. 9. Analyze PCR results with electrophoresis (see method below) > * — Bacterial streaks can be used in further experiments (testing or plasmid production) if tested positive > ** — As some of the Master Mix volumes could be lost while aliquoting, increase the stock volume up for extra 2-3 samples or 10%. ## Gel Electrophoresis 1. Add appropriate amount of agarose (up to 1-2%) to 50 ml of TAE buffer* 2. Heat the solution in a microwave oven just to the boiling point (the exact heating time is dependant on the microwave power, gel volume and agarose concentration) 3. Remove the flask from the microwave oven and swirl gently. 4. Let the solution cool to approximately 40-50°C 5. Add ethidium bromide to a final concentration of 10 µg/ml 6. Pour the gel into a tray and place in the well comb, let the gel solidify 7. Remove the comb from the gel. 8. Place the gel into the electrophoresis apparatus and fill the apparatus with TAE, the buffer should be slightly higher than the gel, covering it completely. 9. Load 8-50 μL of the sample depending on the purpose of the gel and the well volume. Do not forget to add the DNA size marker or ladder (we used 2 µl of Evrogen 1 kb or 50 bp+ DNA Ladder). 10. Place the electrodes with the cover over the gel apparatus and initiate the electrophoresis. 11. Run under the voltage of 110V. 12. Take out the gel and place it in a transilluminator to visualize the EtBr intercalated DNA. > * — solution of Tris base 40 mM, acetic acid 20 mM, EDTA 1 mM ### Gel extration > Extraction is performed with Evrogen "Cleanup Standard" Kit without any digressions ## One-pot oligos annealing and phosphorylating In a PCR tube on ice combine the following reagents: |Component|Volume| |PNK |0,5 µl | |10x ligase buffer containing Mg2+ and ATP| 1 µl | |oligos| 2+2 µl| |PCR grade (nuclease-free) water| 4.5 µl| Incubate as following: 1. 30’ at 37°С — phosphorylation step 2. 20’ at 75°С — kinase inactivation step 3. 5’ at 95°С — DNA melting step 4. Let anneal by cooling to room temperature in a turned off heater (2-5 hours). ## Restriction > This protocol is provided by Thermo Fisher Prepare the reaction mixture: |Component|Volume| |Nuclease-free water|up to 20 µl| |10X FastDigest® Buffer |2 µl| |FD Enzyme|1 µl| |DNA|to 1 µg| Mix gently and spin down. Incubate in a heat block at 37°C for 5-30 min*. Analyse the sample on gel electrophoresis to either analyse the restriction pattern or select the required restriction fragment. > * — See the Certificate of Analysis for enzyme and substrate-specific incubation time ## Ligation Prepare the ligation mixture: |Component|Volume| |Nuclease-free water|to 20 µl| |10X T4 Ligase Buffer with ATP|2 µl| |T4 DNA Ligase, 5 u/µl|1 µl| |DNA (vector)|10-100 ng| |DNA (insert)|variable (10–3:1 insert to backbone molar ratio) | Incubate the mixture at 14°C for 15-30 min. Use 2-8 µl of ligase mixture for transformation ### Golden Gate assembly > This protocol is provided by the New England Biolabs Inc. 1. Prepare reaction mixture: | Component | Volume | | Nuclease-free water |to 20 µl| |10X T4 Ligase Buffer with ATP | 2 µl | | NEB Golden Gate Assembly Mix | 1 µl | | Vector | 75 ng| | Inserts | variable (2:1 molar ratio) | 2. Choose the appropriate assembly protocol | Insert number | Suggested assembly protocol | |For 1 Insert | 37°C, 5 min (cloning) or 37°, 1 hr (library preparation) → 60°C, 5 min | |For 2-4 Inserts | 37°C, 1 hr → 60°C, 5 min | | For 5-10 Inserts | (37°C, 1 min → 16°C, 1 min) x 30 → 60°C, 5 min | | For 11 - 20+ inserts | (37°C, 5 min → 16°C, 5 min) x 30 → 60°C, 5 min | Run the required program using an amplifier Use 2 µl of the resulting mixture for transformation # Fluorescence measurement Fluorescence spectrophotometer Cary Eclipse was used. 1. Pick 3 colonies from the bacterial plate and incubate in 5 ml LB medium for 5 hours 2. Centrifuge the cells for 4 minutes at 3500 g. 3. Discard the supernatant completely and resuspend the bacteria in 1 ml of PBS buffer 4. Measure D600 of diluted samples 5. Dilute the samples to D600 of 0,05 with PBS buffer 6. Be sure to take negative and positive controls: for the negative control use clones transformed with vector without insertion and as a positive one the clone with characterised YFP containing construction. 7. Use positive control to set the parameters of an instrument. We excited the fluorescence at 500 nm and measured emission from 510 to 600 nm (5/5 nm gaps) 8. Put 1 ml of a diluted sample into a plastic cuvette and measure the fluorescence. # Protocols for IPPAS B-256 ## Extraction of genomic DNA 1. Precipitate at maximum speed for 10 minutes. Discard supernatant. The volume of cyanobacteria cells in the tube must be about 0.5 g. 2. Wash cells in 50/100 TE 3 times (resuspend and precipitate at max. speed for 5 minutes, discard supernatant after each set). 3. Resuspend cells in 500 μl of mix of guanidine and phenol pH=8. 4. Incubate probe at 75ºC for 15 minutes. 5. Precipitate at maximum speed for 5 minutes. 6. Add 500 μl of TES buffer and 500 µl of Phenol-Chlorophorm and mix gently. 7. Discard interphase and precipitate. 8. Repeat steps 6-7 until the disappearance of interphase. 9. Add 1:1 volume Isopropanol and 1/10 volume of NaCl. 10. Mix gently and precipitate at maximum speed for 5 minutes, discard supernatant. 11. Add 30-100 µl of mQ and mix gently. ## Optimal cultivation conditions - **Name and content of medium**: Zarrouk *Content of Zarrouk* | Chemical reagent: | Mass, g/l | | *Stock 1* | | | NaHCO3 | 16,8| | K2HPO4×3H2O | 1,0 | | Distilled water | Up to 0.2 l | | *Stock 2* | | | NaNO3 | 2,5 | | K2SO4 | 1,0 | | NaCl | 1,0 | | MgSO4×7H2O | 0,2 | | CaCl2×2H2O | 0,04 | | Fe+EDTA* | 1,0 ml | | Microelement stock 1 | 1,0 ml | | Microelement stock 2 | 1,0 ml | | Distilled water | Up to 0.8 l | | Agar (if required solid media) | 12 g (1,2%) | > Stock 1 and Stock 2 must be autoclaved at 121ºС 15 minutes at 100 kPa **separately**. **Preparation of FeSO4 and EDTA solution**. In 134 ml 1N KOH dissolve 30,2 g EDTA. Dillute solution with water, add 24,9 g FeSO4×7H2O and add up to 1 l distilled water. Blow through the solution air without CO2 in the dark for 12 h. To get rid of СО2, blow air through alkali solution. *Content of microelement stocks for Zarrouk medium* | Chemical reagent: | Mass, g/l | | **Microelement stock 1** | | | H3BO3 | 2,86 | | MnCl2×4H2O | 1,81 | | ZnSO4×7H2O | 0,22 | | CuSO4×5H2O | 0,08 | | MoO3 | 0,015 | | **Microelement stock 2** | | | NH4VO3 | 0,023 | | KCr(SO4)2×12H2O | 0,048 | | NiSO4×7H2O | 0,048 | | Na2WO4×2H2O | 0,018 | | Ti2(SO4)3 | 0,040 | | Co(NO3)2×6H2O | 0,044 | - **СО2 source (gas-air mix**): 1,5-2 % СО2 - **Temperature**: 32ºС - **Lighting**: twenty-four hour - **Illumunation**: 500 µmol photon light m2s-1 - **Type of lamps**: one-sided, LED white, red, blue ## -N and -P experiment **Modifications of