http://2010.igem.org/wiki/index.php?title=Special:Contributions&feed=atom&limit=250&target=Emily+Hicks&year=&month=2010.igem.org - User contributions [en]2024-03-28T19:30:26ZFrom 2010.igem.orgMediaWiki 1.16.5http://2010.igem.org/Team:Calgary/Project/TranscriptionTeam:Calgary/Project/Transcription2010-10-28T03:59:28Z<p>Emily Hicks: </p>
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<h1>Project Descriptions</h1><br />
<br />
<br />
<ul><br />
<li><a href="https://2010.igem.org/Team:Calgary/Project/Transcription">Transcription/Translation Reporter Circuit</a></li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Project/misfolding_overview">Protein Misfolding Reporters</a><br />
<ul><br />
<li><a href="https://2010.igem.org/Team:Calgary/Project/IbpAB">Cytoplasmic Stress Detectors</a></li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Project/CpxP">Periplasmic Stress Detectors</a></li><br />
</ul><br />
</li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Project/Controls">Testing Our System</a></li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Project/Achievements">Achievements</a></li><br />
</ul><br />
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<span id="bodytitle"><h1>Transcription/Translation Circuit</h1></span><br />
<br />
<h2 style="color:#0066CC">Overview</h2><br />
<p><br />
Transcription and translation are essential processes for protein expression. Problems that arise during these processes could lead to improper protein formation. Issues that can occur include shortage in length, folding problems, low or no expression, etc. These issues are accentuated in synthetic biology as foreign genes are implemented into prokaryotes such as <i>Escherichia coli</i>. The transcription translation detector circuit was developed in order to test whether or not a gene of interest is being correctly transcribed and translated.</p><br /><br />
<br />
<h3>How the Circuit Works</h3><br />
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The gene of interest is fused to a mutant RFP. Downstream of this is GFP with its own ribosomal binding site. If transcription is occurring, the transcript would include the gene of interest, RFP as well as GFP. Because GFP has its own ribosomal biding site, it should be translated if transcription is happening.</p><br />
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If the gene of interest is also being translated, then RFP should also be translated because it is fused to the GOI.the RFP was specially selected from Dr. Lewenza’s lab. This RFP (nicknamed sRFP or special red fluorescent protein) can fold in the cytoplasm, periplasm and the cellular membrane.<br />
</p><br />
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<h2 style="color:#0066CC">Details</h2><br />
<h3>Assumptions:</h3><br />
<p><br />
For this circuit to work, there are several assumptions that must be made. The first of which is a result of a limitation within the design which is that sRFP will not affect the stability of the protein of interest. Both positives and negatives are not ideal because the circuit functions as an indicator, any assistance could lead to false positives or vice versa. Second, AraC is the right promoter for this circuit. Although there are many benefits for using an arabinose inducible promoter, however evolutionary conditions have established optimal expression in natural promoters. Third is folding properties in the periplasm and cytoplasm (Lewenza, et al., 2006) had to be the same such that a sRFP in the cytoplasm will give the same absorbance as one in the periplasm. Fourth would be that the GOI does not contain a “rut” site (Rho utilization site) which would prematurely stop transcription using Rho dependent termination. Fifth would be that <i>E.coli</i> would be the most compatible cell available for protein expression. Much like the second assumption, genes are optimally expressed in its natural host. Transferring these genes into E.coli might decrease the efficiency of protein expression. These are considerations that must be made in order to ensure the success of this circuit towards its utilization within our testing kit. It is definitely more “artificial” compared to the other two mostly because it overrides the necessity for the natural systems within. However if all limitations are accounted for, this could be a very useful tool if coupled with our other systems. </p><br />
<br />
<p>Helpful tips with understanding the circuit: With the way the circuit is developed, a failure of transcription will lead to a failure of translation. Therefore it is impossible to see only red cells, but possible to see green cells. If a brownish color is expressed (a mixture of red and green), this indicates both transcription and translation. Also if only green cells are noticed, then to definitively test whether or not there is something wrong with translation, a user must employ the other two circuits. Meaning positive in the folding circuits indicates the translation mechanism works. However, due to the design of attaching sRFP with the GOI, the GOI misfolding will affect the stability of sRFP.</p><br /><br /><br />
<br />
<h3>Problems that can arise during transcription / translation:</h3><br />
<p>There are numerous problems that can arise in the transcription and translation, especially when trying to turn <i>E.coli</i> into a factory for foreign proteins. Each category of transcription and translation can be broken down into pre, during, and post. Although some aspects between post-transcription and pre-translation are slightly grey, there are parts of it that are quite clear. For example, the attachment of the 30S subunit from rRNA would be considered pre-translational but not post-transcriptional. This section describes some of the possible transcription/translation issues and the following responses by the system. </p><br /><br />
<br />
<h4 style="color:#003366">Transcription</h4><br />
<p><b>Pre-transcription</b></p><br />
<p><em>Transcription factors</em></p><br />
<p>One of the main ideas of synthetic biology is the expression of proteins from foreign enzymes. For example, GFP comes from Aequorea Victoria (Andersen, et al., 1998) . One of the considerations is whether or not foreign circuits have the corresponding transcriptions within <i>E.coli</i>. If these transcription factors have a profound effect on whether or not transcription can occur (Kleinert, et al., 2003) , then natural promoters might be hindered or lack the necessary transcription factors for expression. Therefore it is necessary to include an arabinose promoter (pBad/araC), a well characterized and working promoter in <i>E.coli</i>. (iGEM registry,2003) </p><br />
<br />
<p>If the problem of the foreign circuit lies in the promoter, the circuit can be used to detect this simply through inserting the RBS+GOI into the circuit and compare this with inserting the foreign promoter + RBS + GOI. If there is expression without the foreign promoter, and no expression with it, then there could be a repressor bounded to the operon of the circuit. If there is expression in both then a third circuit can be constructed with just the foreign promoter + RBS + GOI without the arabinose promoter. If there is no expression in the third, then the foreign promoter lacks the necessary transcription factors to operate in the host <i>E.coli</i>.</p> <br />
<br />
<p><em>Promoter strength</em></p><br />
<p>This is not a problem with natural promoters however this is an issue faced by many synthetic biologist when matching a promoter with a GOI. More is not always better, over expression of protein could lead to higher amounts of aggregation and longer folding time.(Brock, 2010) Choosing the pBAD/araC promoter is beneficial because induction varies with arabinose concentrations. Therefore it is possible to use a 96 well plate with varying levels of arabinose to promote induction at various strengths. A plate reader can then be used to read absorbance levels to find the optimal amount of indicator expressed.</p><br /><br />
<br />
<p><b>Transcription</b></p><br />
<p><em>Repressor/amount of inducer</em></p> <br />
<p>The ratio of inducer to plasmid copy number would be a problem when trying to express a foreign protein in <i>E.coli</i>. Much like issue with transcription factors, the circuit was designed to include an arabinose promoter that way it is possible to control the concentration of the inducer arabinose. In that case there will be no shortage in the concentration of inducer because the promoter is well characterized meaning its induction is known.</p><br />
<br />
<p><em>Hair pin loop/rho dependent termination</em></p><br />
<p>The formation of premature hair pin loops and rho utilization sites formed from within the gene are potential methods of premature stops to transcription. Hair pin loops are typically 7 to 20 amino acids long (Lewin, 2007) and ruts sites are 22-116 base pairs.(Banerjee, et al., 2007) The more likely of the two when forming an accidental termination site would be a hair pin loop. This relies on the palindrome formation with high concentrations of guanine and cysteine which results in a RNA pulling from the DNA. Our system would detect premature termination of RNA would result in no signal with our GFP signal.</p><br /><br />
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<p><b>Post-transcription</b></p><br />
<p><em>mRNA shape degradation</em></p><br />
<p>Although transcription occurs, mRNA instability results in the degradation of the mRNA. The circuit would suggest that the GFP report was not expressed. Despite transcription occurring completely, the most logical approach would be to group this under issues with transcription, also because pre-translational steps have not occurred yet.</p><br /><br />
<br />
<h4 style="color:#003366">Translation</h4><br />
<p><b>Pre-translation</b></p><br />
<p>No current issues arise from this step.</p><br /><br />
<p><b>Translation</b></p><br />
<p><em>Multi codon usage</em></p> <br />
