Team:DTU-Denmark/Regulatory sytems

From 2010.igem.org

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<li><a href="https://2010.igem.org/Team:DTU-Denmark/Regulatory_sytems#lambda">Lambda Phage</a></li>
<li><a href="https://2010.igem.org/Team:DTU-Denmark/Regulatory_sytems#lambda">Lambda Phage</a></li>
<li><a href="https://2010.igem.org/Team:DTU-Denmark/Regulatory_sytems#gifsy">Gifsy Phages</a></li>
<li><a href="https://2010.igem.org/Team:DTU-Denmark/Regulatory_sytems#gifsy">Gifsy Phages</a></li>
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<li><a href="https://2010.igem.org/Team:DTU-Denmark/Regulatory_sytems#antitermination">Anti-Termination systems</a></li>
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<li><a href="https://2010.igem.org/Team:DTU-Denmark/Switch#Applications">Applications</a></li>
<li><a href="https://2010.igem.org/Team:DTU-Denmark/Switch#Applications">Applications</a></li>
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<br><li><a href="https://2010.igem.org/Team:DTU-Denmark/SPL">Synthetic Promoter Library</a></li><br>
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<br><li><a href="https://2010.igem.org/Team:DTU-Denmark/SPL">Synthetic Promoter Library</a>
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<li ><a href="https://2010.igem.org/Team:DTU-Denmark/Modelling">Modelling</a>
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<ul><font size="2">
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<li><a href="https://2010.igem.org/Team:DTU-Denmark/Modelling#Introduction">Introduction</a></li>
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<li><a href="https://2010.igem.org/Team:DTU-Denmark/SPL#standard">The DTU SPL Standard</a>
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<li><a href="https://2010.igem.org/Team:DTU-Denmark/Modelling#Approach">Modelling Approach</a></li>
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<li><a href="https://2010.igem.org/Team:DTU-Denmark/Modelling#SPL">Modelling SPL</a></li>
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<li><a href="https://2010.igem.org/Team:DTU-Denmark/SPL#strategy">Strategy</a></li>
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<li><a href="https://2010.igem.org/Team:DTU-Denmark/Modelling#AntiRepressors">Modelling Anti-Repressors</a></li>
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<li><a href="https://2010.igem.org/Team:DTU-Denmark/SPL#design">Primer Design</a></li>
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<li><a href="https://2010.igem.org/Team:DTU-Denmark/SPL#protocol">Protocol</a></li>
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</ul>
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</li>
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<li><a href="https://2010.igem.org/Team:DTU-Denmark/SPL#advantages">Advantages</a></li>
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</li><br>
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<li ><a href="https://2010.igem.org/Team:DTU-Denmark/Modelling">Modeling</a>
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<ul><font size="2">
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<li><a href="https://2010.igem.org/Team:DTU-Denmark/Modelling#Approach">Modeling Approach</a></li>
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<a name="lambda"></a><h1>The lambda phage</h1>
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<p align="justify">The temperate bacteriophage lambda, <i>E. coli</i>, is the best-studied phage with regard to phage structure and regulation. Temperate phages are able to choose between the lytic cycle and the lysogenic cycle as described in Figure 1. </p>
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<b>The lambda phage</b><br><br>
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<a name="lambda"></a>
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<p align="justify">The temperate bacteriophage lambda found to infect <i>E. coli</i>, is the best-studied phage with regard to phage structure and regulation. Once lambda infects a cell, it can chose to either follow the lytic cycle or the lysogenic cycle described in Figure 1.</p>
<table class="http://upload.wikimedia.org/wikipedia/commons/5/5a/Phage2.JPG" align="center">
<table class="http://upload.wikimedia.org/wikipedia/commons/5/5a/Phage2.JPG" align="center">
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  <caption align="bottom"><p align="justify"><b>Figure 1</b>: The two distinct development pathways of a prophage life cycle [3].<br>In the lytic pathway the phage uses the bacterial molecular machinery to make many viral copies for infection of other cells before lysing the host bacterium. In contrast to the lytic pathway, the phage integrates its DNA into the bacterial genome in the lysogenic pathway. The lysogenic states is very stable, which means that the prophage can be replicated along with the bacterial genome for generations.<br>
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  <caption align="bottom"><p align="justify"><b>Figure 1</b>: The two distinct development pathways of a prophage life cycle [wikipedia].<br>In the lytic pathway the phage uses the bacterial molecular machinery to make many viral copies for infection of other cells before lysing the host bacterium. In contrast to the lytic pathway, the phage integrates its DNA into the bacterial genome in the lysogenic pathway. The lysogenic state is very stable, which means that the prophage can be replicated along with the bacterial genome for generations.<br>
Despite the stability of the lysogenic state, the lytic state is readily induced when the bacteria are irradiated with ultraviolet light.</p></caption>
Despite the stability of the lysogenic state, the lytic state is readily induced when the bacteria are irradiated with ultraviolet light.</p></caption>
<tr><td><img src="http://upload.wikimedia.org/wikipedia/commons/5/5a/Phage2.JPG"  width="400px"></td></tr>
<tr><td><img src="http://upload.wikimedia.org/wikipedia/commons/5/5a/Phage2.JPG"  width="400px"></td></tr>
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<p align="justify">The organization of the phage chromosome is shown in Figure 2.</p>
