Team:Copenhagen/Project/Cyanobacteria

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<table id="graa" cellpadding=20px><td width="900px" height="100%" valign="top" ><p align="justify"><h2>How is it sustainable?</h2>
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<table cellpadding=20px><td width="660px" height="100%" valign="top" ><p align="justify"><h2>Cyano Bacteria</h2>
 
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Background information
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The sustainability of our system lies with the type of bacteria that we use – cyanobacteria (Synechococcus spp.). These bacteria have a metabolism that is uncommon among bacteria as they, just like plants, perform photosynthesis. This means that they convert CO<sub>2</sub> into sugar and oxygen which has at least two great advantages:
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1) The bacteria use CO<sub>2</sub> as their sole source of carbon and thus one does not need to feed them any sugar – they make the sugars they need themselves – as long as they have access to CO<sub>2</sub>. Thus, they are both easy and cheap to culture. <br><br>
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2) Their consumption of CO<sub>2</sub> reduces the abundance of this gas in the atmosphere and thus they help in the reversion of the development of global warming. The metabolism of these bacteria makes CyanoDelux a sustainable light source. The bacteria should be able to grow (and produce light each night) for long periods of time, only in the presence of salt water (which is their natural habitat) and with access to atmospheric air.
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<b>So, how will we do it?</b>
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We want to take advantage of bioluminescence, a biochemical process that naturally occurs in several organisms, including a range of bacteria, but that does not normally occur in cyanobacteria. In many organisms, bioluminescence is due to the activity of the enzyme luciferase which catalyzes a redox reaction involving three substrates and which, as a byproduct, produces blue-green light (~490 nm). The luciferase enzyme is encoded on the lux operon in bacteria, which in its minimal form comprises five genes: LuxC,-D,-A,-B, and –E, of which A and B encode the two subunits of the luciferase and CDE accessory proteins that are needed to restore the substrates of the enzyme. We wish to genetically engineer Synechococcus Elongatus Pcc 7002 cyanobacteria so that they become bioluminescent only when it is dark. This we plan to accomplish using two different approaches, involving two independent gene constructs which both include the luxCDABE cassette.
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<b>Our constructs</b>
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Cyanobacteria follow a circadian rhythm which means that there are differences in their activities in the day compared to in the night. Therefore, the expression levels of certain genes vary depending on the time of day. We wanted to take advantage of this phenomenon by choosing a promoter that is especially active during night and inactive during the day and put it in front of the luxCDABE cassette (kindly provided by T. Knight). As such that the lux genes are only transcribed and the bacteria only produce light during night. We chose the promoter lrtA from Synechocystis sp. PCC 6803 (BBa_K390008) which is associated with the electron transport chain of photosynthesis in this organism. When it is dark and photosynthesis is shut off the electron acceptors of the electron transport chain are in their reduced state and the lrtA promoter is active. But in the presence of light, when photosynthesis is turned on, the promoter is inactive.
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<table id="graa"><tr><img src="https://static.igem.org/mediawiki/2012/e/e7/Ec.JPG" width="600px" align="center" style="border:1px solid black;"></tr></table>
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In addition to this construct, we will also create a control construct with the luxCDABE cassette which will also be light-sensitive, but by a different mechanism which has already been developed and has been shown to be effective in E. coli (Ohlendorff et. al, 2012). Unlike with the lrtA promoter, we already know that this light-sensitive system should function (at least in E. coli) and therefore it serves as a control construct. The promoter ProC (BBa_J23100) drives constitutive expression of the two genes YF1 (BBa_K592004) and FixJ (BBa_K592005). YF1 is a fusion protein consisting of domains from two different proteins, a blue-light sensing domain and a histidine kinase domain. In the presence of light, the kinase activity of YF1 is repressed and in darkness this enzyme is active. Thus, during night is phosphorylates FixJ. In its phosphorylated form FixJ binds to the downstream promoter FixK2 (BBa_K592006) which activates it. The FixK2 promoter then drives expression of the luxCDABE cassette which, like with the experimental construct, leads to the production of light exclusively in darkness. The genes encoding FixJ and YF1 and the promoter FixK2 were all generously provided by the Uppsala 2011 iGEM team.
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<table id="graa"><img src="https://static.igem.org/mediawiki/2012/f/f6/Cc.JPG" width="600px" align="center" style="border:1px solid black;"></table>
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<b>Reference:</b>
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Ohlendorf, R., Vidavski, R., Eldar, A., Moffat, K., and Möglich, A.  2012.  From dusk till dawn: one-plasmid systems for light-regulated gene expression.  J. Mol. Biol.  416:534-42.
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<table cellpadding=20px><td width="660px" height="100%" valign="top" ><p align="justify"><h2>Bioluminescence</h2>
 
