Difference between revisions of "Part:BBa K200000"

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Together, the RcsB~P homodimer and the RcsB~P+RcsA heterodimer are thought to regulate ~5% of the E.coli genome <cite>4</cite>. It is important to note that RcsB is a global regulator found in many [http://en.wikipedia.org/wiki/Enterobacteria Enterobacteria]. In Salmonella, up to 38 genes were found to be up-regulated by RcsB including ftsZ which affects the rate of cell division. <cite>1</cite> The majority of upregulated genes are associated with enhancing structural integrety and acid resistance. 22 genes were found to be downregualted by RcsB in the same microarray experiment.<cite>3</cite>
 
Together, the RcsB~P homodimer and the RcsB~P+RcsA heterodimer are thought to regulate ~5% of the E.coli genome <cite>4</cite>. It is important to note that RcsB is a global regulator found in many [http://en.wikipedia.org/wiki/Enterobacteria Enterobacteria]. In Salmonella, up to 38 genes were found to be up-regulated by RcsB including ftsZ which affects the rate of cell division. <cite>1</cite> The majority of upregulated genes are associated with enhancing structural integrety and acid resistance. 22 genes were found to be downregualted by RcsB in the same microarray experiment.<cite>3</cite>
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===<b>Colanic Acid Biosynthesis:</b>===
 
===<b>Colanic Acid Biosynthesis:</b>===
  
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The overexpression of RcsB is akin to fitting a stethoscope to the end of a megaphone. In essence, the sensitivity of the RCS Phosphorelay system becomes such that the smallest of membrane perturbations becomes sufficient to trigger colanic acid production. Thus, the background noise switches the RCS Phosphorelay system on permanently.
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The RcsB~P+RcsA heterodimer acts as a positive regulator for genes responsible for colanic acid production. <cite>2</cite> This is via the activation of the ugd/cps operon which is required for capsule synthesis. <br> Colanic acid is a heteropolymer containing glucose, galactose, fucose, and glucuronic acid. The overexpression of RcsB is akin to fitting a stethoscope to the end of a megaphone. In essence, the sensitivity of the RCS Phosphorelay system becomes such that the smallest of membrane perturbations becomes sufficient to trigger colanic acid production. Thus, the background noise switches the RCS Phosphorelay system on permanently.
  
 
There are a total of 19 genes involved in colonic acid biosynthesis, the best characterised of which are shown below.  
 
There are a total of 19 genes involved in colonic acid biosynthesis, the best characterised of which are shown below.  
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===RcsB and the positive feedback loop===
 
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RcsB~P is able to heterodimerise with the protein RcsA. The RcsB~P+RcsA complex acts as a transcription factor that induces the expression of a number of genes. One of these genes codes for RcsA, this initiates a positive feedback loop.
 
  
 
<html>
 
<html>
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</p><p class="ecoparagraph">
  
