Difference between revisions of "Team:CLSB-UK/Project/Synechocystis"

 
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<li style="background-color:#000000"> <a href="https://2016.igem.org/Team:CLSB-UK/Project/BPV_Cell">
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<h2> Synechocystis </h2>
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<h2> Engineering <i>Synechocystis</i> </h2>
  
 
<h4> Overview </h4>
 
<h4> Overview </h4>
  
 
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Synechocystis PCC 6803 is a species of cyanobacteria which is used as a model organism in numerous different areas of biology. Its genome was one of the first to be completely sequenced and is freely available online, initially on Cyanobase but now in many places. The functions of a significant proportion of its genes are understood. As such, it is simple enough to target genes that code for particular functions and it is possible to alter specific systems with considerable precision. Together with the fact that synechocystis is fairly easy to transform, this makes it an ideal organism for use in synthetic biology and it has attracted substantial interest in the last few years as a potential producer of biofuels. Nonetheless, its use is not particularly widespread in iGEM and there are very few parts that are specific to it. Therefore any team that chooses to use it faces substantial obstacles, such as the lack of suitable plasmids, promoters and ribosome binding sites.
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Our genetic approach to improving the efficiency of BPVs is to increase the rate at which electrons released in photolysis are exported to the anode, and the two methods by which we attempt to do so can be grouped into two categories:
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<li>The synthesis of lipid soluble redox mediators</li>
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<li>The production of membrane proteins capable of transporting electrons</li>
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Along the way, we encountered several problems with using <i>Synechocystis</i> PCC sp. 6803 as a model organism, so we targeted some of our modification at making it easier to modify.
 
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<img src="https://static.igem.org/mediawiki/2016/6/6d/T--CLSB-UK--Synechocystis.jpg">
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<span class="label"><b>Figure 1.</b>Our culture of <i>Synechocystis</i> PCC sp. 6803</span>
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<span class="label"><b>Figure 1.</b><i>Synechocystis</i> PCC6803 growing on BG11 medium.</span>
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<h4> Photosynthesis </h4>
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<h4> Redox Mediators </h4>
  
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<p>Redox mediators are compounds that can exist in oxidised and reduced states, enabling them to transport electrons and deposit them. Lipid soluble redox mediators are able to transfer electrons across the lipid bilayer by taking electrons in on one side of the membrane and releasing them on the other side. Adding lipid soluble redox mediators have been shown to increase the efficiency, so we looked to engineer cyanobacteria to produce their own lipid soluble mediators in large enough quantities to increase exoelectrogenesis efficiency themselves. The redox mediators we looked at are quinones, phenazines and riboflavin. Quinones require a very complicated metabolic pathway for their synthesis and the synthesis of phenazines has eluded <a href="https://2013.igem.org/Team:Bielefeld-Germany/Project/Phenazine">iGEM teams</a> in the past. However we were able to make significant headway in our attempts to modify <i>Synechocystis</i> PCC sp. 6803 to produce <a href="https://2016.igem.org/Team:CLSB-UK/Project/Riboflavin">Riboflavin</a> this year.</p>
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Cyanobacteria are the only known prokaryotes that carry out plant-like oxygenic photosynthesis. It is thought that plants’ chloroplasts are, in fact, derived from endosymbiotic cyanobacteria. However, photosynthesis in cyanobacteria is unusual in several respects. In particular, the cellular machinery involved in it is organized in an atypical manner. In cyanobacteria, the electron transport chains for photosynthesis and respiration are both embedded in the same membrane system. Electrons are shuttled back and forth between the two electron transport chains and various components are involved in both processes. These are: Plastoquinone (PQ), the cytochrome b­<sub>6</sub>f complex, Plastocyanin (PC) and cytochrome c533. These act much like switches on a railway, shifting electrons between the two electron transport chains.  
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<h4> Inefficiencies </h4>
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<h4> Membrane Proteins </h4>
  