<p align="justify">The organization of the phage chromosome is shown in Figure 2.</p>
<table class="http://upload.wikimedia.org/wikipedia/commons/e/e4/Bacteriophage_lambda_genome.png" align="center">
<table class="http://upload.wikimedia.org/wikipedia/commons/e/e4/Bacteriophage_lambda_genome.png" align="center">
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  <caption align="bottom"><p align="justify"><b>Figure 2</b>: Schematic representation of the genome of the bacteriophage lambda [5].</p></caption>
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  <caption align="bottom"><p align="justify"><b>Figure 2</b>: Schematic representation of the genome of the bacteriophage lambda [wikipedia].</p></caption>
<tr><td><img src="http://upload.wikimedia.org/wikipedia/commons/e/e4/Bacteriophage_lambda_genome.png"  width="400px"></td></tr>
<tr><td><img src="http://upload.wikimedia.org/wikipedia/commons/e/e4/Bacteriophage_lambda_genome.png"  width="400px"></td></tr>
</table><br>
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<h3>The Lysogenic Pathway</h3>
<p align="justify">The rightward promoter/operator region in lambda prophages forms a switch with the two genes <i>cro</i> and <i>cI</i> under mutually exclusive expression in the lambdoid phage.</p>
<p align="justify">The rightward promoter/operator region in lambda prophages forms a switch with the two genes <i>cro</i> and <i>cI</i> under mutually exclusive expression in the lambdoid phage.</p>
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<p align="justify">This regulatory region consists of the promoters pRM and pR and the three sub-operator sites, OR1, OR2 and OR3, in-between the promoter. The interesting feature about these two promoters is that they are orientated in a back-to-back fashion and are also called divergent or bidirectional promoters. The sub-operator sites are shared regulatory regions that influence the expression of two oppositely oriented genes. This type of promoter arrangement is common in prokaryotes and is also found in humans and other higher species. The pR promoter is very strong and has greater similarity to the promoter consensus sequence than the very weak pRM promoter [6].</p>
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<p align="justify">This regulatory region consists of the promoters pRM and pR and the three sub-operator sites, OR1, OR2 and OR3, in-between the promoter. The interesting feature about these two promoters is that they are orientated in a back-to-back fashion and are also called divergent or bidirectional promoters. The sub-operator sites are shared regulatory regions that influence the expression of two oppositely oriented genes. This type of promoter arrangement is common in prokaryotes and is also found in humans and other higher species. The pR promoter is very strong and has greater similarity to the promoter consensus sequence than the very weak pRM promoter [Strainic et al.,1999].</p>
<p align="justify">The <i>cI</i> gene encodes cI, the lambda repressor protein. The presence of this protein stabilizes the lysogenic state and causes immunity to superinfections by other lambda phages. cI has a dual function by acting as repressor and activator. It represses transcription from the pR promoter while up-regulating its own expression from the pRM promoter.</p>
<p align="justify">The <i>cI</i> gene encodes cI, the lambda repressor protein. The presence of this protein stabilizes the lysogenic state and causes immunity to superinfections by other lambda phages. cI has a dual function by acting as repressor and activator. It represses transcription from the pR promoter while up-regulating its own expression from the pRM promoter.</p>
<p align="justify">A dimer is formed by the cI repressor and binds to DNA in the helix-turn-helix binding motif. The cI repressor binds to all three operator sites in the order OR1 = OR2 > OR3, because of its different intrinsic affinities for the operator sites.</p>
<p align="justify">A dimer is formed by the cI repressor and binds to DNA in the helix-turn-helix binding motif. The cI repressor binds to all three operator sites in the order OR1 = OR2 > OR3, because of its different intrinsic affinities for the operator sites.</p>
<p align="justify">The repressor binds with highest affinity to OR1 and that stimulates binding of more cI to OR2 by a mechanism called positive cooperative binding. Binding to OR1 and OR2 blocks binding of RNA polymerase to the pR promoter, so switching to the lytic cycle is prevented. At high repressor concentrations cI down-regulates its own expression by binding to OR3, so all three operator sites are occupied and expression from the pRM promoter is limited i.e cI, while the expression from cro genes still is inhibited. It is important to understand, that regulation of the switch is solely dependent on repressor concentrations and not by other regulatory proteins e.g. anti-repressors. The Cro protein does not bind with positive cooperation to the three operator sites whilst cI does. The result is that cI and the lysogenic state is more stable, because it can outcompete the cro protein. </p>
<p align="justify">The repressor binds with highest affinity to OR1 and that stimulates binding of more cI to OR2 by a mechanism called positive cooperative binding. Binding to OR1 and OR2 blocks binding of RNA polymerase to the pR promoter, so switching to the lytic cycle is prevented. At high repressor concentrations cI down-regulates its own expression by binding to OR3, so all three operator sites are occupied and expression from the pRM promoter is limited i.e cI, while the expression from cro genes still is inhibited. It is important to understand, that regulation of the switch is solely dependent on repressor concentrations and not by other regulatory proteins e.g. anti-repressors. The Cro protein does not bind with positive cooperation to the three operator sites whilst cI does. The result is that cI and the lysogenic state is more stable, because it can outcompete the cro protein. </p>
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<p align="justify">The lytic pathway of lambda is induced by the SOS response after DNA damage in <i>E. coli</i> by e.g. UV light. This is achieved when the repressor protein cI is cleaved by a protein expressed during SOS response, RecA [4].</p>