<p>When inserting foreign genes into <i>E.coli</i>, the ratios of tRNAs in <i>E.coli</i> in comparison to the foreign source can vary. Shortages in tRNA can lead to problems with rate and accuracy of translation. (Ran and Higgs, 2010) Kinetics is a factor of rate of protein formation, decreased concentrations of necessary tRNAs results in slower formation of proteins. Based on the research by Drummond and Wilke, lack of accuracy is caused by mistranslation causing higher amount of misfolding.(Drummond and Wilke, 2008) If the GOI’s multi codon usage disagrees with the host <i>E.coli</i>, there would be aggregation which will inhibit protein expression. The circuit can detect that there are problems with translation because sRFP would be form aggregate bodies with the protein of interest (POI). </p><br />
<br />
<br />
<p><em>Premature stop codon</em><p><br />
<p>Stop codons inhibit translation. The circuit would indicate the presence of a premature stop codon because the sRFP would not be translated therefore no signal would be present. </p><br />
<br />
<p><em>RBS compatibility</em></p><br />
<p>The ribosome binding site allows the attachment of the ribosome. Differences in ribosome strength could change the translation frequencies. This leaves room for protein misfolding. Because of the specificity of the RBS to the expression of the gene, as well as the potential of affecting the triple nucleotide site which could shift the reading frame. The circuit was designed in such a way that the user is capable of attaching their own RBS.</p><br />
<br />
<p><em>Copy number</em></p><br />
<p>Copy number refers to the number of plasmids that can exist within on <i>E.coli</i> cell.(iGEM Registry, 2009) Although this does not change the rate of transcription (polymerase per second, PoPs) like promoter strength, the effects are similar. Increasing the concentration of slower folding proteins could result in aggregation due to exposed hydrophobic segments . (Ran and Higgs, 2010) The circuit will detect this as an issue with translation as this could affect the protein.</p><br /><br />
<br />
<p><b>Post-translation</b></p><br />
<p><em>Lack of chaperones</em></p><br />
<p>The lack of essential chaperones could result in protein misfolding. <i>E.coli</i> may not have the necessary chaperones to correct the conformation of the POI. The formation of misfolded protein will cause the aggregation of sRFP, therefore indicating an error in translation.</p><br /><br /> <br />
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<h2 style="color:#0066CC">Design of the Circuit</h2><br />
<center><a href="http://s872.photobucket.com/albums/ab287/iGEMCalgary_2010/?action=view&current=transcriptiontranslationcircuit.png" target="_blank"><img src="http://i872.photobucket.com/albums/ab287/iGEMCalgary_2010/transcriptiontranslationcircuit.png" border="0" alt="Photobucket"></a></center><br />
<p>pBad/araC Promoter- This promoter was chosen because it allow for variable strength without replacing the promoter (if the circuit had a promoter library). Because more is not always better, the user can customize optimal levels of promoter strength in protein expression. This part is also highly characterized (iGEM Registry, 2003).</p><br />
<br />
<p>Multiple Cloning Sites- We are using modified biobrick prefix and suffix. What this means is that these sites are not separating the biobrick parts from the sequences, rather they are located between the arabinose promoter and the sRFP.</p><br />
<p>ccdB- A place holder that was added for selection in addition to antibiotic selection. The circuit will contain a suicide ccdB gene as a placeholder for the GOI. If this is not removed, the cell which has this transformed plasmid will die. This will ensure that the only cells present on the plate will only express the genes intended to be there.</p> <br />
<p>RBS- We have decided not include a RBS within this sequence to allow customizability. Natural RBS are known to indicate optimal PoPs plus issues with this would indicate compatibility problems on the part of the RBS and GOI. This would also eliminate any issues regard reading frame shifts of the RBS to the GOI for those that are biobricking new parts.</p> <br />
<p>sRFP (special red fluorescent protein)- This is part of the translation portion of the circuit. This indicator was chosen because it can fold in the cytoplasm, periplasm and membranes.(Lewenza, et al., 2006) One of the limitations of this circuit is that the GOI must be fused to sRFP in order for translation detection to occur. This means additional time on the part of the user to rebiobrick the end portion of the GOI such that the stop codons are removed. Current studies by Lewenza, et al. reveals that RFP can be localized in the cytoplasm as well as the outer membrane.</p> <br />
<br />
<h2 style="color:#0066CC">Reference:</h2><br />
<br />
<p>1. Andersen, J. B., Sternberg, C., Poulsen, L. K., Bjorn, S. P., Givskov, M., Molin, S., et al. (1998). New Unstable Variants of Green Fluorescent Protein for Studies of Transient Gene Expression in Bacteria. Appl. Envir. Microbiol., 64(6), 2240-2246. Retrieved from http://aem.asm.org/cgi/content/abstract/64/6/2240.</p><br />
<p>2. Banerjee, S., Chalissery, J., Bandey, I., & Sen, R. (2006). Rho-dependent transcription termination: more questions than answers. Journal of microbiology (Seoul, Korea), 44(1), 11-22. Retrieved from: http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1838574&tool=pmcentrez&rendertype=abstract.</p><br />
<p>3. Drummond, D. A., & Wilke, C. O. (2008). Mistranslation-induced protein misfolding as a dominant constraint on coding-sequence evolution. Cell, 134(2), 341-52. doi: 10.1016/j.cell.2008.05.042.</p><br />
<p>4. Help:plasmid backbones/features. (2008). Retrieved from http://partsregistry.org/Help:Plasmid_backbones/Features<p><br />
<br />
<p>5. Kleinert, H., Schwarz, P. M., & Förstermann, U. (n.d.). Regulation of the expression of inducible nitric oxide synthase. Biological chemistry, 384(10-11), 1343-64. doi: 10.1515/BC.2003.152.</p><br />
<p>6. Kosuri, S. (2003, December 5). Part:bba_i0500. Retrieved from http://partsregistry.org/Part:BBa_I0500</p><br />
<br />
<p>7. Lewin, Benjamin (2007). Genes IX. Sudbury, MA: Jones and Bartlett Publishers.</p><br />
<p>8. Lewenza, S., Vidal-Ingigliardi, D., & Pugsley, A. P. (2006). Direct visualization of red fluorescent lipoproteins indicates conservation of the membrane sorting rules in the family Enterobacteriaceae. Journal of bacteriology, 188(10), 3516-24. doi: 10.1128/JB.188.10.3516-3524.2006.</p><br />
<p>9. Madigan, M.T., Martinko, J.M., Dunlap, P.V., & Clark, D.P. (2008). Brock biology of microorganisms (12th edition). San Francisco, California: Benjamin Cummings.</p><br />
<br />
<p>10. Ran, W., & Higgs, P. G. (2010). The influence of anticodon-codon interactions and modified bases on codon usage bias in bacteria. Molecular biology and evolution, 27(9), 2129-40. doi: 10.1093/molbev/msq102.</p><br />
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</html></div>Emily Hickshttp://2010.igem.org/Team:Calgary/Project/TranscriptionTeam:Calgary/Project/Transcription2010-10-28T03:58:46Z<p>Emily Hicks: </p>
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<h1>Project Descriptions</h1><br />
<br />
<br />
<ul><br />
<li><a href="https://2010.igem.org/Team:Calgary/Project/Transcription">Transcription/Translation Reporter Circuit</a></li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Project/misfolding_overview">Protein Misfolding Reporters</a><br />
<ul><br />
<li><a href="https://2010.igem.org/Team:Calgary/Project/IbpAB">Cytoplasmic Stress Detectors</a></li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Project/CpxP">Periplasmic Stress Detectors</a></li><br />
</ul><br />
</li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Project/Controls">Testing Our System</a></li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Project/Achievements">Achievements</a></li><br />
</ul><br />
<br />
</div><br />
<br />
<div class="mainbody"><br />
<br />
<span id="bodytitle"><h1>Transcription/Translation Circuit</h1></span><br />
<br />
<h2 style="color:#0066CC">Overview</h2><br />
<p><br />
Transcription and translation are essential processes for protein expression. Problems that arise during these processes could lead to improper protein formation. Issues that can occur include shortage in length, folding problems, low or no expression, etc. These issues are accentuated in synthetic biology as foreign genes are implemented into prokaryotes such as <i>Escherichia coli</i>. The transcription translation detector circuit was developed in order to test whether or not a gene of interest is being correctly transcribed and translated.</p><br /><br />
<br />
<h3>How the Circuit Works</h3><br />
<object width="425" height="344"><param name="movie" value="http://www.youtube.com/v/3-e35n_PxeA?hl=en&fs=1"></param><param name="allowFullScreen" value="true"></param><param name="allowscriptaccess" value="always"></param><embed src="http://www.youtube.com/v/3-e35n_PxeA?hl=en&fs=1" type="application/x-shockwave-flash" allowscriptaccess="always" allowfullscreen="true" width="425" height="344"></embed></object><br />
<table><br />
<tr><br />
<td><br />
<p><br />
The gene of interest is fused to a mutant RFP. Downstream of this is GFP with its own ribosomal binding site. If transcription is occurring, the transcript would include the gene of interest, RFP as well as GFP. Because GFP has its own ribosomal biding site, it should be translated if transcription is happening.</p><br />
</td><br />
</tr><br />
</table> <br />
<br/><br />
<table><br />
<tr><br />
<td><br />
<p><br />
If the gene of interest is also being translated, then RFP should also be translated because it is fused to the GOI.the RFP was specially selected from Dr. Lewenza’s lab. This RFP (nicknamed sRFP or special red fluorescent protein) can fold in the cytoplasm, periplasm and the cellular membrane.<br />
</p><br />
</td><br />
</tr><br />
</table><br /><br /><br />
<br />
<h2 style="color:#0066CC">Details</h2><br />
<h3>Assumptions:</h3><br />
<p><br />
