Team:Reading/Experiments

Experiments

This section documents the designs and protocols for experiments we carried out during our project

Competition assay

The aim of the competition assay was to determine which bacteria strain, either triple mutant or wild type was fitter and therefore more able to compete for nutrients and resources. This gives us an indication of how one strain may grow compared to another and to see the impact of the modifications made to the mutant strain. This also allows us to consider the safety of the use of the mutant, by providing insight into how well the mutant bacteria can survive in normal conditions if released into the wild when competing against wild type bacteria. This would allow us to consider the potential impacts of the accidental release of the mutant on the environment.

The process involved inoculating a single conical flask containing BG-11 medium with both the wild type and triple mutant to see which would survive in the flask and outcompete the other strain. This would be shown by then attempting to grow a loop of culture on kanamycin agar plates. This is used as the triple mutant contains the genes for kanamycin resistance, as is present on the plasmid whereas the wild type is susceptible to kanamycin as it lack the genes required for resistance and so would not grow on kanamycin resistance plates. The growth on the kanamycin plates (measured in colony forming units per ml (cfu/ml)) is then compared to the growth on a BG-11 plate without kanamycin, which gives an indication of the total number of cfu/ml from the culture. This gives an indication of which strain outcompeted the other.

The design of the experiment is as follows, observe aseptic technique throughout, 6803 refers to Synechocystis sp. PCC 6803:

  1. Add approx. 200ml of BG-11 medium to a sterile conical flask.
  2. Inoculate flask with 10µl of stock Wild type 6803, and 10µl of stock mutant 6803.
  3. Incubate flask in incubator on a shaker, at 58rpm and 30°C, under bright white light. Allow bacteria time to grow, monitoring the growth over time by observing the colour of the medium and by taking OD750 readings.
  4. Once the flask had reached a suitable cell density (e: g hundreds of millions of cells per ml), spread 1ml of culture over the surface of two plates, one of BG-11 and agar, and one which additionally contains the antibiotic Kanamycin (mutants should be marked with a Kanamycin resistance gene).
  5. Estimate the colony forming units per ml (cfu/ml) on each plate.
  6. The difference in cfu/ml on each plate will give an indication of which strain of 6803, wild type or mutant, will out-compete the other when placed in the same environment.

Ferricyanide assay to measure electron output of various strains of Synechocystis sp. PCC 6803

[Fe(CN)6]3- + e- ⇌ [Fe(CN)6]4-

Mountain View

  • Ferricyanide is a complex ion composed of an Fe3+ ion chelated by six CN- ions, which has an octahedral geometry, and a net charge of 3-. The structure of the ion is shown to the right.
  • Treatment of the ferricyanide ion with ferrous salts gives Prussian Blue (pigment)
  • Ferricyanide reduction measures activity of membraneous reductases
  • Ferricyanide oxidised by Fe3+ in the presence of Ferene-S (acidic conditions)
  • Chromogenic Ferene-S/Fe2+ complex is formed upon reduction, which has a net 4- charge. This reaction is shown above.

This reaction is used investigate the ability of the bacteria to reduce the external ferricyanide ion, so is a measure of the electron output from the cell. The terminal oxidases accept electrons as part of the electron transport chain as the final acceptor. Our triple mutant has been modified to remove the three terminal oxidases from the bacteria and so would theoretically mean more electrons are released out of the cell, instead of being transferred to an oxidase.

The assay is used to determine how many electrons are being released by the cell. We shall be using potassium ferricyanide as a membrane-impermeable electron acceptor. The ferricyanide is reduced in the presence of electrons and will produce a colour change, which can then be measured by using a spectrophotometer. The extent of this colour change can then be used to show the number of electrons being liberated from the cell surface and therefore can give us an indication of the efficiency of the bacteria in producing electrons. This would be compared then to the wild type as a control. This can be compared to the voltage readings from the fuel cell using the triple mutant as well as other mutants with different mutations.

The method for the assay is as follows.

  • Harvest cells from log phase (0.4 - 0.6 a.u).
  • Centrifuge the cells to produce supernatant by centrifugation at 1800g using a low speed centrifuge for a few seconds.
  • This will produce a supernatant which is then extracted and is resuspended in a flask with 2.4ml of BG-11 3µl of 1mM Potassium Ferricyanide in then added and the flask is then filled to 3ml with BG-11.
  • The spectrophotometer is blanked using 1ml of BG-11, which is added to a cuvette and the value is zeroed.
  • 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
  • This is repeated at 680nm and 750nm using a fresh sample each time and zeroing the sample prior to use.
  • 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.
  • The data is measured four times over 12 hours at 8am, 12pm, 4pm and 8pm.
  • Ferricyanide concentrations were calculated from the absorbance at 420 nm of the supernatant (1 Au = 1 mM).
  • Used to determine rate of ferricyanide reduction in pmol [Fe(CN)6]3− min−1.

    • Public perception and awareness study: Methods and Questionaire

    References
    • 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).
    • National Center for Biotechnology Information. PubChem Compound Database; CID=439210, https://pubchem.ncbi.nlm.nih.gov/compound/439210 (accessed Aug. 25, 2015).
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