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<a name="gifsy"></a><h1>The Gifsy phages: Gifsy1 and Gifsy2</h1>
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<h3>The Lytic Pathway</h3>
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<p align="justify">The lytic pathway of lambda is induced by the SOS response after DNA damage in <i>E. coli</i> by e.g. UV light. This is achieved when the repressor protein cI is cleaved by a protein expressed during SOS response, RecA [Lemire et al.,2008]. After induction of the lytic cycle, transcription through the pL and pR promoters is instigated. This results in the expression of the N protein from the pL promoter. The N protein participates in anti-termination, a process that prevents termination of transcription by termination signal. This allows transcription through termination signals located downstream of the early genes.</p>
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<p align="justify">The <i>nut</i>-site plays an important role in anti-termination as it promotes the formation of an anti-termination complex on the nascent mRNA, consisting of the lambda N protein and four <i>E. coli</i> proteins, NusA, NusB, NusE and NusG. The <i>nut</i>-site is made up of regions, the conserved <i>boxA</i> region and the variable <i>boxB</i> region.
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NusA and the N protein is necessary for anti-termination effect. NusA native to e-coli binds to the spacer region, between boxA and boxB, and the N protein binds to boxB. NusA has a 50% greater affinity for <i>nutL</i>, found downstream of the pL promoter, compared to <i>nutR</i>, found downstream of the pR promoter. This mode of anti-termination is also found in other lambda-related phages such as P21 and P22. Under normal conditions, these N analog proteins show specificity for <i>nut</i>-sites of the respective phages, but this specificity is lost when the N proteins are overexpressed.</p>
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<table class="https://static.igem.org/mediawiki/2010/0/05/Anti-termination-complex.PNG" align="center">
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<caption align="bottom"><p align="justify"><b>Figure 3</b>: The anti-termination complex is illustrated. The anti-termination complex consists of RNA-polymerase, the hosts proteins, NusA, NusB, NusE and NusG and (Burmann et.al. 2010).</p></caption>
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<tr><td><img src="https://static.igem.org/mediawiki/2010/0/05/Anti-termination-complex.PNG"  width="250px"></td></tr>
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<p align="justify">Expression of the Q protein is observed as a result of the anti-termination by the N protein during transcription from the pR promoter. Q is another protein that is capable of causing anti-termination. However, unlike the N-protein, it prevents termination of transcription by the binding to DNA sequences just upstream of the late pR' promoter, resulting in transcription of the late genes.</p>
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<p align="justify">Few papers describe and test the distance between the nut-site and the termination steam loop. NusA and N have been found to be sufficient for anti-termination under in vitro conditions, however, N is unable of singularly inducing anti-termination. In the tested construct, <i>nutL</i> was placed 200nt upstream of the lambda right terminator (Whalen et.al.1988). The required distance between <i>boxB</i>, within the nut-site, to the terminator stem loop has yet to be determined but it was placed 26 bp from the beginning of the terminator stem loop.</p>
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<b>The Gifsy phages: Gifsy1 and Gifsy2</b><br><br>
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<a name="gifsy"></a>
<p align="justify">Gifsy 1 and Gifsy 2 are temperate phages present in the vast majority of <i>Salmonella enterica</i> serovar Typhimurium strains; the genomic positioning of the prophages is illustrated in Figure 2. This strain of pathogenic bacteria infects a broad spectrum of animal species, from reptiles to mammals [1].</p>
<p align="justify">Gifsy 1 and Gifsy 2 are temperate phages present in the vast majority of <i>Salmonella enterica</i> serovar Typhimurium strains; the genomic positioning of the prophages is illustrated in Figure 2. This strain of pathogenic bacteria infects a broad spectrum of animal species, from reptiles to mammals [1].</p>
<table class="https://static.igem.org/mediawiki/2010/3/35/RegSysFig1_DTU.png" align="center">
<table class="https://static.igem.org/mediawiki/2010/3/35/RegSysFig1_DTU.png" align="center">
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  <caption align="bottom"><p align="justify"><b>Figure 2</b>: The positions of the prophage inserts into the Salmonella genome [1].</p></caption>
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  <caption align="bottom"><p align="justify"><b>Figure 2</b>: The positions of the prophage inserts into the Salmonella genome [Lemire et al., 2008].</p></caption>
<tr><td><img src="https://static.igem.org/mediawiki/2010/3/35/RegSysFig1_DTU.png"></td></tr>
<tr><td><img src="https://static.igem.org/mediawiki/2010/3/35/RegSysFig1_DTU.png"></td></tr>
</table><br>
</table><br>
<p align=”justify”>Similar to the lambda phage, the Gifsy phages follow either lytic cycle or lysogenic cycle after infecting <i>S. enterica</i>.</p>
<p align=”justify”>Similar to the lambda phage, the Gifsy phages follow either lytic cycle or lysogenic cycle after infecting <i>S. enterica</i>.</p>
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<p align="justify">Salmonella strains harbor different subsets of prophages and therefore have differences in prophage distribution, lysogeny thereby contributes to the genetic diversity of Salmonella genomes. Since phages are able to switch between these two different developmental states, they are a very interesting example of a natural genetic switch and this is why we choose to use the key regulatory elements from Gifsy-1 and Gifsy-2 prophages to construct our own bistable switch in <i>E.coli</i> [1].</p>