For this circuit to work, there are several assumptions that must be made. The first of which is a result of a limitation within the design which is that sRFP will not affect the stability of the protein of interest. Both positives and negatives are not ideal because the circuit functions as an indicator, any assistance could lead to false positives or vice versa. Second, AraC is the right promoter for this circuit. Although there are many benefits for using an arabinose inducible promoter, however evolutionary conditions have established optimal expression in natural promoters. Third is folding properties in the periplasm and cytoplasm (Lewenza, et al., 2006) had to be the same such that a sRFP in the cytoplasm will give the same absorbance as one in the periplasm. Fourth would be that the GOI does not contain a “rut” site (Rho utilization site) which would prematurely stop transcription using Rho dependent termination. Fifth would be that <i>E.coli</i> would be the most compatible cell available for protein expression. Much like the second assumption, genes are optimally expressed in its natural host. Transferring these genes into E.coli might decrease the efficiency of protein expression. These are considerations that must be made in order to ensure the success of this circuit towards its utilization within our testing kit. It is definitely more “artificial” compared to the other two mostly because it overrides the necessity for the natural systems within. However if all limitations are accounted for, this could be a very useful tool if coupled with our other systems. </p><br />
<br />
<p>Helpful tips with understanding the circuit: With the way the circuit is developed, a failure of transcription will lead to a failure of translation. Therefore it is impossible to see only red cells, but possible to see green cells. If a brownish color is expressed (a mixture of red and green), this indicates both transcription and translation. Also if only green cells are noticed, then to definitively test whether or not there is something wrong with translation, a user must employ the other two circuits. Meaning positive in the folding circuits indicates the translation mechanism works. However, due to the design of attaching sRFP with the GOI, the GOI misfolding will affect the stability of sRFP.</p><br /><br /><br />
<br />
<h3>Problems that can arise during transcription / translation:</h3><br />
<p>There are numerous problems that can arise in the transcription and translation, especially when trying to turn <i>E.coli</i> into a factory for foreign proteins. Each category of transcription and translation can be broken down into pre, during, and post. Although some aspects between post-transcription and pre-translation are slightly grey, there are parts of it that are quite clear. For example, the attachment of the 30S subunit from rRNA would be considered pre-translational but not post-transcriptional. This section describes some of the possible transcription/translation issues and the following responses by the system. </p><br /><br />
<br />
<h4 style="color:#003366">Transcription</h4><br />
<p><b>Pre-transcription</b></p><br />
<p><em>Transcription factors</em></p><br />
<p>One of the main ideas of synthetic biology is the expression of proteins from foreign enzymes. For example, GFP comes from Aequorea Victoria (Andersen, et al., 1998) . One of the considerations is whether or not foreign circuits have the corresponding transcriptions within <i>E.coli</i>. If these transcription factors have a profound effect on whether or not transcription can occur (Kleinert, et al., 2003) , then natural promoters might be hindered or lack the necessary transcription factors for expression. Therefore it is necessary to include an arabinose promoter (pBad/araC), a well characterized and working promoter in <i>E.coli</i>. (iGEM registry,2003) </p><br />
<br />
<p>If the problem of the foreign circuit lies in the promoter, the circuit can be used to detect this simply through inserting the RBS+GOI into the circuit and compare this with inserting the foreign promoter + RBS + GOI. If there is expression without the foreign promoter, and no expression with it, then there could be a repressor bounded to the operon of the circuit. If there is expression in both then a third circuit can be constructed with just the foreign promoter + RBS + GOI without the arabinose promoter. If there is no expression in the third, then the foreign promoter lacks the necessary transcription factors to operate in the host E.coli.</p> <br />
<br />
<p><em>Promoter strength</em></p><br />
<p>This is not a problem with natural promoters however this is an issue faced by many synthetic biologist when matching a promoter with a GOI. More is not always better, over expression of protein could lead to higher amounts of aggregation and longer folding time.(Brock, 2010) Choosing the pBAD/araC promoter is beneficial because induction varies with arabinose concentrations. Therefore it is possible to use a 96 well plate with varying levels of arabinose to promote induction at various strengths. A plate reader can then be used to read absorbance levels to find the optimal amount of indicator expressed.</p><br /><br />
<br />
<p><b>Transcription</b></p><br />
<p><em>Repressor/amount of inducer</em></p> <br />
<p>The ratio of inducer to plasmid copy number would be a problem when trying to express a foreign protein in <i>E.coli</i>. Much like issue with transcription factors, the circuit was designed to include an arabinose promoter that way it is possible to control the concentration of the inducer arabinose. In that case there will be no shortage in the concentration of inducer because the promoter is well characterized meaning its induction is known.</p><br />
<br />
<p><em>Hair pin loop/rho dependent termination</em></p><br />
<p>The formation of premature hair pin loops and rho utilization sites formed from within the gene are potential methods of premature stops to transcription. Hair pin loops are typically 7 to 20 amino acids long (Lewin, 2007) and ruts sites are 22-116 base pairs.(Banerjee, et al., 2007) The more likely of the two when forming an accidental termination site would be a hair pin loop. This relies on the palindrome formation with high concentrations of guanine and cysteine which results in a RNA pulling from the DNA. Our system would detect premature termination of RNA would result in no signal with our GFP signal.</p><br /><br />
<br />
<br />
<p><b>Post-transcription</b></p><br />
<p><em>mRNA shape degradation</em></p><br />
<p>Although transcription occurs, mRNA instability results in the degradation of the mRNA. The circuit would suggest that the GFP report was not expressed. Despite transcription occurring completely, the most logical approach would be to group this under issues with transcription, also because pre-translational steps have not occurred yet.</p><br /><br />
<br />
<h4 style="color:#003366">Translation</h4><br />
<p><b>Pre-translation</b></p><br />
<p>No current issues arise from this step.</p><br /><br />
<p><b>Translation</b></p><br />
<p><em>Multi codon usage</em></p> <br />
<p>When inserting foreign genes into <i>E.coli</i>, the ratios of tRNAs in <i>E.coli</i> in comparison to the foreign source can vary. Shortages in tRNA can lead to problems with rate and accuracy of translation. (Ran and Higgs, 2010) Kinetics is a factor of rate of protein formation, decreased concentrations of necessary tRNAs results in slower formation of proteins. Based on the research by Drummond and Wilke, lack of accuracy is caused by mistranslation causing higher amount of misfolding.(Drummond and Wilke, 2008) If the GOI’s multi codon usage disagrees with the host <i>E.coli</i>, there would be aggregation which will inhibit protein expression. The circuit can detect that there are problems with translation because sRFP would be form aggregate bodies with the protein of interest (POI). </p><br />
<br />
<br />
<p><em>Premature stop codon</em><p><br />
<p>Stop codons inhibit translation. The circuit would indicate the presence of a premature stop codon because the sRFP would not be translated therefore no signal would be present. </p><br />
<br />
<p><em>RBS compatibility</em></p><br />
<p>The ribosome binding site allows the attachment of the ribosome. Differences in ribosome strength could change the translation frequencies. This leaves room for protein misfolding. Because of the specificity of the RBS to the expression of the gene, as well as the potential of affecting the triple nucleotide site which could shift the reading frame. The circuit was designed in such a way that the user is capable of attaching their own RBS.</p><br />
<br />
<p><em>Copy number</em></p><br />
<p>Copy number refers to the number of plasmids that can exist within on <i>E.coli</i> cell.(iGEM Registry, 2009) Although this does not change the rate of transcription (polymerase per second, PoPs) like promoter strength, the effects are similar. Increasing the concentration of slower folding proteins could result in aggregation due to exposed hydrophobic segments . (Ran and Higgs, 2010) The circuit will detect this as an issue with translation as this could affect the protein.</p><br /><br />
<br />
<p><b>Post-translation</b></p><br />
<p><em>Lack of chaperones</em></p><br />
<p>The lack of essential chaperones could result in protein misfolding. <i>E.coli</i> may not have the necessary chaperones to correct the conformation of the POI. The formation of misfolded protein will cause the aggregation of sRFP, therefore indicating an error in translation.</p><br /><br /> <br />
<br />
<h2 style="color:#0066CC">Design of the Circuit</h2><br />
<center><a href="http://s872.photobucket.com/albums/ab287/iGEMCalgary_2010/?action=view&current=transcriptiontranslationcircuit.png" target="_blank"><img src="http://i872.photobucket.com/albums/ab287/iGEMCalgary_2010/transcriptiontranslationcircuit.png" border="0" alt="Photobucket"></a></center><br />
<p>pBad/araC Promoter- This promoter was chosen because it allow for variable strength without replacing the promoter (if the circuit had a promoter library). Because more is not always better, the user can customize optimal levels of promoter strength in protein expression. This part is also highly characterized (iGEM Registry, 2003).</p><br />
<br />
<p>Multiple Cloning Sites- We are using modified biobrick prefix and suffix. What this means is that these sites are not separating the biobrick parts from the sequences, rather they are located between the arabinose promoter and the sRFP.</p><br />
<p>ccdB- A place holder that was added for selection in addition to antibiotic selection. The circuit will contain a suicide ccdB gene as a placeholder for the GOI. If this is not removed, the cell which has this transformed plasmid will die. This will ensure that the only cells present on the plate will only express the genes intended to be there.</p> <br />
