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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<div style="background-color:#000000"> Zoom&nbsp;Out </div>
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<h2> A Brief Introduction to Synechocystis </h2>
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<h2> Engineering <i>Synechocystis</i> </h2>
  
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<h4> Overview </h4>
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This is a short but wide-ranging introduction to synechocystis and cyanobacteria in general. It focuses on those points that are relevant to our project and outlines the basics of photosynthesis and genetic modification in cyanobacteria. It also sketches some of the problems that we faced with using synechocystis as our organism (rather than, say, E.coli). This may be helpful to other teams. Certainly, we encountered numerous problems and were often forced to learn by trial and error. Perhaps future teams may avoid similar mistakes by reading this.
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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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<b>Overview</b>
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<li>The synthesis of lipid soluble redox mediators</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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Synechocystis PCC 6803 is a common 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. Several repositories exist, of which the first was CyanoBase. Moreover, 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. Consequently, 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. As such, 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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<span class="label"><b>Figure 1.</b>Our culture of <i>Synechocystis</i> PCC sp. 6803</span>
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<b>Photosynthesis</b>
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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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<h4> Membrane Proteins </h4>
  
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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 b6f 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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<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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<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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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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Aside from this important difference, photosynthesis in cyanobacteria is similar to photosynthesis 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 act as a 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 id="ref"> References </h4>
  
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<ul><li>[1] http://parts.igem.org/Part:BBa_J23119</li></ul>
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Besides the inefficiency of Rubisco, there are three other major rate-limiting factors that affect photosynthesis in synechocystis. First, the availability of PQ. Second, the availability of PC. Third, 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 factors, any gains would be split between the two processes. Thus, the best way to increase 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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<b>Genetic modification and practical problems</b>
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It is relatively simple, in theory, to genetically modify synechocystis since it is naturally able to integrate exogenous DNA into its genome by homologous recombination. However, there are several practical problems associated with this process. First, synechocystis is dodecaploid. All twelve of its chromosomes must take up any foreign DNA in order for it to be fully integrated into the genome. This is a time-consuming process; It is easier to insert replicative rather than integrative plasmids into the organism. The most effective methdo for doing this is natural transformation. Of course, any genes introduced into the organism by this method cannot be passed on to future generations. Nonetheless, for projects that are time-constrained, this is a more sensible approach. It means that one can establish whether or not a particular alteration is effective without having to wait a long time. Since our project only required a proof of concept, we opted to use a replicative plasmid to insert DNA into our synechocystis samples. The lack of suitable vectors meant that our plasmid was not, in fact, BioBrick compatible – though there are some suitable plasmids listed in the registry, none of them were actually available to use.
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The second major difficulty with synechocystis concerns its rate of growth. Synechocystis grow particularly slowly and so is not ideally suited to experiments with a particular time limit. This is a serious problem and we decided to try and address it by over-expressing the CmpA gene, which is involved in bicarbonate transport (see here for more detail). It has been shown that the introduction of additional bicarbonate transporters leads to a two-fold increase in carbon uptake, growth and biomass production. We hoped to achieve a similar effect by specific, carefully-targeted modification of the bicarbonate transporters. A detailed explanation of why more efficient bicarbonate transport leads to a faster rate of growth can be found on the CmpA page.
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Synechocystis must also be stored under very specific conditions. In particular, it must be kept at a constant temperature of around -80C. Though many labs will have facilities capable of achieving such temperatures, some will not. We had to make use of Imperial College, London’s superior technology to store our cultures. Those teams that do not possess such facilities themselves (mostly high school teams should be aware of this.
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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