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<p align="justify">Salmonella strains harbor different subsets of prophages and therefore have differences in prophage distribution, lysogeny thereby contributes to the genetic diversity of Salmonella genomes. Since phages are able to switch between these two different developmental states, they are a very interesting example of a natural genetic switch and this is why we choose to use the key regulatory elements from Gifsy-1 and Gifsy-2 prophages to construct our own bistable switch in <i>E.coli</i> [Lemire et al.,2008].</p>
<h3>Chromosomal organization</h3>
<h3>Chromosomal organization</h3>
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<p align="justify">The overall gene organization of Gifsy-1 and Gifsy-2 prophages is typical of the lambdoid phage family that is illustrated in Figure 2 [1].</p>
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<p align="justify">The overall gene organization of Gifsy-1 and Gifsy-2 prophages is typical of the lambdoid phage family that is illustrated in Figure 2 [Lemire et al.,2008].</p>
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<p align="justify">The regulation region of the lambda phage, also called immunity region, is of particular interest to us. This region includes the three promoters pR, pRM and pL, the left- and rightward operator and the <i>cro</i> and <i>cI</i> gene responsible for controlling the switch between lysogenic and lytic growth. The Gifsy chromosome and its immunity region are organized in a very similar way to that of the lambda phage as illustrated by Figure 4 [1].</p> <br>
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<p align="justify">The regulation region of the lambda phage, also called immunity region, is of particular interest to us. This region includes the three promoters pR, pRM and pL, the left- and rightward operator and the <i>cro</i> and <i>cI</i> gene responsible for controlling the switch between lysogenic and lytic growth. The Gifsy chromosome and its immunity region are organized in a very similar way to that of the lambda phage as illustrated by Figure 4 [Lemire et al.,2008].</p> <br>
<table class="https://static.igem.org/mediawiki/2010/4/43/RegSysFig3_DTU.png" align="center">
<table class="https://static.igem.org/mediawiki/2010/4/43/RegSysFig3_DTU.png" align="center">
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  <caption align="bottom"><p align="justify"><b>Figure 4</b>: The immunity region in Gifsy phages is illustrated [1].</p></caption>
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  <caption align="bottom"><p align="justify"><b>Figure 4</b>: The immunity region in Gifsy phages is illustrated [Lemire et al.,2008].</p></caption>
<tr><td><img src="https://static.igem.org/mediawiki/2010/4/43/RegSysFig3_DTU.png"  width="400px"></td></tr>
<tr><td><img src="https://static.igem.org/mediawiki/2010/4/43/RegSysFig3_DTU.png"  width="400px"></td></tr>
</table><br>
</table><br>
<h3>Regulation of promoters and repressors</h3>
<h3>Regulation of promoters and repressors</h3>
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<p align="justify">GogR and GtgR are repressor proteins found in Gifsy-1 and Gifsy-2, respectively. These repressor proteins are analogous to the lambda repressor protein, cI, previously described. The Gifsy repressors (136 aa) are much smaller than the lambda repressor cI (237 aa) and lack the typical cleavage motif [1]. The mechanism by which the repressors GogR and GtgR regulate the Gifsy promoters, pR and pRM is analogous to that of cI in the lambda phage with the exception of the mode of lytic induction.</p>
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<p align="justify">GogR and GtgR are repressor proteins found in Gifsy-1 and Gifsy-2, respectively. These repressor proteins are analogous to the lambda repressor protein, cI, previously described. The Gifsy repressors (136 aa) are much smaller than the lambda repressor cI (237 aa) and lack the typical cleavage motif [Lemire et al.,2008]. The mechanism by which the repressors GogR and GtgR regulate the Gifsy promoters, pR and pRM is analogous to that of cI in the lambda phage with the exception of the mode of lytic induction.</p>
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<p align="justify">As previously mentioned, GogR and GtgR lack the cI cleavage site and are inactivated by binding of small anti-repressor proteins, called AntO and AntT. The Gifsy genes encoding these proteins are located outside the immunity region and are under the direct control of the LexA protein. This protein is the major regulator of the SOS regulon and this regulon is activated by the cleavage of LexA by RecA. The interesting point is that Gifsy and lambda prophage regulation is an integral part of the SOS response i.e. once the SOS response is triggered, the lytic pathway is also induced [1,7].</p>
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<p align="justify">As previously mentioned, GogR and GtgR lack the cI cleavage site and are inactivated by binding of small anti-repressor proteins, called AntO and AntT. The Gifsy genes encoding these proteins are located outside the immunity region and are under the direct control of the LexA protein. This protein is the major regulator of the SOS regulon and this regulon is activated by the cleavage of LexA by RecA. The interesting point is that Gifsy and lambda prophage regulation is an integral part of the SOS response i.e. once the SOS response is triggered, the lytic pathway is also induced [Lemire et al.,2008].</p>
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<h3>References</h3>
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<h3> Phage Repressor System </h3>
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<p align="justify"> Maya Lisa anja </p>
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<h4> Alpha-repressor</h4>
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<p align="justify">The C1-repressor is responsible for repressing transcription of the lytic genes, thereby maintaining the stable lysogenic state. The induction of the lytic state is caused by activated RecA, which stimulates the self-cleavage of the C1-repressor. We will be using the C1-repressor in our system. </p>