<p>RBS- We have decided not include a RBS within this sequence to allow customizability. Natural RBS are known to indicate optimal PoPs plus issues with this would indicate compatibility problems on the part of the RBS and GOI. This would also eliminate any issues regard reading frame shifts of the RBS to the GOI for those that are biobricking new parts.</p> <br />
<p>sRFP (special red fluorescent protein)- This is part of the translation portion of the circuit. This indicator was chosen because it can fold in the cytoplasm, periplasm and membranes.(Lewenza, et al., 2006) One of the limitations of this circuit is that the GOI must be fused to sRFP in order for translation detection to occur. This means additional time on the part of the user to rebiobrick the end portion of the GOI such that the stop codons are removed. Current studies by Lewenza, et al. reveals that RFP can be localized in the cytoplasm as well as the outer membrane.</p> <br />
<br />
<h2 style="color:#0066CC">Reference:</h2><br />
<br />
<p>1. Andersen, J. B., Sternberg, C., Poulsen, L. K., Bjorn, S. P., Givskov, M., Molin, S., et al. (1998). New Unstable Variants of Green Fluorescent Protein for Studies of Transient Gene Expression in Bacteria. Appl. Envir. Microbiol., 64(6), 2240-2246. Retrieved from http://aem.asm.org/cgi/content/abstract/64/6/2240.</p><br />
<p>2. Banerjee, S., Chalissery, J., Bandey, I., & Sen, R. (2006). Rho-dependent transcription termination: more questions than answers. Journal of microbiology (Seoul, Korea), 44(1), 11-22. Retrieved from: http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1838574&tool=pmcentrez&rendertype=abstract.</p><br />
<p>3. Drummond, D. A., & Wilke, C. O. (2008). Mistranslation-induced protein misfolding as a dominant constraint on coding-sequence evolution. Cell, 134(2), 341-52. doi: 10.1016/j.cell.2008.05.042.</p><br />
<p>4. Help:plasmid backbones/features. (2008). Retrieved from http://partsregistry.org/Help:Plasmid_backbones/Features<p><br />
<br />
<p>5. Kleinert, H., Schwarz, P. M., & Förstermann, U. (n.d.). Regulation of the expression of inducible nitric oxide synthase. Biological chemistry, 384(10-11), 1343-64. doi: 10.1515/BC.2003.152.</p><br />
<p>6. Kosuri, S. (2003, December 5). Part:bba_i0500. Retrieved from http://partsregistry.org/Part:BBa_I0500</p><br />
<br />
<p>7. Lewin, Benjamin (2007). Genes IX. Sudbury, MA: Jones and Bartlett Publishers.</p><br />
<p>8. Lewenza, S., Vidal-Ingigliardi, D., & Pugsley, A. P. (2006). Direct visualization of red fluorescent lipoproteins indicates conservation of the membrane sorting rules in the family Enterobacteriaceae. Journal of bacteriology, 188(10), 3516-24. doi: 10.1128/JB.188.10.3516-3524.2006.</p><br />
<p>9. Madigan, M.T., Martinko, J.M., Dunlap, P.V., & Clark, D.P. (2008). Brock biology of microorganisms (12th edition). San Francisco, California: Benjamin Cummings.</p><br />
<br />
<p>10. Ran, W., & Higgs, P. G. (2010). The influence of anticodon-codon interactions and modified bases on codon usage bias in bacteria. Molecular biology and evolution, 27(9), 2129-40. doi: 10.1093/molbev/msq102.</p><br />
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<li><a href="https://2010.igem.org/Team:Calgary/Notebook/Safety">Safety</a></li><br />
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<span id="bodytitle"><h1>iGEM Safety Questionnaire</h1></span><br />
<p><br />
As a safety precaution, all iGEM team members have been fully trained in WHIMIS as well as in introductory Biosfatey.<br />
</p><br /><br />
<p><br />
<em>Would any of your project ideas raise safety issues in terms of:</em><br /> <br />
<em>researcher safety</em><br />
<em>public safety, or</em> <br />
<em>environmental safety?</em> <br />
</p><br />
<p><br />
No, not directly. The goal of our project is to create a tool that can help solve protein expression problems in future projects both within the context of the iGEM competiion and beyond. For this reason, our project only really poses safety issues if it was to be used to aid in the expression of toxic or otherwise dangerous proteins. Part of our ethical analysis included a discussion of some of the specific ethical, social and economic issues raised by our project. Check out our ethics page for more information.<br />
</p><br />
<br /><br />
<p><em>Do any of the new BioBrick parts (or devices) that you made this year raise any safety issues?</em></p> <br />
<p><br />
No, none of the parts that we made alone raise any safety issues. Again, our project only has the possibility of posing safety issues in regards to the final product the user of our tool kit is trying to produce.<br />
</p><br />
<br /><br />
<p><br />
<em>Is there a local biosafety group, committee, or review board at your institution?</em><br />
</p> <br />
<p><br />
Yes, we have an office of medical bioethics at our Univeristy. Our project has never come up with this office. Due to the fact that we are using non-pathogenic bacteria and that our project poses no direct safety issues to researchers the public or the environment, they have no concerns with our project at present.</p><br />
<br /><br />
<p><br />
<em>Do you have any other ideas how to deal with safety issues that could be useful for future iGEM competitions? How could parts, devices and systems be made even safer through biosafety engineering?</em></p><br />
<br />
<p><br />
One of the major safety issues with the Registry of Standard Biological parts is that it is open source. Sequences are readily available to anyone that wishes to access hem. Although this is a very helpful resource, it also has the potential to cause harm if the sequences are for toxic or dangerous gene products. idea that our team thought of was to start reviewing sequences that are submitted the registry, and comparing them to a database of pathogenic sequences to ensure that it is not used for the distribution of any harmful sequences. This would be similar to the process that gene synthesis companies who are members of the IGSC use to ensure they don't make any pathogenic or otherwise dangerous sequences.<br />
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<h2>The Protein Man!</h2><br />
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<p>Hey iGEM-ers,</p><br />
<p><br />
This is Protein Man checking in! I hope the iGEM competition is not stressing you out too much! Remember, stress causes protein misfolding, and my job is to promote proper protein expression. I will be the mascot of the 2010 iGEM Calgary team, whose project is about stress detection in our favourite bug E. coli or any other bugs that y’all might be using for your project.</p><br />
<p><br />
<br />
I wish you all good luck and keep an eye out for me at the iGEM jamboree! And ladies, you can totally aggregate around me to relieve your stress. *wink</p><br />
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<p>Checking out,</p><br />
<p><br />
Protein man!</p><br />
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<h2>T-Shirts</h2><br />
<p>T-shirts were printed by Apparel Ink. T-shirts were printed in yellow. Photos of T-shirt coming soon!<br />
<li>6455 Macleod Trail South</li><br />
<li>Calgary,AB. T2H 0K8</li><br />
<li>Ph:403-255-1150</li><br />
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<h2>The Protein Man!</h2><br />
<br />
<br />
<br />
<p>Hey iGEM-ers,</p><br />
<p><br />
This is Protein Man checking in! I hope the iGEM competition is not stressing you out too much! Remember, stress causes protein misfolding and my job is to promote proper protein expression. I will be the mascot of the 2010 iGEM Calgary team, whose project is about stress detection in our favourite bug E. coli or any other bugs that y’all might be using for your project.</p><br />
<p><br />
<br />
I wish you all good luck and keep an eye out for me at the iGEM jamboree and ladies you can aggregate around me to relive your stress.</p><br />
<br />
<p>Checking out,</p><br />
<p><br />
Protein man!</p><br />
<br />
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<li><a href="https://2010.igem.org/Team:Calgary/Project/Transcription">Transcription/Translation Reporter Circuit</a></li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Project/misfolding_overview">Protein Misfolding Reporters</a><br />
<ul><br />
<li><a href="https://2010.igem.org/Team:Calgary/Project/IbpAB">Cytoplasmic Stress Detectors</a></li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Project/CpxP">Periplasmic Stress Detectors</a></li><br />
</ul><br />
</li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Project/Controls">Testing Our System</a></li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Project/Achievements">Achievements</a></li><br />
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<div class="mainbody"><br />
<br />
<span id="bodytitle"><h1>The Project</h1></span><br />
<br />
<h2 style="color:#0066CC">Overview</h2><br />
<p>Many synthetic biology projects involve the expression of recombinant proteins in microorganisms such as <i>E. coli</i>. The problems encountered with many synthetic biology projects often involve problems with protein expression. It is often very difficult to recognize the problem and pinpoint where it lies. The goal of the University of Calgary 2010 iGEM team is to build a protein expression "troubleshooting kit". This kit will contain two systems with which target genes can be inserted. In the resulting cell growth, fluorescent protein production will be used to determine whether there is a problem with protein expression as well as indicate where the protein expression is failing.</p><br />
<br />
<br />
<p><br />
Protein expression happens in three steps: the transcription of cell DNA to mRNA, the translation of mRNA into an amino acid sequence, and the folding of that amino acid sequence into a protein.</p><br />
<br />
<br />
<br />
<img src="http://i872.photobucket.com/albums/ab287/iGEMCalgary_2010/Slide1.png?t=1288229208"></img><br />
<br />
<br /><br />
<br />
<h2 style="color:#0066CC">Sites of Failure</h2><br />
<p><br />
Protein expression can fail at any point along these three steps. Our system uses two circuits to detect at which step possible errors have occured. The first circuit has a fluorescent reporter (GFP) that is produced when DNA is transcribed into mRNA and another (RFP) that is produced when mRNA is translated into a functional protein. When both reporter proteins are expressed in the cell, it indicates both transcription and translation are occurring. The second circuit involves reporter systems that are activated as a result of protein misfolding. Two native stress-activated promoters from <i>E. coli</i> were engineered upstream to fluorescent reporters that will respond to periplasmic and cytoplasmic protein misfolding. If the protein of interest misfolds in either area of the cell, one of the promoters will be activated and the corresponding fluorescence will be observed.<br />
