Difference between revisions of "Part:BBa K200000"
(→‎Characterisation)
 
(36 intermediate revisions by 4 users not shown)
Line 2: Line 2:
 
<partinfo>BBa_K200000 short</partinfo>
 
<partinfo>BBa_K200000 short</partinfo>
  
−
RcsB is a receiver protein in a two component phosphorelay system, which acts as a positive regulator of the capsule genes resulting in colanic acid production. <cite>2</cite> This is via the activation of the ugd/cps operon which is required for capsule synthesis. <br>
+
===<b>R</b>egulation for <b>C</b>apsular <b>S</b>ynthesis (RCS) Phosphorelay System===
  
−
It is important to note that RcsB is a central protein in a series of downstream pathways, including the protein ftsZ that affects cell division. <cite>1</cite>
+
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.
 +
[[Image:PhosRelay.png|left|400px]]
  
 +
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.
  
 +
===<b>RcsB as a transcription factor</b>===
 +
 +
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>
 +
 +
 +
 +
 +
 +
 +
 +
===<b>Colanic Acid Biosynthesis:</b>===
 +
 +
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.
 +
 +
<html>
 +
</p><p class="ecoparagraph">
 +
<map name="MAP79413">
 +
<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> -105.5 (ranging from -99 to -112)<br>
 +
 +
<b>Evidence:</b> Binding of purified proteins [Wehland00], Site mutation [Wehland00]');" onmouseout="return nd();">
 +
<area coords="32,55,64,72" onmouseover="return overlib('RcsAB transcriptional dual regulator activates transcription from associated promoter<br><b>Distance to transcription start site:</b> -78.5 (ranging from -72 to -85)<br>
 +
 +
<b>Evidence:</b> Gene expression analysis [Ebel99], Automated inference based on similarity to consensus sequences [Ebel99]');" onmouseout="return nd();">
 +
<area coords="274,41,338,72" onmouseover="return overlib(' <b>Gene:</b>  wzb    G7106    b2061    ECK2055<br><b>Location:</b> 2,133,679 <- 2,134,122  <br> <b>Product:</b> tyrosine phosphatase');" onmouseout="return nd();">
 +
<area coords="745,41,816,72" onmouseover="return overlib(' <b>Gene:</b>  wcaB    G7103    b2058    ECK2052<br><b>Location:</b> 2,130,091 <- 2,130,579  <br> <b>Product:</b> predicted colanic acid biosynthesis acyl transferase');" onmouseout="return nd();">
 +
<area coords="631,41,745,72"  onmouseover="return overlib(' <b>Gene:</b>  wcaA    G7104    b2059    ECK2053<br><b>Location:</b> 2,130,582 <- 2,131,421  <br> <b>Product:</b> predicted colanic acid biosynthesis glycosyl transferase');" onmouseout="return nd();">
 +
<area coords="122,41,274,72" onmouseover="return overlib(' <b>Gene:</b>  wza    G7107    b2062    ECK2056<br><b>Location:</b> 2,134,128 <- 2,135,267  <br> <b>Product:</b> lipoprotein required for capsular polysaccharide translocation through the outer membrane, subunit of capsular polysaccharide export apparatus');" onmouseout="return nd();">
 +
<area coords="338,41,619,72"  onmouseover="return overlib(' <b>Gene:</b>  wzc    G7105    b2060    ECK2054<br><b>Location:</b> 2,131,514 <- 2,133,676  <br> <b>Product:</b> tyrosine kinase involved in colanic acid biosynthesis, subunit of capsular polysaccharide export apparatus');" onmouseout="return nd();">
 +
<area coords="16,0,135,89"  onmouseover="return overlib('<b>Operon:</b> wzap (Experim. ev.)  <BR><b>Tr.Activators:</b> (RcsAB)');" onmouseout="return nd();">
 +
<area coords="0,0,818,89" onmouseover="return overlib('<b>Operon</b>: wza-wzb-wzc-wcaAB<br><b>Synonyms:</b> OP00290, cps, cpsB, cpsGB');" onmouseout="return nd();">
 +
</map>
 +
<img src="https://static.igem.org/mediawiki/2009/8/89/Op1.gif" usemap="#MAP79413" border="0">
 +
 +
</html>
 +
 +
 +
 +
<html>
 +
</p><p class="ecoparagraph">
 +
 +
<map name="MAP91795">
 +
<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();">
 +
<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();">
 +
<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();">
 +
<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();">
 +
<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();">
 +
<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();">
 +
<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();">
 +
<area coords="0,0,642,89" onmouseover="return overlib('<b>Operon</b>: yjbEFGH');" onmouseout="return nd();">
 +
</map>
 +
<img src="https://static.igem.org/mediawiki/2009/2/28/Op11.gif" usemap="#MAP91795" border="0">
 +
 +
</html>
  
−
<!-- Add more about the biology of this part here
 
 
===Usage and Biology===
 
===Usage and Biology===
 +
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>
  
 
<!-- -->
 
<!-- -->
Line 20: Line 77:
 
<partinfo>BBa_K200000 parameters</partinfo>
 
<partinfo>BBa_K200000 parameters</partinfo>
 
<!-- -->
 
<!-- -->
 +
 +
 +
===Characterisation===
 +
[[Image:EM.jpg  | 650px]] <br>As you can see from the diagram, the control cells have a smooth cell membrane.  <br>
 +
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>
 +
<br>
 +
We have in addition conducted a functionality test to see if colanic acid encapsulation protects the cell from acidic environments. <br>
 +
[[Image:gra.jpg]]<br>
 +
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> 
 +
<br>
 +
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===
 
<biblio>
 
<biblio>
 
#1 pmid=1597415
 
#1 pmid=1597415
−
</biblio>
 
−
 
−
<biblio>
 
 
#2 pmid=2404948
 
#2 pmid=2404948
 +
#3 pmid=19124574
 +
 
</biblio>
 
</biblio>
 +
 +
<!-- Uncomment this to enable Functional Parameter display
 +
===Functional Parameters===
 +
<partinfo>BBa_K143032 parameters</partinfo>
 +
<!-- -->
 +
  
 
<!-- Uncomment this to enable Functional Parameter display  
 
<!-- Uncomment this to enable Functional Parameter display  

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>