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<p>Another way to enable the transfer of electrons from the general metabolism of the cell to the outside is to alter the membrane of the cell by adding proteins that allow this electron transfer to take place. The two that we looked at in some detail are C type cytochromes and porins. However Cytochromes, as well as being part of an extensive operon themselves, require several other to add the heme groups necessary for their function, and we deemed it too difficult to attempt to overproduce them. On the other hand a part for our porin OprF is available through the registry so we made significant advances using this <a href="https://2016.igem.org/Team:CLSB-UK/Project/Porins">porin</a>.</p>
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The process of photosynthesis itself is much the same as in plants. Importantly, it suffers from similar shortcomings. In particular, Rubisco is the central carbon-fixing enzyme in cyanobacteria as in plants. Rubisco is, of course, very inefficient as it has a high affinity for O2 as well as CO2. In fact, cyanobacteria have evolved a specific mechanism to combat this problem. Rubisco is sheltered within intracellular compartments called carboxysomes that concentrate CO2 and prevent oxygenation. It is currently thought that the carboxysome acts as a diffusion barrier to CO2, preventing CO2 from escaping and excluding O2. However, the carboxysome evolved millions of years ago and so is not fully adapted to modern atmospheric conditions. This means that it operates at sub-optimum efficiency. Thus, photosynthesis is not as efficient as it might be.
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<h4> General <i>Synechocystis</i> improvements</h4>
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<p>The three main problems we encountered in using <i>Synechocystis</i> PCC sp. 6803 were its slow growth rate, its wildly varying ploidy, and the lack of available Biobricks for use with it as a model organism. We attempted to tackle all of these problems alongside improving the electron export. We had huge success synthesising and expressing the <a href="https://2016.igem.org/Team:CLSB-UK/Project/CmpA">bicarbonate transporter CmpA</a>, which markedly improved the growth rate of the bacteria. We also were able to characterize the RFP <a href="https://2016.igem.org/Team:CLSB-UK/Project/AmilCP">AmilCP</a> in <i>Synechocystis</i> PCC sp. 6803 and to conclude that in fact it is not a useful reporter protein for this model organism. We found the <a href="https://2016.igem.org/Team:CLSB-UK/Project/pDF">replicative plasmid pDF-lac</a> and used it to circumvent the polyploidy issues with great success.
 
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There are three other major rate-limiting factors that affect photosynthesis in Synechocystis: the availability of PQ; the availability of PC; and the availability of Ferredoxin/ NADP. The first two factors affect Photosystem I; the latter affects Photosystem II. However, there is no quick or simple way to increase the availability of any of these. Moreover, since PQ and PC are also involved in respiration, for which they are also rate-limiting factor, any gains would be split between the two processes. Thus, the best way to increased the efficiency of photosynthesis in synechocystis is to increase the availability of CO2 to Rubisco, which is the weakest link in photosynthesis.
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We used the promoter BBa_J23119, a consensus sequence in <i>E.coli</i><a href="https://2016.igem.org/wiki/index.php?title=Team:CLSB-UK/Project/Synechocystis/GM#ref"><sup>[1]</sup></a>. We hoped that it would be a very strong promoter when binding to the similar (but not identical) RNA polymerase in <i>Synechocystis</i>. However, whilst we observed expected results in E.coli, we were not able to characterize this promoter in <i>Synechocystis</i> due to the presence of a promoter region already present in our replicative plasmid (the genes were expressed regardless of the strength of the promoter).</p>
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<h4> Problems </h4>
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<h4 id="ref"> References </h4>
  