</p><br />
<br />
<h3>Transcription</h3><br />
<table><br />
<tr><br />
<td><br />
<center><img style="width:200px;" src="http://i872.photobucket.com/albums/ab287/iGEMCalgary_2010/DNAmRNA001-1.png"></img></center><br />
</td><br />
<td><br />
<br />
<p>Transcription is the process by which the cell makes messenger RNA (mRNA) from DNA using RNA polymerase. The mRNA sequence is complementary to the DNA sequence that is being transcribed and uses adenine, cytosine, guanine, and uracil nucleotides (uracil replacing thymine). This mRNA sequence is later used as a template for the amino acid sequence.</p><br />
<br />
</td><br />
</tr><br />
</table><br /><br />
<br />
<br />
<h3>Translation</h3><br />
<table><br />
<tr><br />
<td><br />
<img style="width:200px;" src="http://i872.photobucket.com/albums/ab287/iGEMCalgary_2010/2-1.png"></img><br />
</td><br />
<td><br />
<p>Translation is the process by which the cell forms a polypeptide chain using the mRNA sequence produced during transcription as a template. A ribosome translates the mRNA in three-nucleotide segments which each code for a diffferent amino acid. This polypeptide chain later folds into a functional protein conformation. </p><br />
</td><br />
</tr><br />
</table><br /><br />
<br />
<h3>Folding</h3><br />
<table><br />
<tr><br />
<td><br />
<img style="width:200px;" src="http://i872.photobucket.com/albums/ab287/iGEMCalgary_2010/3-1.png"></img><br />
</td><br />
<td><br />
<p>Protein folding is the process by which a polypeptide chain folds into a functional protein. Amino acids in proteins have a property pertaining to water in which some have high afinity with water (hydrophilic) and some only repel water (hydrophobic). Due to the cells typically containing high quantities of water, the amino acid chain folds in order to hide the hydrophobic amino acids from the cell content. To increase the probability of a successful conformation being made, chaperone proteins are often used by the cell.</p><br />
</td><br />
</tr><br />
</table><br /><br />
<br />
<h2 style="color:#0066CC">Our circuit and conventional transcriptional and translational tests</h2><br />
<p>The problem faced by many researchers today is difficulty in locating which step of the protein expression process is malfunctioning. iGEM teams are additionally constrained by a deadline. Our Toolkit has made it possible to pinpoint the exact process in which errors are occurring (transcription, translation, cytoplasmic protein folding or periplasmic protein folding), over the period of a few days.</p><br />
<br />
<h3>Overview of Conventional Methods</h3> <br />
<p>The most commonly used tests for transcription and translation are the Northern and Western Blot respectively. Northern Blot can be used to detect transcription because it relies on the isolation of mRNA. mRNA is typically extracted from a sample using oligo (dT) – cellulose chromatography. Essentially, this method exploits the poly-A tail characteristic of mRNA. In living cells, there are three types of naturally occurring RNA: rRNA, tRNA and mRNA. mRNA has a segment of (~250) Adenine nucleotides on the 3’ end that enhances both the lifetime and translatability of mRNA. The sample can be ran through a column containing oligo dT or deoxyribose Thymine nucleotides. These thymine nucleotides act as a sort of ‘primer’ such as primers in a PCR binding with the Poly-A tail. This ‘double stranded’ mRNA complex can be eluted out with a slight pH fluctuation. The isolated mRNA is then ran on a gel allowing it to segregate by size, and then blotted onto a nylon membrane. A positively charged nylon membrane is often more effective as the negatively charged nucleic acids have a higher affinity for it. The blotted membrane is then transferred to a transfer buffer containing RNA probes complimentary to the RNA sequence of interest. RNA is very unstable and is often degraded by factors such as high temperatures therefore the blotting process needs occur in the presence of formamide which helps lower the probe-RNA interaction temperature. Formamide is highly corrosive to the skin and an extreme teratogen. The membrane can then be examined under UV light, which will allow the RNA-probe complexes to fluoresce. This indicates that the DNA sequence of interest is being translated. </p><br />
<br />
<h3>Discussion of Disadvantages of Conventional Methods</h3><br />
<p>From this procedure, we can see that there are limitations and disadvantages of using Northern Blot. Firstly, it is an extremely selective procedure. Concentrations of buffers, solvents and probes need to be optimal for the reaction to occur. Additionally, primer and probe sequences need to be exact. The entire Northen Blot procedure can take up to 8 days, which can be big issue for iGEM teams with stringent deadlines. Another major limitation is due the unstability of RNA, if RNA samples are even slightly degraded, the quality of the data and the ability to quantitate expression are severely compromised. For example, even a single cleavage in 20% of 4 kb target molecules will decrease the returned signal by 20%. Thus, RNase-free reagents and techniques are essential. The obvious final disadvatage is that Northern requires the use of harmful chemicals that need to be handled carefully.</p><br />
<br />
<h3>Advantages of Our Circuit</h3> <br />
<p>Our transcription/translation circuit has many advantages over the Norther Blot. For one, it does not require the user to handle RNA and therefore it eliminates concerns regarding mRNA degradation. Another advantage would be that it does not require the use of harmful and carcinogenic chemicals, as it is just a simple construction of two DNA sequences that can then be transformed into cells. Our system also eliminates the specificity of primers and probes by eliminating the use of them together.</p><br />
<br />
<p>Our project is broken up into three smaller sections. Click any of the links on the side to learn more about each individual section.</p><br />
</div><br />
<br />
</div><br />
<br />
</body><br />
</html></div>Emily Hickshttp://2010.igem.org/Team:Calgary/ProjectTeam:Calgary/Project2010-10-28T03:09:16Z<p>Emily Hicks: </p>
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<h1>Project Descriptions</h1><br />
<br />
<br />
<ul><br />
<li><a href="https://2010.igem.org/Team:Calgary/Project/Transcription">Transcription/Translation Reporter Circuit</a></li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Project/misfolding_overview">Protein Misfolding Reporters</a><br />
<ul><br />
<li><a href="https://2010.igem.org/Team:Calgary/Project/IbpAB">Cytoplasmic Stress Detectors</a></li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Project/CpxP">Periplasmic Stress Detectors</a></li><br />
</ul><br />
</li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Project/Controls">Testing Our System</a></li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Project/Achievements">Achievements</a></li><br />
</ul><br />
<br />
</div><br />
<br />
<div class="mainbody"><br />
<br />
<span id="bodytitle"><h1>The Project</h1></span><br />
<br />
<h2 style="color:#0066CC">Overview</h2><br />
<p>Many synthetic biology projects involve the expression of recombinant proteins in microorganisms such as <i>E. coli</i>. The problems encountered with many synthetic biology projects often involve problems with protein expression. It is often very difficult to recognize the problem and pinpoint where it lies. The goal of the University of Calgary 2010 iGEM team is to build a protein expression "troubleshooting kit". This kit will contain two systems with which target genes can be inserted. In the resulting cell growth, fluorescent protein production will be used to determine whether there is a problem with protein expression as well as indicate where the protein expression is failing.</p><br />
<br />
<br />
<p><br />
Protein expression happens in three steps: the transcription of cell DNA to mRNA, the translation of mRNA into an amino acid sequence, and the folding of that amino acid sequence into a protein.</p><br />
<br />
<br />
<br />
<img src="http://i872.photobucket.com/albums/ab287/iGEMCalgary_2010/Slide1.png?t=1288229208"></img><br />
<br />
<br /><br />
<br />
<h2 style="color:#0066CC">Sites of Failure</h2><br />
<p><br />
Protein expression can fail at any point along these three steps. Our system uses two circuits to detect at which step possible errors have occured. The first circuit has a fluorescent reporter (GFP) that is produced when DNA is transcribed into mRNA and another (RFP) that is produced when mRNA is translated into a functional protein. When both reporter proteins are expressed in the cell, it indicates both transcription and translation are occurring. The second circuit involves reporter systems that are activated as a result of protein misfolding. Two native stress-activated promoters from <i>E. coli</i> were engineered upstream to fluorescent reporters that will respond to periplasmic and cytoplasmic protein misfolding. If the protein of interest misfolds in either area of the cell, one of the promoters will be activated and the corresponding fluorescence will be observed.<br />
</p><br />
<br />
<h3>Transcription</h3><br />
<table><br />
<tr><br />
<td><br />
<center><img style="width:200px;" src="http://i872.photobucket.com/albums/ab287/iGEMCalgary_2010/DNAmRNA001.png"></img></center><br />
</td><br />
<td><br />
<br />
<p>Transcription is the process by which the cell makes messenger RNA (mRNA) from DNA using RNA polymerase. The mRNA sequence is complementary to the DNA sequence that is being transcribed and uses adenine, cytosine, guanine, and uracil nucleotides (uracil replacing thymine). This mRNA sequence is later used as a template for the amino acid sequence.</p><br />
<br />
</td><br />
</tr><br />
</table><br /><br />
<br />
<br />
<h3>Translation</h3><br />
<table><br />
<tr><br />
<td><br />