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<h1> Anti-Termination system </h1>
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<h3> Termination </h3>
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<p align="justify"> INTRO TERMINATION
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Factor dependent and independent.
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Figure, involded factors natural
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<h3> Antitermination </h3>
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<p align="justify">INTRO ANTITERMINATION N-PROTEIN
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The N-protein is able to suppress transcription termination at both factor-dependent and factor-independent termination sites. N anti-termination is strongly stimulated by the NusA protein. Unlike the N-protein, the Q-protein specifically binds to a DNA sequence immediately upstream of the pR´ promoter.
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N-LAMBDE FACTOR DEPENDENT: Anti-Termination is the process by which the termination of gene transcription is prevented. Such control of gene transcription can be found in the phage Lambda system. The mechanism is controlled by proteins, such as the lambda N or lambda Q-proteins. The expression of early genes and late genes are both regulated by the anti-termination mechanism, controlled by the lambda N-protein and the lambda Q-protein, respectively.
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<p align="justify"><b>N-Protein</b>
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The mechanism of N-protein nut-site termination have been studied heavily the last years and the current best  descriped mechanism  have been done in a couple of reviews (XXXXX,XXXX,XXXX)  The anti-termination function by introduction of the N-protein (or equivalent) that interacts with nusA and disrupt the termination.
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Of known systems can be mentioned lambda, p21, p22 FUNCTION XXXXXXXXXXXX shown in the figure below. 
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In the known systems the nut site is placed from XXX bp to XXX bp upstream of the termination steam loop. (REFERENCES !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! IMPORTANT  - read two gottersman 2010 – crystallography papers)))) figures from papers on antitermination.</p>
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Few papers descripe and test the the actual  needed distance from the termination steam loop. From other systems and mechanism it is known that XXX bp is needed for regulation of RNAP or DNAP. (MOGENS ABOUT REFERENCES AND SYSTEMS SEE MICRO-BIO-TEXT BOOKS_) .</p>
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<ul>
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<li> Lemire, S., N. Figueroa-Bossi, and L. Bossi. 2008. Prophage contribution to Salmonella virulence and diversity, p. 159-192. In M. Hensel and H. Schmidt (ed.), Horizontal gene transfer in the evolution of bacterial pathogenesis.. Cambridge University, Cambridge, England.</li>
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<li> Lemire, S., Figueroa-Bossi, N. & Bossi, L. A Singular Case of Prophage Complementation in Mutational Activation of recET Orthologs in Salmonella enterica Serovar Typhimurium. J. Bacteriol. 190, 6857-6866 (2008).</li>
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<li> Promoter Interference in a Bacteriophage Lambda Control Region: Effects of a Range of Interpromoter Distances
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Michael G. Strainic, Jr., Jennifer J. Sullivan, Julio Collado-Vides, and Pieter L. deHaseth</li>
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<li> Bacteriophage lambda. July 2010, 10:55 UTC. In Wikipedia: The Free Encyclopedia. Wikimedia Foundation Inc. Encyclopedia on-line. Available from http://en.wikipedia.org/wiki/Bacteriophage_lambda. Internet. Retrieved 10 August 2010.</li>
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<li> Bacteriophage lysogenic and lytic cycle. 21 October 2008, 22:34 UTC. In Wikipedia: The Free Encyclopedia. Wikimedia Foundation Inc. Encyclopedia on-line. Available from http://en.wikipedia.org/wiki/File:Phage2.JPG. Internet. Retrieved 10 August 2010.</li>
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<li> Schematic representation of the genome of the bacteriophage lambda. 21 March 2009, 14:58 UTC. In Wikipedia: The Free Encyclopedia. Wikimedia Foundation Inc. Encyclopedia on-line. Available from http://en.wikipedia.org/wiki/File:Bacteriophage_lambda_genome.png. Internet. Retrieved 10 August 2010.</li>
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<li> Lou, C. et al. Synthesizing a novel genetic sequential logic circuit: a push-on push-off switch. Mol Syst Biol 6,  (2010).</li>
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<li> Hensel, M. & Schmidt, H. Horizontal gene transfer in the evolution of pathogenesis. P 5 (Cambridge University Press: 2008).</li>
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<li> Burmann. B., Schweimer. K., Luo. X., Wahl. M., Stitt. B., Gottesmann. M., Rösch. P., " A NusE:NusG Complex Links Transcription and Translation" Science 2010.</li>
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<li> Nudler. E., Gottesman. M.E., "Transcription termination and anti-termination in E.coli", Genes to Cells (2002) </li>
 +
<li> Prasch. S., Jurk. M., Washburn. R.S., Gottesman. M.E., Wöhrl. B., Rösch. P., "Rna-binding specificity of E. coli NusA" Nucleic Acids Research 2009.</li>
 +
<li> Whalen. W., Ghosh. B., Das. A., "NusA protein is necessary and sufficient in vitro for phage lambda N gene product to suppress a rho-independent terminator placed downstream of nutL. Proc.Natl. Acad sci. 1988</li>
 +
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<p align="justify">The N protein were isolated from salmonella genomic DNA with specific designed primers. We used the natural occurring RBS site, as a High expression of N have shown non specific anti-termination effect on a global scale on the genome. [[#References References]]</p>
 