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<p class="ecoparagraph">
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<map name="MAP91795">
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<map name="MAP91791">
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<area coords="41,55,72,72" onmouseover="return overlib('IHF transcriptional dual regulator activates transcription from associated promoter<br><b>Distance to transcription start site:</b> -71 (ranging from -77 to -65)<br><b>Evidence:</b> Binding of purified proteins [Murtin98], Human inference based on similarity to consensus sequences [Murtin98]');" onmouseout="return nd();">
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<area coords="147,18,177,35" onmouseover="return overlib('H-NS transcriptional dual regulator represses transcription from associated promoter<br>The exact location of this binding site is unknown or not specified.<br><b>Evidence:</b> Gene expression analysis<br>The nature of binding of this protein is not well known.');" onmouseout="return nd();">
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<area coords="0,55,32,72" onmouseover="return overlib('RcsAB transcriptional dual regulator activates transcription from associated promoter<br><b>Distance to transcription start site:</b> -147.5 (ranging from -154 to -141)<br><b>Evidence:</b> Gene expression analysis [Ferrieres07], Human inference based on similarity to consensus sequences [Ferrieres07]');" onmouseout="return nd();">
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<area coords="0,74,32,91" onmouseover="return overlib('RcsAB transcriptional dual regulator activates transcription from associated promoter<br><b>Distance to transcription start site:</b> -166.5 (ranging from -173 to -160)<br><b>Evidence:</b> Gene expression analysis [Ebel99], Automated inference based on similarity to consensus sequences [Ebel99]');" onmouseout="return nd();">
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<area coords="98,41,137,72" onmouseover="return overlib(' <b>Gene:</b>  yjbE    EG11923    b4026    ECK4018<br><b>Location:</b> 4,233,929 -> 4,234,171  <br> <b>Product:</b> predicted protein');" onmouseout="return nd();">
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<area coords="34,74,66,91" onmouseover="return overlib('RcsAB transcriptional dual regulator activates transcription from associated promoter<br><b>Distance to transcription start site:</b> -125.5 (ranging from -132 to -119)<br><b>Evidence:</b> Binding of cellular extracts, Site mutation [Wehland00], Binding of purified proteins [Wehland00]');" onmouseout="return nd();">
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<area coords="154,41,247,72" onmouseover="return overlib(' <b>Gene:</b>  yjbF    EG11924    b4027    ECK4019<br><b>Location:</b> 4,234,285 -> 4,234,923  <br> <b>Product:</b> predicted lipoprotein');" onmouseout="return nd();">
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<area coords="147,0,184,17" onmouseover="return overlib('GadE transcriptional activator activates transcription from associated promoter<br>The exact location of this binding site is unknown or not specified.<br><b>Evidence:</b> Binding of purified proteins [Hommais04], Gene expression analysis [Hommais04]');" onmouseout="return nd();">
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<area coords="246,41,353,72" onmouseover="return overlib(' <b>Gene:</b>  yjbG   EG11925   b4028   ECK4020<br><b>Location:</b> 4,234,920 -> 4,235,657  <br> <b>Product:</b> conserved protein');" onmouseout="return nd();">
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<area coords="104,60,165,91" onmouseover="return overlib(' <b>Gene:</b>  rcsA   EG10820   b1951   ECK1949<br><b>Synonyms:</b> cpsR<br><b>Location:</b> 2,021,992 -> 2,022,615   <br> <b>Product:</b> positive DNA-binding transcriptional regulator of capsular polysaccharide synthesis, activates its own expression, subunit of RcsAB transcriptional dual regulator');" onmouseout="return nd();">
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<area coords="352,41,640,72"  onmouseover="return overlib(' <b>Gene:</b>  yjbH    EG11926    b4029    ECK4021<br><b>Location:</b> 4,235,657 -> 4,237,753   <br> <b>Product:</b> predicted porin');" onmouseout="return nd();">
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<area coords="24,0,184,108" onmouseover="return overlib('<b>Operon:</b> rcsAp (Experim. ev.)  <BR><b>Tr.Activators:</b> (GadE RcsAB) <BR><b>Tr.Inhibitors:</b> (Hns)');" onmouseout="return nd();">
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<area coords="22,0,143,89" onmouseover="return overlib('<b>Operon:</b> yjbEp (No experim. ev.)  <BR><b>Tr.Activators:</b> (RcsAB IHF)');" onmouseout="return nd();">
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<area coords="0,0,184,108" onmouseover="return overlib('<b>Operon</b>: rcsA');" onmouseout="return nd();">
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<area coords="0,0,642,89" onmouseover="return overlib('<b>Operon</b>: yjbEFGH');" onmouseout="return nd();">
 
</map>
 
</map>
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<img src="https://static.igem.org/mediawiki/2009/8/89/Op8.gif" usemap="#MAP91791" border="0">
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<img src="https://static.igem.org/mediawiki/2009/2/28/Op11.gif" usemap="#MAP91795" border="0">
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</p>
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</html>
 
</html>
  
 
===Usage and Biology===
 
===Usage and Biology===
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&nbsp;Colanic acid is an exopolysaccharide secreted by some bacterial species to protect themselves from the environment that surrounds them.
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RcsB was used alongside [[Part:BBa_K200002 |YgiV]] and [[Part:BBa_K200003 |Rfal]] by the Imperial iGEM 2009 [http://2009.igem.org/Team:Imperial_College_London <i>The E.ncapsulator</i>] team to upregulate colanic acid production as part of the auto-encapsulation mechanism. <br>
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<br><br>The gene was used alongside [[Part:BBa_K200002 |YgiV]] and [[Part:BBa_K200003 |Rfal]] by the Imperial iGEM 2009 [http://2009.igem.org/Team:Imperial_College_London <i>The E.ncapsulator</i>] team to upregulate colanic acid production as part of the auto-encapsulation mechanism. <br>
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<partinfo>BBa_K200000 parameters</partinfo>
 