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Bioluminescence is light that is produced by a chemical reaction and then emitted from the living organism in which the reaction takes place.
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<table id="graa" cellpadding=20px><td width="900px" height="100%" valign="top" ><p align="justify"><h2>Bioluminescence</h2>
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One of the more known organisms to produce this kind of light are fireflies, but many other, such as bacteria, are able to emit light as well. Bioluminescence is used in nature for many different purposes. Some use it to scare of enemies, others to attract mates and still others use it as camouflage.
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Bioluminescence is light produced by a chemical reaction and subsequently emitted from the living organism in which the reaction takes place.  
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Luciferase is the enzyme responsible for the chemical reaction that produces light. Luciferase catalyzes the oxidation of the compound luciferin which results in an unstable intermediate that will emit light as it decays to its ground state.
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As in nature, bioluminescence can be used for many different purposes in biotechnology as well. A common use is different imaging methods and expression analyses.  
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One of the more known organisms to produce this kind of light are fireflies, but many other, such as bacteria, are able to emit light as well. Bioluminescence is used in nature for many different purposes. Some use it to scare of enemies, others as camouflage or even to attract mates.  
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A. fischeri are gram negative bacteria that can live in symbiosis with for example squid and thereby make them glow. The bacteria contain an operon which is a collection of genes that are all controlled by the same promoter and therefore expressed simultaneously. The Lux operon in A. fischeri contains the genes Lux A, Lux B, Lux C, Lux D and Lux E whose joint function is to produce the enzyme luciferase and regenerate the components necessary for the enzyme to work. The expression of the genes is controlled by a promotor that can be induced by many different stimulants for example a certain wavelength of light, a high colony concentration or the presence of a certain protein.
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Luciferase is the major light-producing enzyme. It catalyzes the oxidation of the compound luciferin into a unstable intermediate which by emitting light will decay into a favorable ground state.
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As in nature, bioluminescence has a broad range of applications in biotechnology as well. A common use is different imaging methods and expression analyses.  
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In our project the lux operon will be placed in the cyanobacteria PCC 7002 and a night induced promotor (name and biobrick number) will control the transcription and thereby the production of light.
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A. fischeri are gram negative bacteria that can live in symbiosis with for example squid and thereby make them glow. The bacteria contain an operon which is a collection of genes that are all controlled by the same promoter and therefore expressed simultaneously.  The Lux operon in A. fischeri contains the genes Lux A, Lux B, Lux C, Lux D and Lux E whose joint function is to produce the enzyme luciferase and regenerate the components necessary for the enzyme to work. The gene expression is controlled by an inducible-promotor meaning that different stimulants such as certain wavelength of light, a high colony concentration or the presence of a certain protein, can initiate the light production.
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In our project the lux operon will be placed in the cyanobacteria PCC 7002 and a night induced promotor lrtA
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<a href="http://partsregistry.org/Part:BBa_K390008" style="text-decoration:none; color:blue;"> BBa_K390008</a> will control the transcription and thereby the production of light.<br></p>
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Latest revision as of 11:40, 26 September 2012

How is it sustainable?

The sustainability of our system lies with the type of bacteria that we use – cyanobacteria (Synechococcus spp.). These bacteria have a metabolism that is uncommon among bacteria as they, just like plants, perform photosynthesis. This means that they convert CO2 into sugar and oxygen which has at least two great advantages:

1) The bacteria use CO2 as their sole source of carbon and thus one does not need to feed them any sugar – they make the sugars they need themselves – as long as they have access to CO2. Thus, they are both easy and cheap to culture.

2) Their consumption of CO2 reduces the abundance of this gas in the atmosphere and thus they help in the reversion of the development of global warming. The metabolism of these bacteria makes CyanoDelux a sustainable light source. The bacteria should be able to grow (and produce light each night) for long periods of time, only in the presence of salt water (which is their natural habitat) and with access to atmospheric air.