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<h3>nut sites Engineering</h3>
 
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<p align="justify">Severel papers analyse the function of the nut-site. It has been shown that this mechanism can be manipulated in different manners and that the function can be canceled and reactivated by counter-mutations in XXXXX (REFFFFF). Futher it has been shown that the specificity of the N-proteins can be changed from lambda to P22 by only a few mutations showing a possible coevolution, or possible interactions to increase possible genomic randomization
 
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Latest revision as of 03:35, 28 October 2010

Welcome to the DTU iGEM wiki!



The lambda phage

The temperate bacteriophage lambda found to infect E. coli, is the best-studied phage with regard to phage structure and regulation. Once lambda infects a cell, it can chose to either follow the lytic cycle or the lysogenic cycle described in Figure 1.

Figure 1: The two distinct development pathways of a prophage life cycle [wikipedia].
In the lytic pathway the phage uses the bacterial molecular machinery to make many viral copies for infection of other cells before lysing the host bacterium. In contrast to the lytic pathway, the phage integrates its DNA into the bacterial genome in the lysogenic pathway. The lysogenic state is very stable, which means that the prophage can be replicated along with the bacterial genome for generations.
Despite the stability of the lysogenic state, the lytic state is readily induced when the bacteria are irradiated with ultraviolet light.


The organization of the phage chromosome is shown in Figure 2.

Figure 2: Schematic representation of the genome of the bacteriophage lambda [wikipedia].


The Lysogenic Pathway

The rightward promoter/operator region in lambda prophages forms a switch with the two genes cro and cI under mutually exclusive expression in the lambdoid phage.