<partinfo>BBa_K200000 parameters</partinfo>
 
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===Characterisation===
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[[Image:EM.jpg  | 650px]] <br>As you can see from the diagram, the control cells have a smooth cell membrane.  <br>
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However, for those cells where RcsB (BBa_K200000) is induced, there is an obvious outer coating of colanic acid that forms on the outside of the cells, encapsulating the cell fully. <br>
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<br>
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We have in addition conducted a functionality test to see if colanic acid encapsulation protects the cell from acidic environments. <br>
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[[Image:gra.jpg]]<br>
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Overnight cultures were incubated in pH 2 buffer solutions for 4 hours. Cells were plated before and after the experiment to determine the colony forming units. From the graph, it shows that cells encapsulated with colanic acid have a much improved cell viability, with colony forming units almost 2 orders of magnitude higher than control cells.  This provides a clear demonstration of the protective effects of colanic acid expression via RcsB.<br> 
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<br>
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With the additional genes YgiV and RfaI, we expect the cell viability to increase further as an even more complete protective capsule is being produced.<br>
  
 
===References===
 
===References===

Latest revision as of 16:17, 31 October 2009

Colanic acid global regulator -> RcsB

Regulation for Capsular Synthesis (RCS) Phosphorelay System

RcsB is a transcription factor that forms part of a phosphorelay system. As the name suggests, a phosphorelay system consists of a series of nodes down which a phosphate group is passed. The transfer of this phosphate group down along the Rcs relay system is initiated by membrane stress. Once the phosphate group has been passed to RcsB, it can bind to DNA and act as a transcription factor.

PhosRelay.png

The Rcs phosphorelay is composed of two membrane-anchored proteins, RcsC and RcsD, and a cytoplasmic protein, RcsB. In response to a signal, the sensor kinase RcsC autophosphorylates and the phosphoryl group is transferred to RcsB, via RcsD. RcsB can both homodimerise and heterodimerise with RcsA, these two complexes serve to regulate different operons.

RcsB as a transcription factor

Together, the RcsB~P homodimer and the RcsB~P+RcsA heterodimer are thought to regulate ~5% of the E.coli genome 4. It is important to note that RcsB is a global regulator found in many [http://en.wikipedia.org/wiki/Enterobacteria Enterobacteria]. In Salmonella, up to 38 genes were found to be up-regulated by RcsB including ftsZ which affects the rate of cell division. 1 The majority of upregulated genes are associated with enhancing structural integrety and acid resistance. 22 genes were found to be downregualted by RcsB in the same microarray experiment.3




Colanic Acid Biosynthesis:

The RcsB~P+RcsA heterodimer acts as a positive regulator for genes responsible for colanic acid production. 2 This is via the activation of the ugd/cps operon which is required for capsule synthesis.
Colanic acid is a heteropolymer containing glucose, galactose, fucose, and glucuronic acid. The overexpression of RcsB is akin to fitting a stethoscope to the end of a megaphone. In essence, the sensitivity of the RCS Phosphorelay system becomes such that the smallest of membrane perturbations becomes sufficient to trigger colanic acid production. Thus, the background noise switches the RCS Phosphorelay system on permanently.

There are a total of 19 genes involved in colonic acid biosynthesis, the best characterised of which are shown below.


Usage and Biology

RcsB was used alongside YgiV and Rfal by the Imperial iGEM 2009 [http://2009.igem.org/Team:Imperial_College_London The E.ncapsulator] team to upregulate colanic acid production as part of the auto-encapsulation mechanism.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]



Characterisation

EM.jpg
As you can see from the diagram, the control cells have a smooth cell membrane.
However, for those cells where RcsB (BBa_K200000) is induced, there is an obvious outer coating of colanic acid that forms on the outside of the cells, encapsulating the cell fully.

We have in addition conducted a functionality test to see if colanic acid encapsulation protects the cell from acidic environments.
Gra.jpg
Overnight cultures were incubated in pH 2 buffer solutions for 4 hours. Cells were plated before and after the experiment to determine the colony forming units. From the graph, it shows that cells encapsulated with colanic acid have a much improved cell viability, with colony forming units almost 2 orders of magnitude higher than control cells. This provides a clear demonstration of the protective effects of colanic acid expression via RcsB.

With the additional genes YgiV and RfaI, we expect the cell viability to increase further as an even more complete protective capsule is being produced.

References

<biblio>

  1. 1 pmid=1597415
  2. 2 pmid=2404948
  3. 3 pmid=19124574

</biblio>