So, how will we do it?
We want to take advantage of bioluminescence, a biochemical process that naturally occurs in several organisms, including a range of bacteria, but that does not normally occur in cyanobacteria. In many organisms, bioluminescence is due to the activity of the enzyme luciferase which catalyzes a redox reaction involving three substrates and which, as a byproduct, produces blue-green light (~490 nm). The luciferase enzyme is encoded on the lux operon in bacteria, which in its minimal form comprises five genes: LuxC,-D,-A,-B, and –E, of which A and B encode the two subunits of the luciferase and CDE accessory proteins that are needed to restore the substrates of the enzyme. We wish to genetically engineer Synechococcus Elongatus Pcc 7002 cyanobacteria so that they become bioluminescent only when it is dark. This we plan to accomplish using two different approaches, involving two independent gene constructs which both include the luxCDABE cassette.

Our constructs
Cyanobacteria follow a circadian rhythm which means that there are differences in their activities in the day compared to in the night. Therefore, the expression levels of certain genes vary depending on the time of day. We wanted to take advantage of this phenomenon by choosing a promoter that is especially active during night and inactive during the day and put it in front of the luxCDABE cassette (kindly provided by T. Knight). As such that the lux genes are only transcribed and the bacteria only produce light during night. We chose the promoter lrtA from Synechocystis sp. PCC 6803 (BBa_K390008) which is associated with the electron transport chain of photosynthesis in this organism. When it is dark and photosynthesis is shut off the electron acceptors of the electron transport chain are in their reduced state and the lrtA promoter is active. But in the presence of light, when photosynthesis is turned on, the promoter is inactive.



In addition to this construct, we will also create a control construct with the luxCDABE cassette which will also be light-sensitive, but by a different mechanism which has already been developed and has been shown to be effective in E. coli (Ohlendorff et. al, 2012). Unlike with the lrtA promoter, we already know that this light-sensitive system should function (at least in E. coli) and therefore it serves as a control construct. The promoter ProC (BBa_J23100) drives constitutive expression of the two genes YF1 (BBa_K592004) and FixJ (BBa_K592005). YF1 is a fusion protein consisting of domains from two different proteins, a blue-light sensing domain and a histidine kinase domain. In the presence of light, the kinase activity of YF1 is repressed and in darkness this enzyme is active. Thus, during night is phosphorylates FixJ. In its phosphorylated form FixJ binds to the downstream promoter FixK2 (BBa_K592006) which activates it. The FixK2 promoter then drives expression of the luxCDABE cassette which, like with the experimental construct, leads to the production of light exclusively in darkness. The genes encoding FixJ and YF1 and the promoter FixK2 were all generously provided by the Uppsala 2011 iGEM team.


Reference:
Ohlendorf, R., Vidavski, R., Eldar, A., Moffat, K., and Möglich, A. 2012. From dusk till dawn: one-plasmid systems for light-regulated gene expression. J. Mol. Biol. 416:534-42.

Bioluminescence

Bioluminescence is light produced by a chemical reaction and subsequently emitted from the living organism in which the reaction takes place. One of the more known organisms to produce this kind of light are fireflies, but many other, such as bacteria, are able to emit light as well. Bioluminescence is used in nature for many different purposes. Some use it to scare of enemies, others as camouflage or even to attract mates. Luciferase is the major light-producing enzyme. It catalyzes the oxidation of the compound luciferin into a unstable intermediate which by emitting light will decay into a favorable ground state. As in nature, bioluminescence has a broad range of applications in biotechnology as well. A common use is different imaging methods and expression analyses. A. fischeri are gram negative bacteria that can live in symbiosis with for example squid and thereby make them glow. The bacteria contain an operon which is a collection of genes that are all controlled by the same promoter and therefore expressed simultaneously. The Lux operon in A. fischeri contains the genes Lux A, Lux B, Lux C, Lux D and Lux E whose joint function is to produce the enzyme luciferase and regenerate the components necessary for the enzyme to work. The gene expression is controlled by an inducible-promotor meaning that different stimulants such as certain wavelength of light, a high colony concentration or the presence of a certain protein, can initiate the light production. In our project the lux operon will be placed in the cyanobacteria PCC 7002 and a night induced promotor lrtA BBa_K390008 will control the transcription and thereby the production of light.

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