<img style="width:200px;" src="http://i872.photobucket.com/albums/ab287/iGEMCalgary_2010/transcriptiontranslation001.png"></img><br />
</td><br />
<td><br />
<p>Translation is the process by which the cell forms a polypeptide chain using the mRNA sequence produced during transcription as a template. A ribosome translates the mRNA in three-nucleotide segments which each code for a diffferent amino acid. This polypeptide chain later folds into a functional protein conformation. </p><br />
</td><br />
</tr><br />
</table><br /><br />
<br />
<h3>Folding</h3><br />
<table><br />
<tr><br />
<td><br />
<img style="width:200px;" src="http://i872.photobucket.com/albums/ab287/iGEMCalgary_2010/translationprotein001.png"></img><br />
</td><br />
<td><br />
<p>Protein folding is the process by which a polypeptide chain folds into a functional protein. Amino acids in proteins have a property pertaining to water in which some have high afinity with water (hydrophilic) and some only repel water (hydrophobic). Due to the cells typically containing high quantities of water, the amino acid chain folds in order to hide the hydrophobic amino acids from the cell content. To increase the probability of a successful conformation being made, chaperone proteins are often used by the cell.</p><br />
</td><br />
</tr><br />
</table><br /><br />
<br />
<h2 style="color:#0066CC">Our circuit and conventional transcriptional and translational tests</h2><br />
<p>The problem faced by many researchers today is difficulty in locating which step of the protein expression process is malfunctioning. iGEM teams are additionally constrained by a deadline. Our Toolkit has made it possible to pinpoint the exact process in which errors are occurring (transcription, translation, cytoplasmic protein folding or periplasmic protein folding), over the period of a few days.</p><br />
<br />
<h3>Overview of Conventional Methods</h3> <br />
<p>The most commonly used tests for transcription and translation are the Northern and Western Blot respectively. Northern Blot can be used to detect transcription because it relies on the isolation of mRNA. mRNA is typically extracted from a sample using oligo (dT) – cellulose chromatography. Essentially, this method exploits the poly-A tail characteristic of mRNA. In living cells, there are three types of naturally occurring RNA: rRNA, tRNA and mRNA. mRNA has a segment of (~250) Adenine nucleotides on the 3’ end that enhances both the lifetime and translatability of mRNA. The sample can be ran through a column containing oligo dT or deoxyribose Thymine nucleotides. These thymine nucleotides act as a sort of ‘primer’ such as primers in a PCR binding with the Poly-A tail. This ‘double stranded’ mRNA complex can be eluted out with a slight pH fluctuation. The isolated mRNA is then ran on a gel allowing it to segregate by size, and then blotted onto a nylon membrane. A positively charged nylon membrane is often more effective as the negatively charged nucleic acids have a higher affinity for it. The blotted membrane is then transferred to a transfer buffer containing RNA probes complimentary to the RNA sequence of interest. RNA is very unstable and is often degraded by factors such as high temperatures therefore the blotting process needs occur in the presence of formamide which helps lower the probe-RNA interaction temperature. Formamide is highly corrosive to the skin and an extreme teratogen. The membrane can then be examined under UV light, which will allow the RNA-probe complexes to fluoresce. This indicates that the DNA sequence of interest is being translated. </p><br />
<br />
<h3>Discussion of Disadvantages of Conventional Methods</h3><br />
<p>From this procedure, we can see that there are limitations and disadvantages of using Northern Blot. Firstly, it is an extremely selective procedure. Concentrations of buffers, solvents and probes need to be optimal for the reaction to occur. Additionally, primer and probe sequences need to be exact. The entire Northen Blot procedure can take up to 8 days, which can be big issue for iGEM teams with stringent deadlines. Another major limitation is due the unstability of RNA, if RNA samples are even slightly degraded, the quality of the data and the ability to quantitate expression are severely compromised. For example, even a single cleavage in 20% of 4 kb target molecules will decrease the returned signal by 20%. Thus, RNase-free reagents and techniques are essential. The obvious final disadvatage is that Northern requires the use of harmful chemicals that need to be handled carefully.</p><br />
<br />
<h3>Advantages of Our Circuit</h3> <br />
<p>Our transcription/translation circuit has many advantages over the Norther Blot. For one, it does not require the user to handle RNA and therefore it eliminates concerns regarding mRNA degradation. Another advantage would be that it does not require the use of harmful and carcinogenic chemicals, as it is just a simple construction of two DNA sequences that can then be transformed into cells. Our system also eliminates the specificity of primers and probes by eliminating the use of them together.</p><br />
<br />
<p>Our project is broken up into three smaller sections. Click any of the links on the side to learn more about each individual section.</p><br />
</div><br />
<br />
</div><br />
<br />
</body><br />
</html></div>Emily Hickshttp://2010.igem.org/Team:Calgary/ExtrasTeam:Calgary/Extras2010-10-28T03:00:43Z<p>Emily Hicks: </p>
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<h1>Extras</h1><br />
<br />
<ul><br />
<li><a href="#tshirt">T-Shirts</a></li><br />
<li><a href="#proteinman">The Protein Man</a></li><br />
</ul><br />
<br />
<br />
<br />
</div><br />
<br />
<div class="mainbody"><br />
<br />
<span id="bodytitle"><h1>Extras</h1></span><br />
<br />
<h2>T-Shirts</h2><br />
<p>T-shirts were printed by Apparel Ink. T-shirts were printed in yellow. Photos of T-shirt coming soon!<br />
<li>6455 Macleod Trail South</li><br />
<li>Calgary,AB. T2H 0K8</li><br />
<li>Ph:403-255-1150</li><br />
</p><br />
<img class="autodlogo" src="http://i872.photobucket.com/albums/ab287/iGEMCalgary_2010/FinalT-shirt.png"></img><br />
<br />
<a name="proteinman"></a><br />
<h2>The Protein Man!</h2><br />
<br />
<br />
<br />
<p>Hey iGEM-ers,</p><br />
<p><br />
This is Protein Man checking in! I hope the iGEM competition is not stressing you out too much! Remember, stress causes protein misfolding and my job is to promote proper protein expression. I will be the mascot of the 2010 iGEM Calgary team, whose project is about stress detection in our favourite bug E. coli or any other bugs that y’all might be using for your project.</p><br />
<p><br />
<br />
I wish you all good luck and keep an eye out for me at the iGEM jamboree and ladies you can aggregate around me to relive your stress.</p><br />
<br />
<p>Checking out,</p><br />
<p><br />
Protein man!</p><br />
<br />
<embed type="application/x-shockwave-flash" src="http://picasaweb.google.com/s/c/bin/slideshow.swf" width="288" height="192" flashvars="host=picasaweb.google.com&hl=en_US&feat=flashalbum&RGB=0x000000&feed=http%3A%2F%2Fpicasaweb.google.com%2Fdata%2Ffeed%2Fapi%2Fuser%2F116832843163741069211%2Falbumid%2F5532924929269156113%3Falt%3Drss%26kind%3Dphoto%26hl%3Den_US" pluginspage="http://www.macromedia.com/go/getflashplayer"></embed><br />
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</html></div>Emily Hickshttp://2010.igem.org/Team:Calgary/Project/misfolding_overviewTeam:Calgary/Project/misfolding overview2010-10-28T02:43:33Z<p>Emily Hicks: </p>
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<h1>Project Descriptions</h1><br />
<br />
<br />
<ul><br />
<li><a href="https://2010.igem.org/Team:Calgary/Project/Transcription">Transcription/Translation Reporter Circuit</a></li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Project/misfolding_overview">Protein Misfolding Reporters</a><br />
<ul><br />
<li><a href="https://2010.igem.org/Team:Calgary/Project/IbpAB">Cytoplasmic Stress Detectors</a></li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Project/CpxP">Periplasmic Stress Detectors</a></li><br />
</ul><br />
</li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Project/Controls">Testing Our System</a></li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Project/Achievements">Achievements</a></li><br />
</ul><br />
<br />
</div><br />
<br />
<div class="mainbody"><br />
<br />
<span id="bodytitle"><h1>Misfolding detection circuit overview</h1></span><br />
<br />
<h2 style="color:#0066CC">How Does Protein Misfolding Occur?</h2><br />
<p><br />
Protein misfolding can occur as a result of a variety of factors. Overproduction of proteins in the cell is a good example. When proteins are overproduced, the cell can become overwhelmed and lack the necessary resources such as chaperones in order to deal with the large amount of protein. Proteins can also misfold due to mutations that occur in the coding region of the protein that can alter the amino acid sequence thereby interrupting the native structure of the protein. This can cause it to misfold into a non-functional state. Proteins can also misfold due to cellular stress such as changes in pH, temperature and changes in media. Localization can also be an issue. If a periplasmic protein lacks a signal sequence for example, it could misfold in the cytoplasm because the conditions are different in the two cellular compartments.<br />
</p><br />
<br /><br />
<br />
<h2 style="color:#0066CC">Why do we care?</h2><br />
<p><br />
Protein misfolding is an important topic in many regards. Many diseases, particularly neurodegenerative disorders such as prion diseases and Alzhemier's disease result from misfolding proteins. The production of recombinant proteins in prokaryotes such as E. Coli can also pose a problem. Non-native proteins are more susceptible to misfolding than native proteins. This can complicate many lab projects such as the design of peptide based drugs. With this in mind, a detection system for protein misfolding could be a very useful tool.<br />
</p><br />
<br /><br />
<br />
<h2 style="color:#0066CC">How does our system detect protein misfolding?</h2><br />
<h3>Current methods</h3><br />
<p><br />
