Team:Calgary/Project/IbpAB

From 2010.igem.org

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<li><a href="http://2010.igem.org/Team:Calgary/Project/Controls">Testing our system</a></li>
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<li><a href="http://2010.igem.org/Team:Calgary/Project/Controls">Testing Our System</a></li>
<li><a href="http://2010.igem.org/Team:Calgary/Project/Achievements">Achievements</a></li>
<li><a href="http://2010.igem.org/Team:Calgary/Project/Achievements">Achievements</a></li>
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<h3 style="color:#0066CC"> The sigma 32 pathway and activation triggers</h3>
 
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<img src="http://i872.photobucket.com/albums/ab287/iGEMCalgary_2010/ibpab-1.png"></img> </td>
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<td> The cytoplasmic stress detector has a fusion of sigma 32 activated heat shock promoter which allows a higher output compared to the ibpAB promoter and FxsA promoter </td>
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<td> Our cytoplasmic stress reporter circuit uses a fusion of two sigma32-dependent heat shock promoter upstream of Green Fluorescent Protein (GFP). </td>
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The ibpAB promoter contorls the trasncription of two small proteins: ibpA and ibpB.  These are small heat shock proteins called inclusion body binding proteins. In the presence of inclusion bodies within the cytoplasm, they are thought to form mixed complexes, ibpA allowing ibpB to bind to the inclusoon body at higher temperatures.  The binding of these proteins to the misfolded protein lowers its hydrophobicity, preventing further binding of exposed peptide chains, thus stabilizing the protein and mediating its refolding by the DnaK/DnaJ/GrpE chaperone protein system (Matuszewska at al., 2005).  Transcription levels from this promoter have been found to increase the most upon heat shock as compared to other heat shock promoters (Chuang et al., 1993).
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The ibpAB promoter controls the trasncription of two small proteins: ibpA and ibpB.  These are small heat shock proteins called inclusion body binding proteins. In the presence of inclusion bodies within the cytoplasm, they are thought to form mixed complexes, ibpA allowing ibpB to bind to the inclusoon body at higher temperatures.  The binding of these proteins to the misfolded protein lowers its hydrophobicity, preventing further binding of exposed peptide chains, thus stabilizing the protein and mediating its refolding by the DnaK/DnaJ/GrpE chaperone protein system (Matuszewska at al., 2005).  Transcription levels from this promoter have been found to increase the most upon heat shock as compared to other heat shock promoters (Chuang et al., 1993).
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<p><i>B: MalE31 induction with IPTG; C: MalE31 induction and reporter reading with just ibpAB promoter; D: MalE31 induction and reporter reading with just fxsA promoter; E: MalE31 induction and reporter reading with ibpAB/FxsA fusion promoter (Kraft et al, 2006)</i></p>
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<p>B: MalE31 induction with IPTG; C: MalE31 induction and reporter reading with just ibpAB promoter; D: MalE31 induction and reporter reading with just fxsA promoter; E: MalE31 induction and reporter reading with ibpAB/FxsA fusion promoter (Kraft et al, 2006)</p>
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<h2 style="color:#0066CC">References</h2>
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Kraft, M., Knupfer, U., Wenderoth, R., Pietschmann, P., Hock, B., & Horn, U. (2007). An online monitoring system based on a synthetic sigma32-dependent tandem promoter for visualization of insoluble proteins in the cytoplasm of escherichia coli. Applied Microbiology and Biotechnology, 75(2), 397-406. </p>
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Latest revision as of 03:34, 28 October 2010

Cytoplasmic Stress Detectors

How does a native E. coli cell combat protein related stress in the Cytoplasm?

There are several heat shock pathways in E. coli which are actively transcribed in response to cellular stress. There are housekeeping genes called sigma factors that are responsible for maintaining homeostasis in the cell and helping with protein folding. In the cytoplasm, stress, in particular misfolded protein, is largely regulated through the sigma32 pathway. Normally, sigma factor 32 is bound to heat shock proteins such as GroE and DnaK. In the presence of misfolding protein however, these heat shock proteins bind to the misfolded proteins, levaing sigma 32 free to form a complex with RNA Polymerase. This allows for transcription from various sigma-32 dependent promoters, driving the expression of anything downstream,. Many studies have found sigma-32 dependent promoters to be very effective at measuring levels of cytoplasm protein misfolding in E. Coli. One such promoter is the ibpAB promoter, which controls a heat shock operon in E. Coli.

iGEM Calgary cytoplasmic stress detection circuit

Our cytoplasmic stress reporter circuit uses a fusion of two sigma32-dependent heat shock promoter upstream of Green Fluorescent Protein (GFP).

The ibpAB Promoter

The ibpAB promoter controls the trasncription of two small proteins: ibpA and ibpB. These are small heat shock proteins called inclusion body binding proteins. In the presence of inclusion bodies within the cytoplasm, they are thought to form mixed complexes, ibpA allowing ibpB to bind to the inclusoon body at higher temperatures. The binding of these proteins to the misfolded protein lowers its hydrophobicity, preventing further binding of exposed peptide chains, thus stabilizing the protein and mediating its refolding by the DnaK/DnaJ/GrpE chaperone protein system (Matuszewska at al., 2005). Transcription levels from this promoter have been found to increase the most upon heat shock as compared to other heat shock promoters (Chuang et al., 1993).

We chose to use the ibpAB promoter in our system in order to monitor cytoplasm misfolding . We specifically chose to use a fusion promoter, which fuses fxsa, another heat shock promter in E. Coli that is not well known, to the ibpAb promoter. Kraft et al (1997) designed this fusion promoter and found it to be considerably more sensitive to misfoled protein in the cytoplasm than either of the promoters alone. We coupled this promoter with GFP downstream as our reporter. We then proceeded to measure GFP output in the presence of folded and msifolded proteins. For more information please visit our characterization page. .

ibpAB

B: MalE31 induction with IPTG; C: MalE31 induction and reporter reading with just ibpAB promoter; D: MalE31 induction and reporter reading with just fxsA promoter; E: MalE31 induction and reporter reading with ibpAB/FxsA fusion promoter (Kraft et al, 2006)

References

Kraft, M., Knupfer, U., Wenderoth, R., Pietschmann, P., Hock, B., & Horn, U. (2007). An online monitoring system based on a synthetic sigma32-dependent tandem promoter for visualization of insoluble proteins in the cytoplasm of escherichia coli. Applied Microbiology and Biotechnology, 75(2), 397-406.