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<ul><li>[1] http://parts.igem.org/Part:BBa_J23119</li></ul>
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Synechocystis is rarely used in Genetic Modification and there are very few BioBricks available for use in this chassis. There are several reasons for this. Firstly, unlike E.coli, Synechocystis 6803 is not monoploid. In fact its ploidy (number of copies of its chromosome) is entirely dependent on conditions, varying from 1 copy in prolonged Phosphorus-deficient environments to over 50 copies! [1]
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Equally the growth rate is very low. The maximum recorded growth rate of Synechocystis gives a doubling time of 5.13 hours and this is only achieved with almost hourly dilutions to preserve the exponential growth stage, otherwise Synechocystis rapidly reaches a steady population size. [2]
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Despite these two problems, there is no better model cyanobacterial organism, and this means that routine Genetic Modifications that require cyanobacteria are rendered difficult so that many iGEM teams are put off from attempting to harness the potential offered by photosynthesizing bacteria. In light of this iGEM CLSB UK decided to take up the task of improving the ability of Synechocystis PCC 6803 in conjunction with our task to improve BPVs.
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<p>[1](The ploidy level of Synechocystis sp. PCC 6803 is highly variable and is influenced by growth phase and by chemical and physical external parameters.
 
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Zerulla K1, Ludt K1, Soppa J1.)
 
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[2]Characterization of a model cyanobacterium Synechocystis sp. PCC 6803 autotrophic growth in a flat-panel photobioreactor
 
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Authors: Tomáš Zavřel, Maria A. Sinetova, Diana Búzová, Petra Literáková, Jan Červený</p>
 
 
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Latest revision as of 12:55, 18 October 2016

Click on different parts on the picture to find out more!

Engineering Synechocystis

Overview

Our genetic approach to improving the efficiency of BPVs is to increase the rate at which electrons released in photolysis are exported to the anode, and the two methods by which we attempt to do so can be grouped into two categories:

  • The synthesis of lipid soluble redox mediators
  • The production of membrane proteins capable of transporting electrons

Along the way, we encountered several problems with using Synechocystis PCC sp. 6803 as a model organism, so we targeted some of our modification at making it easier to modify.

Figure 1.Our culture of Synechocystis PCC sp. 6803

Redox Mediators

Redox mediators are compounds that can exist in oxidised and reduced states, enabling them to transport electrons and deposit them. Lipid soluble redox mediators are able to transfer electrons across the lipid bilayer by taking electrons in on one side of the membrane and releasing them on the other side. Adding lipid soluble redox mediators have been shown to increase the efficiency, so we looked to engineer cyanobacteria to produce their own lipid soluble mediators in large enough quantities to increase exoelectrogenesis efficiency themselves. The redox mediators we looked at are quinones, phenazines and riboflavin. Quinones require a very complicated metabolic pathway for their synthesis and the synthesis of phenazines has eluded iGEM teams in the past. However we were able to make significant headway in our attempts to modify Synechocystis PCC sp. 6803 to produce Riboflavin this year.

Membrane Proteins

Another way to enable the transfer of electrons from the general metabolism of the cell to the outside is to alter the membrane of the cell by adding proteins that allow this electron transfer to take place. The two that we looked at in some detail are C type cytochromes and porins. However Cytochromes, as well as being part of an extensive operon themselves, require several other to add the heme groups necessary for their function, and we deemed it too difficult to attempt to overproduce them. On the other hand a part for our porin OprF is available through the registry so we made significant advances using this porin.

General Synechocystis improvements

The three main problems we encountered in using Synechocystis PCC sp. 6803 were its slow growth rate, its wildly varying ploidy, and the lack of available Biobricks for use with it as a model organism. We attempted to tackle all of these problems alongside improving the electron export. We had huge success synthesising and expressing the bicarbonate transporter CmpA, which markedly improved the growth rate of the bacteria. We also were able to characterize the RFP AmilCP in Synechocystis PCC sp. 6803 and to conclude that in fact it is not a useful reporter protein for this model organism. We found the replicative plasmid pDF-lac and used it to circumvent the polyploidy issues with great success.

We used the promoter BBa_J23119, a consensus sequence in E.coli[1]. We hoped that it would be a very strong promoter when binding to the similar (but not identical) RNA polymerase in Synechocystis. However, whilst we observed expected results in E.coli, we were not able to characterize this promoter in Synechocystis due to the presence of a promoter region already present in our replicative plasmid (the genes were expressed regardless of the strength of the promoter).

References

  • [1] http://parts.igem.org/Part:BBa_J23119