Difference between revisions of "Part:BBa K1583100"

 
(Plate Reader assay before addition image)
Line 1: Line 1:
−
 
 
__NOTOC__
 
__NOTOC__
 
<partinfo>BBa_K1583100 short</partinfo>
 
<partinfo>BBa_K1583100 short</partinfo>
  
−
CsgA under control of L-rhamnose-inducible promoter
+
<html>
 +
 
 +
This part is meant to express the ''csgA'' gene under control of L-rhamnose-inducible promoter.
 +
 
 +
CsgA is a protein monomer which can aggregate to form amyloid nanowires in natural biofilms of E.coli. This protein is transported as an unfolded protein out of the cell. Outside the cell CsgA proteins self-assemble into nanowires after nucleation on the membrane protein CsgB. CsgC prevents CsgA proteins from self-assembling inside the cell and the transport is ensured by the proteins CsgEFG.
 +
 
 +
This part was designed to be able to control the nanowire formation.
 +
 
 +
<h3>Characterization</h3>
 +
<p>
 +
This part was characterized in three different experiments:
 +
<li> Fluorescence assay </li>
 +
<li> Crystal Violet assay </li>
 +
<li> Transmission electron microscopy </li>
 +
</p>
 +
 
 +
<h3> Fluorescence assay </h3>
 +
<p>
 +
To be able to ensure that CsgA is expressed, we added a gene encoding for GFP (<a href="https://parts.igem.org/Part:BBa_I13504">BBa_I13504</a>) under induction of the same rhamnose promoter (<a href="https://parts.igem.org/Part:BBa_K1583112">BBa_K1583112</a>) to check that the promoter works. Both cells containing CsgA and cells containing CsgA + GFP were induced with 0.2% (w/v) and 0.5% (w/v) rhamnose (Figure 1).
 +
 
 +
We assume that the difference between CsgA and CsgA+GFP in size does not influence transcription and translation of CsgA to much. Using a calibration curve connecting <b>fluorescence</b> [au] and <b>mass</b> [ng] we were able to calculate the expression rate of CsgA with units [proteins/(cell*second)].</p>
 +
 
 +
 
  
−
<!-- Add more about the biology of this part here
+
</p>
−
===Usage and Biology===
+
  
 
<!-- -->
 
<!-- -->

Revision as of 00:06, 18 September 2015

pRha + CsgA

This part is meant to express the ''csgA'' gene under control of L-rhamnose-inducible promoter. CsgA is a protein monomer which can aggregate to form amyloid nanowires in natural biofilms of E.coli. This protein is transported as an unfolded protein out of the cell. Outside the cell CsgA proteins self-assemble into nanowires after nucleation on the membrane protein CsgB. CsgC prevents CsgA proteins from self-assembling inside the cell and the transport is ensured by the proteins CsgEFG. This part was designed to be able to control the nanowire formation.

Characterization

This part was characterized in three different experiments:

  • Fluorescence assay
  • Crystal Violet assay
  • Transmission electron microscopy

    • Fluorescence assay

    To be able to ensure that CsgA is expressed, we added a gene encoding for GFP (BBa_I13504) under induction of the same rhamnose promoter (BBa_K1583112) to check that the promoter works. Both cells containing CsgA and cells containing CsgA + GFP were induced with 0.2% (w/v) and 0.5% (w/v) rhamnose (Figure 1). We assume that the difference between CsgA and CsgA+GFP in size does not influence transcription and translation of CsgA to much. Using a calibration curve connecting fluorescence [au] and mass [ng] we were able to calculate the expression rate of CsgA with units [proteins/(cell*second)].

    Sequence and Features BBa_K1583100 SequenceAndFeatures