Difference between revisions of "Team:Reading/Transformations"

Genetic modifications, with the aim to increase the number of electrons transfered to the anode in our fuel cell, are a major part of our project. Here we detail the transformations we plan to carry out with Synechocystis sp. PCC 6803, and the modifications these will produce.

Bacterial nanowires are electrical conductive protein filaments⁶, which are a form of modified pili that allow electrons to travel away from the cell surface to reduce the cells surroundings. Nanowires were first identified in the bacterium Geobacter sulfurreducens¹, and when the gene PilA1, (coding for pilin, the protein subunit of pili), was deleted, G. sulfurreducens became unable to reduce Fe³⁺ and Mn⁴⁺ in its surroundings¹.

Since their initial discovery, bacterial nanowires have been observed in several species of bacteria, including Synechocystis sp. PCC 6803², which holds much importance for our project.

Much research has been done on nanowires, however mainly in Geobacter species, and there has been little research into how nanowires function in Synechocystis sp. PCC 6803. Because of this, the method by which Synechocystis sp. PCC 6803 transfers electrons to the electrodes in biological photovoltaic devices is very much unknown; endogenous mediators such as Quinones or Flavins may be being produced to shuttle electrons, or nanowires may be providing the major route of electron transportation in Synechocystis sp. PCC 6803³. This casts doubt over the benefits of attempting to modify or induce the over-expression of nanowires in Synechocystis sp. PCC 6803.

We plan to induce hyperpilation in Synechocystis sp. PCC 6803, as we believe this will have several benefits to our photovoltaic even without the added bonus of bacterial nanowires. Hyperpilation may promote the formation of biofilms on the electrode surfaces in our fuel cell, and will aid adhesion of bacterial cells to the carbon fibre surface. We plan to induce hyperpilation by deleting the gene pilT1, an ATPase which controls pilus retaction⁵. When deleted, the bacteria exhibit more numerous long surface pili⁴. We also shall modify Synechocystis sp. PCC 6803 by inserting additional copies of the pilA1 gene, which codes for pilin, the pilus subunit, so that pilA1 is responsible for the biogenesis of pili⁵. The insertion of pilA1 will induce hyperpilation in the bacterium, and if the nanowires in Synechocystis sp. PCC 6803 are formed and function in a similar manner to those in G. Sulfurreducens, should also induce the production of more bacterial nanowires, which will increase the current of electrons from the bacteria to the anode in our fuel cell.

The photosynthetic apparatus in cyanobacteria uses the energy from light to produce high energy electrons for electron transportation. These electrons are themselves supplied by the photolysis of water at the oxygen evolving complex on PSII, so the electron current through the photosynthetic electron transport chain (PETC), and the electrons being supplied to the anode in our fuel cell are all derived from the oxidation of water⁷.

Electrons which are transferred out of the cell to the anode exit from the plastoquinone pool, as well as the soluble electron carriers of the PETC and the respiratory electron transport chain (RETC). To increase the number of electrons being transfered to the anode from the plastoquinone pool, loss to electron ‘sinks’ must be prevented. The primary electron sink from the PETC is from Ferredoxin NADP reductase (FNR). Inactivation of this complex would force cyclic photophosphorylation to occur, in which electrons liberated from PSI are transferred to ferredoxin, and then back to the plastoquinone pool⁸ via ferredoxin-quinone reductase⁹. This would be advantageous as it would greatly increase the number of electrons available to be transferred to the anode. However, complete knockout of FNR would be lethal, as this removes the cells primary method of reforming NADPH, so starves the cell of reducing power.

Instead we must look to removing the terminal oxidases, which are the other major electron sinks in the photosynthetic and respiratory metabolism of cyanobacteria. The three major terminal oxidases⁹, we shall consider are:

• COX – (Cytochrome c oxidase) – located on the thylakoid membrane. It oxidises plastocyanin and Cytochrome c₆ from the lumen of the cell.
• Cyd – (bd – Quinol oxidase) – located on both the cytoplasmic and thylakoid membranes. It oxidises the plastoquinone pool directly.
• ARTO – (Alternative respiratory terminal oxidase) – located on the cytoplasmic membrane. It oxidises the plastoquinone pool directly.

These complexes all oxidise the plastoquinone pool either directly or indirectly, and sink electrons by combing them with protons and oxygen to evolve water⁹. We plan to attempt knockouts of the genes which code for these oxidases^{9 10}. The genes we plan to knockout are:

• COX subunit genes: ctaC, ctaD, ctaE
• Cyd genes: cydA, cydB
• ARTO subunit 2 gene: ctaCII

These knockouts have been carried out in the past^{9 10}, and do not affect the viability of the cells. The normal function of the terminal oxidases in cyanobacterium is to oxidise the plastoquinone pool to prevent oxidative damage due to high light intensities⁹, and are known to be important for Synechocystis sp. PCC 6803 to adapt to and survive rapidly changing light intensities¹⁰. However in our cell electrons will be lost from the plastoquinone pool to the anode, so there should not be excessive oxidative damage due to a heavily reduced plastoquinone pool.

1. Reguera, G. et al. Extracellular electron transfer via microbial nanowires. Nature 435, 1098–1101 (2005).
2. Gorby, Y. A. et al. Electrically conductive bacterial nanowires produced by Shewanella oneidensis strain MR-1 and other microorganisms. Proc. Natl. Acad. Sci. 103, 11358–11363 (2006).
3. Bradley, R. W., Bombelli, P., Rowden, S. J. L. & Howe, C. J. Biological photovoltaics: intra- and extra-cellular electron transport by cyanobacteria. Biochem. Soc. Trans. 40, 1302–1307 (2012).
4. Okamoto, S. & Ohmori, M. The Cyanobacterial PilT Protein Responsible for Cell Motility and Transformation Hydrolyzes ATP. Plant Cell Physiol. 43, 1127–1136 (2002).
5. Yoshihara, S. & Ikeuchi, M. Phototactic motility in the unicellular cyanobacterium Synechocystis sp. PCC 6803. Photochem. Photobiol. Sci. 3, 512–518 (2004).
6. Malvankar, N. S. & Lovley, D. R. Microbial Nanowires: A New Paradigm for Biological Electron Transfer and Bioelectronics. ChemSusChem 5, 1039–1046 (2012).
7. Cereda, A. et al. A Bioelectrochemical Approach to Characterize Extracellular Electron Transfer by Synechocystis sp. PCC6803. PLoS ONE 9, (2014).
8. Vermaas, W. F. Photosynthesis and Respiration in Cyanobacteria. (2001).
9. 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).
10. Lea-Smith, D. J. Ross, N. Zori, M. Bendall, D. S. Dennis, J. S. Scott, S. A. Smith, A. G. Howe, C. J. Thylakoid terminal oxidases are essential for the cyanobacterium Synechocystis sp. PCC 6803 to survive rapidly changing light intensities. Plant physiol. 162, 484-495 (2013).

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