Zarrouk** | Chemical reagent: | Mass, g/l; -N | Mass, g/l; -P | | *Stock 1* | | | | NaHCO3 | 16,8| -//- | | K2HPO4×3H2O | 1,0 | — | | Distilled water | Up to 0.2 l | -//- | | *Stock 2* | | | | NaNO3 | — | 2,5 | | K2SO4 | 1,0 | | NaCl | 1,0 | -//- | | MgSO4×7H2O | 0,2 | -//- | | CaCl2×2H2O | 0,04 | -//- | | Fe+EDTA* | 1,0 ml | -//- | | Microelement stock 1 | 1,0 ml | -//- | | Microelement stock 2 | 1,0 ml | -//- | | Distilled water | Up to 0.8 l | -//- | ** Handling of IPPAS B-256 culture** All manipulations, except biochemical analysis and OD measurement, with culture were carried out **in sterile conditions** in order to prevent contamination. > 9 50-ml flasks with 25 ml of culture (2 flasks for each — control, -N, -P) were stored at 32ºС, 30-40 µmol photon light m2s-1 for 7 days. On the first and on the last days of experiment sterile probes for OD680 and OD750 were measured. Thereafter, the final volume of 3 flask in one probe was mixed, homogenised and analysed from the biochemical point of view. ![https://static.igem.org/mediawiki/2021/c/cb/T--LMSU--flasks.jpeg](N and P starvation experiment) **Fluorescent and light microscopy** For revealing of culture condition and approximate lipid content cell were checked under microscope. > To mark triacylglycerols BODIPY lipophilic fluorescent dye was used (503/512 nm). Protocol of dyeing: 1. Add 1:1 of 8% PFE to the probe and leave it at 4ºC overnight. 2. Precipitate at maximum speed for 10 minutes. Discard supernatant. 3. Wash probes with ~200 µl 50/100 TE 3 times (vortex - precipitate - discard supernatant). 4. Add 90 µl of 50/100 TE and 10 µl of BODIPY (ThermoFisher Scientific), vortex and incubate at room temperature in the dark for 30 minutes. 5. Wash probes with ~100 µl 50/100 TE 3 times (vortex - precipitate - discard supernatant). 6. Add 100 µl of 50/100 TE. # Chemical analysis **Fats ** The sample was dried up in desiccator for 5 hours. Then lipids were isolated by extraction with dried hexane and filtration. The solvent was removed at reduced pressure, and the residue was distilled in vacuum. **Protein** The sample is heated in the presence of concentrated sulphuric acid with copper sulphate as catalyst. The acid breaks down the organic substance by the process of oxidation and reduced nitrogen is liberated in the form of ammonium sulfate. The sample is said to be fully decomposed when we obtain a clear and colorless solution. (Note: Potassium sulfate is added to increase the boiling point of the medium). Then sodium hydroxide is added to convert the ammonium salt to ammonia. The distilled vapors are then trapped in H3BO3 water solution. Ammonium ion concentration in the acid solution, and thus the amount of nitrogen in the sample, is measured via acid–base titration with HCl known concentration. The amount of protein nitrogen is converted to the content protein using coefficient. Universal protein coefficient 6.25 is adopted on the basis that the average content nitrogen consumption in most proteins is 16%. **Carbohydrates** Hydrolyze the sample by 3M water solution HCl. Then neutralize solution (pH=7) by NaOH and isolate by filtration. Pipette out in a conical flask the Benedict’s quantitative reagent. Add Na2CO3 and a few porcelain chips to the flask to prevent bumping. Allow it to boil for 2-3 minutes more and add filtrate drop by drop till the solution becomes colourless. Note down the volume of the filtrate used.