This regulatory region consists of the promoters pRM and pR and the three sub-operator sites, OR1, OR2 and OR3, in-between the promoter. The interesting feature about these two promoters is that they are orientated in a back-to-back fashion and are also called divergent or bidirectional promoters. The sub-operator sites are shared regulatory regions that influence the expression of two oppositely oriented genes. This type of promoter arrangement is common in prokaryotes and is also found in humans and other higher species. The pR promoter is very strong and has greater similarity to the promoter consensus sequence than the very weak pRM promoter [Strainic et al.,1999].

The cI gene encodes cI, the lambda repressor protein. The presence of this protein stabilizes the lysogenic state and causes immunity to superinfections by other lambda phages. cI has a dual function by acting as repressor and activator. It represses transcription from the pR promoter while up-regulating its own expression from the pRM promoter.

A dimer is formed by the cI repressor and binds to DNA in the helix-turn-helix binding motif. The cI repressor binds to all three operator sites in the order OR1 = OR2 > OR3, because of its different intrinsic affinities for the operator sites.

The repressor binds with highest affinity to OR1 and that stimulates binding of more cI to OR2 by a mechanism called positive cooperative binding. Binding to OR1 and OR2 blocks binding of RNA polymerase to the pR promoter, so switching to the lytic cycle is prevented. At high repressor concentrations cI down-regulates its own expression by binding to OR3, so all three operator sites are occupied and expression from the pRM promoter is limited i.e cI, while the expression from cro genes still is inhibited. It is important to understand, that regulation of the switch is solely dependent on repressor concentrations and not by other regulatory proteins e.g. anti-repressors. The Cro protein does not bind with positive cooperation to the three operator sites whilst cI does. The result is that cI and the lysogenic state is more stable, because it can outcompete the cro protein.

The Lytic Pathway

The lytic pathway of lambda is induced by the SOS response after DNA damage in E. coli by e.g. UV light. This is achieved when the repressor protein cI is cleaved by a protein expressed during SOS response, RecA [Lemire et al.,2008]. After induction of the lytic cycle, transcription through the pL and pR promoters is instigated. This results in the expression of the N protein from the pL promoter. The N protein participates in anti-termination, a process that prevents termination of transcription by termination signal. This allows transcription through termination signals located downstream of the early genes.

The nut-site plays an important role in anti-termination as it promotes the formation of an anti-termination complex on the nascent mRNA, consisting of the lambda N protein and four E. coli proteins, NusA, NusB, NusE and NusG. The nut-site is made up of regions, the conserved boxA region and the variable boxB region. NusA and the N protein is necessary for anti-termination effect. NusA native to e-coli binds to the spacer region, between boxA and boxB, and the N protein binds to boxB. NusA has a 50% greater affinity for nutL, found downstream of the pL promoter, compared to nutR, found downstream of the pR promoter. This mode of anti-termination is also found in other lambda-related phages such as P21 and P22. Under normal conditions, these N analog proteins show specificity for nut-sites of the respective phages, but this specificity is lost when the N proteins are overexpressed.

Figure 3: The anti-termination complex is illustrated. The anti-termination complex consists of RNA-polymerase, the hosts proteins, NusA, NusB, NusE and NusG and (Burmann et.al. 2010).


Expression of the Q protein is observed as a result of the anti-termination by the N protein during transcription from the pR promoter. Q is another protein that is capable of causing anti-termination. However, unlike the N-protein, it prevents termination of transcription by the binding to DNA sequences just upstream of the late pR' promoter, resulting in transcription of the late genes.

Few papers describe and test the distance between the nut-site and the termination steam loop. NusA and N have been found to be sufficient for anti-termination under in vitro conditions, however, N is unable of singularly inducing anti-termination. In the tested construct, nutL was placed 200nt upstream of the lambda right terminator (Whalen et.al.1988). The required distance between boxB, within the nut-site, to the terminator stem loop has yet to be determined but it was placed 26 bp from the beginning of the terminator stem loop.


The Gifsy phages: Gifsy1 and Gifsy2

Gifsy 1 and Gifsy 2 are temperate phages present in the vast majority of Salmonella enterica serovar Typhimurium strains; the genomic positioning of the prophages is illustrated in Figure 2. This strain of pathogenic bacteria infects a broad spectrum of animal species, from reptiles to mammals [1].

Figure 2: The positions of the prophage inserts into the Salmonella genome [Lemire et al., 2008].


Similar to the lambda phage, the Gifsy phages follow either lytic cycle or lysogenic cycle after infecting S. enterica.

Salmonella strains harbor different subsets of prophages and therefore have differences in prophage distribution, lysogeny thereby contributes to the genetic diversity of Salmonella genomes. Since phages are able to switch between these two different developmental states, they are a very interesting example of a natural genetic switch and this is why we choose to use the key regulatory elements from Gifsy-1 and Gifsy-2 prophages to construct our own bistable switch in E.coli [Lemire et al.,2008].

