Team:Reading/Protocols

Protocols

Note: This section is an account of all laboratory protocols used by the team this year. The references at the bottom of the page provide the sources for the protocols, however we have modified several of them to our needs.

Growth of Synechocystis sp. PCC 6803

  1. Thaw stock of 6803 on ice
  2. 100ml of BG-11 medium added to sterile conical flask aseptically
  3. Add 10µl or 5 loopfuls of 6803 to flask
  4. Incubate at ~30°C and on shaker at ~58rpm, under constant illumination
  5. Check growth each day by measuring OD750

Taking optical density of culture to measure growth
We measured the OD750 to match that used in research papers on Synechocystis1.

  1. Set spectrophotometer to measure at OD750
  2. Blank with 1ml of BG-11
  3. Take OD750 of 1ml of culture
  4. If OD750 over 1 a.u, dilute 250µl of culture in 750µl of BG-11 (1/4 dilution)
To calculate cell density, we used the equation, 1 a.u = 1.6x108 cells per ml of culture2.

Solar water disinfection (SODIS)
SODIS is an extremely cheap and simple method of sterilising water, requiring only sunlight, and Polyethylene terephthalate (PET) plastic bottles3. We used water from Whiteknights lakeon the University of Reading campus.

  1. Pass water through a filter into a PET bottle to remove particulates
  2. Seal bottle and place in sunlight. Leave bottle exposed to intense sunlight for a minimum of 6 hours

Miniprep: Isolation of plasmid DNA from bacteria
We used the Thermo Scientific GeneJET Plasmid Miniprep Kit4 for our miniprep. This protocol closely follows protocol A, which is detailed in the booklet included in the kit.

  1. Pellet cells from 1.5ml of culture by centrifugation at 4000rpm for 2 minutes, and remove as much supernatant as possible and discard, leaving only the pellet.
  2. Resuspend pellet in 250µl of Resuspension Solution by vortexing breifly until pellet no longer visable.
  3. Lyse cells by adding 250µl of Lysis Solution and mixing thoroughly by inverting the tube 4-6 times until solution becomes viscous and clear
  4. Add 350µl of Neutralisation Solution and mix immediately and gently until solution becomes cloudy.
  5. Centrifuge for 5 minutes at 12,000rpm
  6. Transfer supernatant to a GeneJET spin column. Avoid disturbing white precipitate.
  7. Centrifuge for 1 minute at 12,000rpm and discard the flow-through.
  8. Add 500µl of Wash Solution to GeneJET spin column, centrifuge for 1 minute at 12,000rpm, and discard flow-through.
  9. Repeat step 8.
  10. Discard flow-through and centrifuge for 1 minute at 12,000rpm to remove residual Wash Solution.
  11. Transfer GeneJET spin column to a fresh 1.5ml microcentrifuge tube. Add 50µl of Elution Buffer, being careful not to touch the membrane with the pipette.
  12. Incubate at room temperature for 2 minutes and then centrifuge for 2 minutes at 12,000rpm
  13. Discard GeneJET spin column and store purified plasmid DNA at -20°C.

BG-11 Agar plates

BG-11 plates are used to grow Synechocystis sp. PCC 6803 strains. Antibiotics are added as needed for selection. 1.5% agar is used.

  1. Add 7.55 g of agar to 500 ml BG-11 and autoclave to sterilise
  2. If making kanamycin plates cool to ~55℃ and add 50 ug/ml
  3. Agar melted in steamer and kept at 55℃ until needed for pouring

If making a kanamycin cap:

  1. 0.6% agar w/v
  2. Cool BG-11 to ~55ºC and add 0.5mg/ml kan
  3. Add ~3ml to a plain BG-11 plate with transformed colonies on it
  4. Leave for >1 week for Kan selection to occur

Old Ferricyanide Method
  1. Harvest cells from log phase (0.4 - 0.6 a.u)
  2. Centrifuge the cells to produce supernatant by centrifugation at 1800g using a low speed centrifuge for a few seconds.
  3. This will produce a supernatant which is then extracted and is resuspended in a flask with 2.4ml of BG-11
  4. 1ml of 1mM Potassium Ferricyanide in then added and the flask is then filled to 100ml with BG-11
  5. Repeat this for the another flask. Wrap this in aluminium foil to block out light.
  6. The cells are then pelleted at 14000g for 5 minutes. The cells are then resuspended in 2.4ml of BG-11 and then filled to 3ml without adding the Potassium Ferricyanide.
  1. Resuspend in 2.4ml of BG-11 and add 3µl of Potassium Ferricyanide and fill to 3ml of BG-11.
  2. To a fresh cuvette 1ml of sample is added and then placed in the spectrophotometer and the value is recorded at a wavelength of 420nm
  3. This is repeated at 680nm and 750nm using a fresh sample each time and zeroing the sample prior to use.
  4. The data is measured four times over 9.30am, 1230pm, 2.30pm and 5.30pm.
  5. Ferricyanide concentrations were calculated from the absorbance at 420 nm of the supernatant (1 Au = 1 mM).
  6. Used to determine rate of ferricyanide reduction in pmol [Fe(CN)6]3− min−1.
Revised Ferricyanide protocol- TW-L
  1. Harvest cells from log phase (0.4 - 0.6 a.u)
  2. Centrifuge the cells to produce supernatant by centrifugation at 1800g/4725rpm using a low speed centrifuge for a few seconds.
  3. Dispose of supernatant, leaving behind a dark green pellet
  4. Add 2ml of potassium ferricyanide
  5. 4 measurements every 2 hours at least
  6. Ferricyanide concentrations calculated from abs of 420nm of the supernatent (1abs = 1 mM)
    7. Before every measurement:
  1. Remove 1ml of solution from the desired flask and place in a 1.5ml Eppendorff tube
  2. Centrifuge at 10200 rpm for 5 minutes
  3. Remove the supernatant and pipette it into the cuvette
  4. Measure the sample at 420, 680, and 750nm
  5. Dispose of the cuvettes
References
  1. Bradley, R. W., Bombelli, P., Lea-Smith, D. J. & Howe, C. J. Terminal oxidase mutants of the cyanobacterium Synechocystis sp. PCC 6803 show increased electrogenic activity in biological photo-voltaic systems. Phys. Chem. Chem. Phys. PCCP 15, 13611–13618 (2013).
  2. Pojidaeva E, Zichenko V, Shestakov SV, Sokolenko A (2004) Involvement of the SppA1 peptidase in acclimation to saturating light intensities in Synechocystis sp. strain PCC 6803. J Bacteriol 186: 3991–3999.
  3. SODIS. 2015. SODIS Method, Available at: href="http://www.sodis.ch/methode/index_EN
  4. ThermoScientific. 2013. Thermo Scientific GeneJET PCR Purification Kit #K0701, #K0702. [Online] Available at: https://www.lifetechnologies.com/order/catalog/product/K0502?ICID=search-k0502
  5. Bradley, R. W., Bombelli, P., Lea-Smith, D. J. & Howe, C. J. Terminal oxidase mutants of the cyanobacterium Synechocystis sp. PCC 6803 show increased electrogenic activity in biological photo-voltaic systems. Phys. Chem. Chem. Phys. PCCP 15, 13611–13618 (2013). http://pubs.rsc.org/en/content/articlehtml/2013/cp/c3cp52438h
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