Team:Calgary/Project/IbpAB

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<ul>
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<li><a href="http://2010.igem.org/Team:Calgary/Project/Transcription">Transcription/Translation Reporter Circuit</a></li>
<li><a href="http://2010.igem.org/Team:Calgary/Project/Transcription">Transcription/Translation Reporter Circuit</a></li>
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<li>Protein Misfolding Reporters
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<li><a href="http://2010.igem.org/Team:Calgary/Project/misfolding_overview">Protein Misfolding Reporters</a>
<ul>
<ul>
<li><a href="http://2010.igem.org/Team:Calgary/Project/IbpAB">Cytoplasmic Stress Detectors</a></li>
<li><a href="http://2010.igem.org/Team:Calgary/Project/IbpAB">Cytoplasmic Stress Detectors</a></li>
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<li><a href="http://2010.igem.org/Team:Calgary/Project/Controls">System Controls</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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<span id="bodytitle"><h1>Cytoplasmic Stress Detectors</h1></span>
<span id="bodytitle"><h1>Cytoplasmic Stress Detectors</h1></span>
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<h2>How does protein misfolding occur?</h2>
 
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<p>Protein misfolding can occur as a result of several factors. It can be
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<h2 style="color:#0066CC">How does a native <i>E. coli</i> cell combat protein related stress in the Cytoplasm?</h2>
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due to overproduction of the protein in the cell, in which case the cell
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lacks resources such as chaperones to fold the protein fast enough. This
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<p>
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can cause the proteins to misfold and form aggregate bodies. Proteins can
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      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.
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also misfold due to mutations that occur in the coding region of the
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</p>
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protein that can alter the amino acid sequence and thereby interrupting
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the native structure of the protein, causing it to misfold and be
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dysfunctional. Proteins can also misfold due to cellular stress such as a
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change in pH, temperature and change in media. Due to lack of optimal
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folding conditions proteins can form aggregate bodies and cause
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activation of heat shock systems, chaperone systems and proteolytic
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pathways which are involved in either refolding the proteins into their
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native form or degrading the aggregate bodies. Proteins can also misfold
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due to lack of localization. For example: if a periplasmic protein lacked
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a signal sequence it will misfold in the cytoplasm because the conditions
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are different in the two cellular compartments.</p>
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<h2>How does a native <i>E. coli</i> cell combat protein related stress?</h2>
 
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p>
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<h2 style="color:#0066CC">iGEM Calgary cytoplasmic stress detection circuit</h2>
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        There are several heat shock pathways in E. coli which are actively
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transcribed in response to cellular stress. This class of proteins are
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called small heat shock proteins (sHsps). sHsps consist of proteins such
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as ibpA, ibpB, DnaK, DnaJ, GroEL and GroES. Amongst these, IbpA and ibpB
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are two different proteins that are activated as a result of cytoplasmic
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stress response. IbpA and ibpB proteins are chaperones that are
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responsible for refolding aggregated bodies and inclusion bodies into
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their native conformation.</p>
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<h2> iGEM Calgary cytoplasmic stress detection circuit</h2>
 
<table>
<table>
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<img src="http://i872.photobucket.com/albums/ab287/iGEMCalgary_2010/ibpab-1.png"></img> </td>
<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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<h3>The ibpAB Promoter</h3>
<p>
<p>
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In our cytoplasmic stress detector circuit, we decided to fuse two
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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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different promoter regions from two heat shock proteins, which are ibpAB
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and fxsA. In a study done by Kraft et al, they demonstrate that a fusion
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<p>
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of IbpAB/fxsA promoters combined along with T7 DNA has a significantly
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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.
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higher output as a result of heat shock compared to the promoters
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.</p>
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individually. </p>
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<table>
<table>
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<td>
<td>
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<a href="http://s872.photobucket.com/albums/ab287/iGEMCalgary_2010/?action=view&current=ibpAB-2.png" target="_blank"><img src="http://i872.photobucket.com/albums/ab287/iGEMCalgary_2010/ibpAB-2.png" border="0" alt="ibpAB"></a>
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<a href="http://s872.photobucket.com/albums/ab287/iGEMCalgary_2010/?action=view&current=ibpAB-2.png" target="_blank"><img src="http://i872.photobucket.com/albums/ab287/iGEMCalgary_2010/ibpAB-2.png" border="0" alt="ibpAB"></a> </td>
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</tr>
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</table>
<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>
<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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</td>
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<object width="425" height="344"><param name="movie" value="http://www.youtube.com/v/CO_cK2D4l1s?hl=en&fs=1"></param><param name="allowFullScreen" value="true"></param><param name="allowscriptaccess" value="always"></param><embed src="http://www.youtube.com/v/CO_cK2D4l1s?hl=en&fs=1" type="application/x-shockwave-flash" allowscriptaccess="always" allowfullscreen="true" width="425" height="344"></embed></object>
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<td>
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<p>
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This fusion promoter will be connected to the registry part I13504 which
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is RBS-GFP-B0015. The ibpAB/fxsA circuit will be activated in the presence
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of aggregation in the cell. We will be using MalE31 with a signal sequence
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deletion (MalE31&#8710;SS) which was designed by Betton et al. The native
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E. coli protein MalE generally exported into the periplasmic space but
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this mutated protein does not get exported to the periplasmic space due to
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the signal sequence deletion.  Also Betton et al designed MalE31such that
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there are two amino acid changes in the protein and it misfolds. The
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MalE31&#8710;SS protein coding region will be used in order to induce
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cytoplasmic protein stress in E. coli. </p>
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<p>Ideally, this misfolded
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MalE31&#8710;SS should activate the plasmid system containing
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ibpAB/fxsA-I13504 which will produce GFP alerting the researcher that
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their protein is not being expressed in the cell because it is misfolding
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and as a result getting degraded. Our circuit should also be activated
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much faster than the native stress system because the ibpAB/fxsA promoter
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is much more sensitive to the presence of aggregate bodies in the cell.
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The promoter also gives a much higher output compared to the promoters
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individually, which is the case in the E. coli genome which should allow
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us to detect the fluorescence level much faster.
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</p>
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</td>
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</table>
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<h2 style="color:#0066CC">References</h2>
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<p>
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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>
</div>
</div>

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.