Chromosomal organization

The overall gene organization of Gifsy-1 and Gifsy-2 prophages is typical of the lambdoid phage family that is illustrated in Figure 2 [Lemire et al.,2008].

The regulation region of the lambda phage, also called immunity region, is of particular interest to us. This region includes the three promoters pR, pRM and pL, the left- and rightward operator and the cro and cI gene responsible for controlling the switch between lysogenic and lytic growth. The Gifsy chromosome and its immunity region are organized in a very similar way to that of the lambda phage as illustrated by Figure 4 [Lemire et al.,2008].


Figure 4: The immunity region in Gifsy phages is illustrated [Lemire et al.,2008].


Regulation of promoters and repressors

GogR and GtgR are repressor proteins found in Gifsy-1 and Gifsy-2, respectively. These repressor proteins are analogous to the lambda repressor protein, cI, previously described. The Gifsy repressors (136 aa) are much smaller than the lambda repressor cI (237 aa) and lack the typical cleavage motif [Lemire et al.,2008]. The mechanism by which the repressors GogR and GtgR regulate the Gifsy promoters, pR and pRM is analogous to that of cI in the lambda phage with the exception of the mode of lytic induction.

As previously mentioned, GogR and GtgR lack the cI cleavage site and are inactivated by binding of small anti-repressor proteins, called AntO and AntT. The Gifsy genes encoding these proteins are located outside the immunity region and are under the direct control of the LexA protein. This protein is the major regulator of the SOS regulon and this regulon is activated by the cleavage of LexA by RecA. The interesting point is that Gifsy and lambda prophage regulation is an integral part of the SOS response i.e. once the SOS response is triggered, the lytic pathway is also induced [Lemire et al.,2008].

References

  • Lemire, S., N. Figueroa-Bossi, and L. Bossi. 2008. Prophage contribution to Salmonella virulence and diversity, p. 159-192. In M. Hensel and H. Schmidt (ed.), Horizontal gene transfer in the evolution of bacterial pathogenesis.. Cambridge University, Cambridge, England.
  • Lemire, S., Figueroa-Bossi, N. & Bossi, L. A Singular Case of Prophage Complementation in Mutational Activation of recET Orthologs in Salmonella enterica Serovar Typhimurium. J. Bacteriol. 190, 6857-6866 (2008).
  • Promoter Interference in a Bacteriophage Lambda Control Region: Effects of a Range of Interpromoter Distances Michael G. Strainic, Jr., Jennifer J. Sullivan, Julio Collado-Vides, and Pieter L. deHaseth
  • Bacteriophage lambda. July 2010, 10:55 UTC. In Wikipedia: The Free Encyclopedia. Wikimedia Foundation Inc. Encyclopedia on-line. Available from http://en.wikipedia.org/wiki/Bacteriophage_lambda. Internet. Retrieved 10 August 2010.
  • Bacteriophage lysogenic and lytic cycle. 21 October 2008, 22:34 UTC. In Wikipedia: The Free Encyclopedia. Wikimedia Foundation Inc. Encyclopedia on-line. Available from http://en.wikipedia.org/wiki/File:Phage2.JPG. Internet. Retrieved 10 August 2010.
  • Schematic representation of the genome of the bacteriophage lambda. 21 March 2009, 14:58 UTC. In Wikipedia: The Free Encyclopedia. Wikimedia Foundation Inc. Encyclopedia on-line. Available from http://en.wikipedia.org/wiki/File:Bacteriophage_lambda_genome.png. Internet. Retrieved 10 August 2010.
  • Lou, C. et al. Synthesizing a novel genetic sequential logic circuit: a push-on push-off switch. Mol Syst Biol 6, (2010).
  • Hensel, M. & Schmidt, H. Horizontal gene transfer in the evolution of pathogenesis. P 5 (Cambridge University Press: 2008).
  • Burmann. B., Schweimer. K., Luo. X., Wahl. M., Stitt. B., Gottesmann. M., Rösch. P., " A NusE:NusG Complex Links Transcription and Translation" Science 2010.
  • Nudler. E., Gottesman. M.E., "Transcription termination and anti-termination in E.coli", Genes to Cells (2002)
  • Prasch. S., Jurk. M., Washburn. R.S., Gottesman. M.E., Wöhrl. B., Rösch. P., "Rna-binding specificity of E. coli NusA" Nucleic Acids Research 2009.
  • Whalen. W., Ghosh. B., Das. A., "NusA protein is necessary and sufficient in vitro for phage lambda N gene product to suppress a rho-independent terminator placed downstream of nutL. Proc.Natl. Acad sci. 1988