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

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<h2> A Brief Introduction to Synechocystis </h2>
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<h2> Synechocystis </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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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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<b>Overview</b>
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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>Synechocystis PCC6803 growing on BG11 medium.</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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<b>Photosynthesis</b>
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<h4> Photosynthesis </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 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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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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<h4> Inefficiencies </h4>
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Aside from this important difference, the process of 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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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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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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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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<h4> Problems </h4>
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<b>Genetic modification and practical problems</b>
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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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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 method 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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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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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. It is also worth briefly mentioning that synechocstis is easy to store. It can be stored under usual lab conditions at a temperature of 1-2 degrees. Most labs will have the facilities to do his and there should be few problems with storage.  
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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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Revision as of 19:39, 13 October 2016

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

Synechocystis

Overview

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.


Figure 1.Synechocystis PCC6803 growing on BG11 medium.

Photosynthesis

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.

Inefficiencies

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.

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.

Problems

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]

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]

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.

[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. Zerulla K1, Ludt K1, Soppa J1.) [2]Characterization of a model cyanobacterium Synechocystis sp. PCC 6803 autotrophic growth in a flat-panel photobioreactor Authors: Tomáš Zavřel, Maria A. Sinetova, Diana Búzová, Petra Literáková, Jan Červený