GFP fusions are a method commonly used to detect protein misfolding. Targeted proteins can be fused to the C-Terminal of reporter genes such as GFP or Luciferase. If the target gene folds correctly, it would permit the reporter gene to also fold correctly, thus giving a measurable output. If the target gene was not able to fold however, the thought is that the reporter gene would not be able to fold correctly either, Arguments have been made however, that the fusion may affect the solubility of the target protein, thus resulting in an ineffective testing system. A more recent system has been the use of a split GFP system. Cabantous et al (2005) describe a system using two fractions of GFP. The smaller part is fused to the target protein. The small size of the fraction of GFP fused to the target protein is thought to not affect the solubility of the protein of interest. Nevertheless, many heterologous proteins often are not suitable for fusion with such reporters due to inaccessible C terminus of the target protein. <br />
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<h3>Our System</h3><br />
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Another method of protein misfolding detecton is thus to look at transcription levels of different heat shock promoters. By monitoring the activity levels of native stress promoters, you cab look more to the cell to report in its own stress levels. Because the reporter itself is decoupled from the stress, there is a minimized chance of the reporter having a stabilizing effect on the misfolding protein.Because transcription from these promoters is drastically increased during times of stress in the cell, these promoters, when coupled with different reporter genes such as GFP or lacZ, can be used as indicators of protein misfolding, as this is a stress for the cell. <br />
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<h3>Our stress promoters</h3><br />
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We chose four stress promoters to look at: three that monitor stress in periplasm of E Coli: <a href="https://2010.igem.org/Team:Calgary/Project/CpxP">Periplasmic Stress Detectors</a>, and one that monitors stress in the cytoplasm of E. Coli: <a href="https://2010.igem.org/Team:Calgary/Project/IbpAB">Cytoplasmic Stress Detectors</a><br />
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</html></div>Emily Hickshttp://2010.igem.org/Team:Calgary/Notebook/CalendarTeam:Calgary/Notebook/Calendar2010-10-28T00:03:08Z<p>Emily Hicks: </p>
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All parts of the project were designed and executed by team members. Three constructs used for testing were provided from the Raivio lab in Edmonton and plasmid DNA of malE and its three mutant forms were supplied from the Betton lab in France. All other constructs were built and tested by team members. <br />
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<p class="tabText" style="padding-left:10px;">Have you ever seen an aggregate protein dance? Look out for iGEM Calgary’s rambunctious mascot “Protein Man” at the Jamboree promoting proper protein expression. <a class="tabLink" href="https://2010.igem.org/Team:Calgary/Extras/Protein_Man">Learn more about him...</a></p><br />
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<p class="tabText" style="padding-left:10px;">Alberta’s very own Jamboree. The three Alberta iGEM teams, the Universities of Alberta, Calgary, and Lethbridge meet to perform a practice project presentation to each other. Experts within their field are also present to give teams challenging questions, and then meet with each team to suggest improvements. <a class="tabLink" href="https://2010.igem.org/Team:Calgary/Community/Conferences#agem">Read more...</a></p><br />
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<p>Our modelling project consists of two components: a mathematical model done in MATLAB and an animation done in Autodesk <i>Maya</i>. We hope to model the formation of inclusion bodies, which are aggregations of misfolded protein that can occur within cells. Using <i>Maya</i>, we also hope to visually show other students one of the proposed aggregation mechanisms described in the literature.<br />
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<p>Our outreach project included educating highschool students about iGEM. Outreach also entailed blog entries and podcast about synthetic life, iGEM and open source as well as Genetically modified foods. The blog entry, podcasts and high school presentations allowed the iGEM students to spread knowledge about iGEM to the general public and to potential future scientists. <a href="https://2010.igem.org/Team:Calgary/Community">Read more here...</a></p><br />
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<h3>Wetlab</h3><br />
<p>You’re stressing me out! This year, in the wetlab, we have been designing a reporter system to detect problems in protein expression. Our system uses a visual output to allow specificity in determining which step in protein expression problems are occurring.</p><br />
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<h3>Modelling</h3><br />
<p>Our modeling project has focused on simulating and modeling the formation of inclusion bodies, aggregates of protein within the cell. We've been exploring the factors that lead to their ormation</p><br />
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<h3>Community</h3><br />
<p>Our human practices section focused on exploring ethical issues in synthetic biology through an ethics paper as well as a podcast focusing on a few key issues. Our outreach initiatives also allowed the iGEM students to spread knowledge about iGEM to the general public and to potential future scientists through activities such as high school presentations and participation in various research symposiums.</p><br />
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<td><a href="https://2010.igem.org/Team:Calgary/Sponsors#bioalb"><img src="https://static.igem.org/mediawiki/2008/7/7b/Bioalberta.jpeg"></img></a></td><br />
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<td><a href="https://2010.igem.org/Team:Calgary/Sponsors#autod"><img src="http://www.innolution.com/img/autodesk_logo.jpg"></img></a></td><br />
<td><a href="https://2010.igem.org/Team:Calgary/Sponsors#neb"><img src="http://i872.photobucket.com/albums/ab287/iGEMCalgary_2010/NEB_logoSmall2.png"></img></a></td><br />
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<td><a href="https://2010.igem.org/Team:Calgary/Sponsors#aihs"><img src="http://i872.photobucket.com/albums/ab287/iGEMCalgary_2010/AlHSLogo.png"></img></a></td><br />
<td><a href="https://2010.igem.org/Team:Calgary/Sponsors#idt"><img src="http://i872.photobucket.com/albums/ab287/iGEMCalgary_2010/IDTLogo.png"></img></a></td><br />
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<td><a href="https://2010.igem.org/Team:Calgary/Sponsors#corning"><img src="http://www.ysbl.york.ac.uk/fbld/2010/Corning100logo.jpg"></img></a></td><br />
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</html></div>Emily Hickshttp://2010.igem.org/Team:Calgary/Parts/CharacterizationTeam:Calgary/Parts/Characterization2010-10-27T23:28:52Z<p>Emily Hicks: </p>
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<h1>Parts</h1><br />
<br />
<ul><br />
<li><a href="https://2010.igem.org/Team:Calgary/Parts/Parts_Submitted">Parts Submitted</a></li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Parts/Characterization">Characterization</a></li><br />
</ul><br />
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<div class="mainbody"><br />
<br />
<span id="bodytitle"><h1>Characterization</h1></span><br />
<br />
<p><br />
<h2 style="color:#0066CC">Experiment 1: Testing of malE and malE31 with literature established reporter constructs</h2><br />
</p><br />
<br />
<h3><u>Protocol:</u></h3><br />
<br />
<p>We obtained degP and cpxR porter constructs from a lab in Edmonton. These constructs contain the promoters upstream of lacZ. These reporters were characterized with NLPE, an outermembrane lipoprotein that activates the cpx pathway. We made TOP10 E. Coli competant cells with plasmids of these constructs and then transformed in malE and malE31 expression constructs. We also transformed in the NLPE expression construct which we received fro the Raivio lab as well. The purpose of this was to verify that the reporters that they ha sent us worked, as they had previously characterized these reporters using NLPE. From plates we made 5 mL LB cultures and induced with 1uL IPTG for cultures contain the NLPE constructs. 75uL of xGal was also added to each culture. The cultures were then grown up overnight in a 37C shaking incubator and observed for color. <br />
</p><br />
<br />
<h3><u>Results</u></h3><br />
<br />
<br />
<p>Figure 1: Image of overnight cultures. From left to right: NLPE in cells with the degP reporter construct, NLPE in cells with the cpxR reporter, malE31 in cells with the degP reporter, malE31 in cells with the cpxR reporter, malE in cells with the degP reporter contruct and malE in cells containing the cpxR reporter.</p><br />
<br />
<br />
<h3><u>Discussion of Results and Conclusion</u></h3><br />
<p><br />
Figure 1 indicates that malE31 is able to activae the cpxR and and degP promoers while malE is not. This allowed us to conlucde that these parts are working as expected. Although these parts are both entered in the registry, the sequences are not complete, so we are submitting new versions of them, constructed ourselves. Once we knew that these parts were functional, we went o to characterize them with our reporter constructs.<br />
</p><br />
<br />
<p><br />
<br />
<h2 style="color:#0066CC">Experiment 2: Characterization of the cpxR promoter's response to folding and misfolding proteins through co-transformation of MalE and MalE31 coupled to arabinose promoter in cpxR reporter competent cells</h2><br />
</p><br />
<br />
<h3><u>Protocol:</u></h3><br />
<br />
<p><br />
Arabinose inducible promoter (I0500) coupled with standard ribosome binding site (B0034) and the respective maltose binding protein were transformed into competent cells containing pCpxR coupled with RFP generator (I13507). These cells were plated and incubated overnight. Colonies from each of the plates were selected and overnight cultures were prepared at 37 C. These 5 ml overnight cultures were then sub-cultured in 20 ml broth. These were shaken for 6-8 hours and aliquoted into 5 ml cultures and induced with varying levels of arabinose(percent). This was incubated in the shaker for 12-14 hours and RFP output was measured using 555 excitation and 632 nm emission frequency.</p><br />
<br />
<h3><u>Results</u></h3><br />
<br />
<br />
</li><a href="http://s872.photobucket.com/albums/ab287/iGEMCalgary_2010/?action=view&current=Unititled-7.png" target="_blank"><img src="http://i872.photobucket.com/albums/ab287/iGEMCalgary_2010/Unititled-7.png" border="0" alt="CpxR"></a></li></td><br />
<br />
<br />
<p>Figure 2: RFP output produced by the CpxR-I13507 system when co-transfected with I0500-B0034-MalE (red) and I0500-B0034-MalE31 (blue) at different arabinose concentrations. RFP levels were measured at 555 nm excitation and 632 nm emission frequencies</p><br />
<br />
<br />
<br />
</li><a href="http://s872.photobucket.com/albums/ab287/iGEMCalgary_2010/?action=view&current=lineofbestfitCpxR.png" target="_blank"><img src="http://i872.photobucket.com/albums/ab287/iGEMCalgary_2010/lineofbestfitCpxR.png" border="0" alt="Photobucket"></a></li><br />
<br />
<p>Figure 3: RFP output produced by the CpxR-I13507 system when co-transfected with I0500-B0034-MalE (red) and I0500-B0034-MalE31 (blue) at different arabinose concentrations. RFP levels were measured at 555 nm excitation and 632 nm emission frequencies.</p><br />
<br />
<h3><u>Discussion of Results and Conclusion</u></h3><br />
<p><br />
Figure 2 and 3 indicate the RFP output normalized with growth ratio (OD) at different levels of arabinose. Figure 1 shows that CpxR-I13507 is activated at the highest level when MalE31, the periplasmic misfolder, is expressed. This occurs around 0.2% arabinose concentration. Similar trends are observed in the case of MalE which is a periplasmic folder. MalE and MalE31 activate the system at different levels. MalE31 has similar trends to MalE but has a higher level of RFP expression. These results prove that MalE and MalE31 can both activate the CpxR system however, MalE31, which misfolds, activates it more rapidly and at a lower level of arabinose concentration compared to MalE. If the line of best fit is studied, it is seen that MalE has very minimal level of Cpx activation. Whereas, malE31 has a linear regression which flattens out as the system reaches its upper threshold of detection. Biologically, this could mean that the MalE31 is activated at levels that saturate the cellular chaperones and cause the system to reach its threshold level of proteolytic and chaperone activities. Another interesting pattern observed is the fact that when MalE is constructed with CpxR-I13507 on the same plasmid (Green), the cell RFP output is much lower compared to cells co-transfected with CpxR-I13507 and I0500-B0034 –MalE. This indicates that insertion of high copy plasmid also induces stress in the periplasmic region of the cell consequently inducing the activation of CpxR system. <br />
</p><br />
<br />
<p><br />
<h2 style="color:#0066CC">Experiment 3: Characetrizing the cpxR promoter's response to varying temperatures over different time periods</h2></p><br />
<br />
<h3><u>Protocol:</u></h3><br />
<p><br />
Top 10 competent cells were transformed with CpxR-I13507 and plated. 5 ml overnight cultures were made from 5 different colonies using LB broth with appropriate antibiotics. Each of these cultures were aliquoted into six different tubes containing 600 µL of culture. These tubes were then placed in hot water baths at 30 C, 37C, 42C, 47C. Measurements were taken every hour for 5 hours after placing the tubes in different temperatures at 555 nm excitation and 632 nm emission.</p><br />
<br />
<br />
</li><a href="http://s872.photobucket.com/albums/ab287/iGEMCalgary_2010/?action=view&current=Untitled-6.png" target="_blank"><img src="http://i872.photobucket.com/albums/ab287/iGEMCalgary_2010/Untitled-6.png" border="0" alt="Temperature induction"></a> </li><br />
<br />
<p>Figure 3: RFP output produced by the CpxR-I13507 system when the system is heat shocked at different temperature for different lengths of time. The RFP output was measured at 555 nm excitation and 632 nm emission frequencies<br />
<br />
</p><br />
<br />
<h3><u>Discussion of Results and Conclusion</u></h3><br />
<p><br />
This graph shows that the CpxR system does respond to temperature activated stress. When the system is placed at 42 C the RFP output is much higher at t=0 compared to the system placed at 37 C or 30 C. This indicates that the system does get activated due to heat shock which matches the literature parameters. At 47 C, the system gets activated faster because the linear regression has a steeper slope. This indicates that the system is being stressed and it produces its downstream product which is RFP in this case and DegP and other chaperones in the genomic DNA much faster in order to cope with periplasmic protein denaturation. Also, it seems that the system gets activated dramatically after 3 hours regardless of the temperature, this could indicate that the system peaks after 3 hours and the genomic CpxR produces enough downstream chaperones and proteases in order for the system to be able to cope with stress which allows the RFP reading to decrease at 4 hours time because the cell reaches homeostasis. This allows the cell to get rid of misfolded protein and other factors that might be contributing to stressing it out and causing the Cpx regulon to be activated. The cell then shows a rapid rise again because it is still under heat shock stress. But, if the cell was placed at 37 degrees, the cell would show a flatline pattern rather than an oscillating pattern.<br />
</p><br />
<p><br />
<h2 style="color:#0066CC">Experiment 4: Characterization of the degP promoter's response to folding and misfolding proteins</h2></P><br />
<br />
<h3><u>Protocol</u></h3><br />
<br />
<br />
<p> Arabinose inducible promoter (I0500) coupled with standard ribosome binding site (B0034) and the respective maltose binding protein were transformed into competent cells containing pDegP coupled with RFP generator (I13507). These cells were plated and incubated overnight. Colonies from each of the plates were selected and overnight cultures were prepared at 37 C. These 5 ml overnight cultures were then subcultured in 20 ml LB broth. These were shaken for 6-8 hours and aliquoted into 5 ml cultures and induced with varying levels of arabinose. This was incubated in the shaker for 12-14 hours and RFP output was measured using 555/632 nm.</p><br />
<br />
<a href="http://s872.photobucket.com/albums/ab287/iGEMCalgary_2010/?action=view&current=DegPinduction.png" target="_blank"><img src="http://i872.photobucket.com/albums/ab287/iGEMCalgary_2010/DegPinduction.png" border="0" alt="Photobucket"></a><br />
<br />
<p> This figure demonstrates that the DegP promoter activated with 15 different concentrations of arabinose. This diagram shows that the DegP promoter (K239000) is not particularly sensitive to misfolding proteins.</p><br />
<br />
<h3><u>Discussion and conclusions</u></h3><br />
<p><br />
<br />
The figure shows that MalE and MalE31 express the DegP promoter in a similar fashion. This is slightly contradictory compared to the literature. The literature claims that the DegP promoter is upregulated in the case of protein misfolding, graph shown . Since MalE and MalE31 have been tested using other experiments described in this page, it is reasonable to conclude that K230009 is not very responsive to protein folding stress, that is , the DNA might not be consistent.<br />
<br />
</p><br />
<br />
<p><br />
<h2 style="color:#0066CC">Experiment 5: Characterization of the ibpAB fusion promoter's response to folding and misfolding proteins</h2></P><br />
<br />
<h3><u>Purpose</u></h3><br />
<br />
<p><br />
The purpose of this assay is to test the output that the cytoplasmic acting fusion promoter (ibpAB-fsxA) will produce with proteins that are known to fold correctly (malEΔSS) and with proteins that are known to misfold (malE31ΔSS) in the cytoplasm. The plasmids containing malEΔSS and malE31ΔSS are coupled to an IPTG inducible promoter and were received from the lab of Jean-Michel Betton.<br />
</p><br />
</p><br />
<br />
</div><br />
<br />
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<br />
</body><br />
</html></div>Emily Hickshttp://2010.igem.org/Team:Calgary/Parts/CharacterizationTeam:Calgary/Parts/Characterization2010-10-27T23:22:36Z<p>Emily Hicks: </p>
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<h1>Parts</h1><br />
<br />
<ul><br />
<li><a href="https://2010.igem.org/Team:Calgary/Parts/Parts_Submitted">Parts Submitted</a></li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Parts/Characterization">Characterization</a></li><br />
</ul><br />
<br />
</div><br />
<br />
<div class="mainbody"><br />
<br />
<span id="bodytitle"><h1>Characterization</h1></span><br />
<br />
<p><br />
<h2 style="color:#0066CC">Experiment 1: Testing of malE and malE31 with literature established reporter constructs</h2><br />
</p><br />
<br />
<h3><u>Protocol:</u></h3><br />
<br />
<p>We obtained degP and cpxR porter constructs from a lab in Edmonton. These constructs contain the promoters upstream of lacZ. These reporters were characterized with NLPE, an outermembrane lipoprotein that activates the cpx pathway. We made TOP10 E. Coli competant cells with plasmids of these constructs and then transformed in malE and malE31 expression constructs. We also transformed in the NLPE expression construct which we received fro the Raivio lab as well. The purpose of this was to verify that the reporters that they ha sent us worked, as they had previously characterized these reporters using NLPE. From plates we made 5 mL LB cultures and induced with 1uL IPTG for cultures contain the NLPE constructs. 75uL of xGal was also added to each culture. The cultures were then grown up overnight in a 37C shaking incubator and observed for color. <br />
</p><br />
<br />
<h3><u>Results</u></h3><br />
<br />
<br />
<p>Figure 1: Image of overnight cultures. From left to right: NLPE in cells with the degP reporter construct, NLPE in cells with the cpxR reporter, malE31 in cells with the degP reporter, malE31 in cells with the cpxR reporter, malE in cells with the degP reporter contruct and malE in cells containing the cpxR reporter.</p><br />
<br />
<br />
<h3><u>Discussion of Results and Conclusion</u></h3><br />
<p><br />
Figure 1 indicates that malE31 is able to activae the cpxR and and degP promoers while malE is not. This allowed us to conlucde that these parts are working as expected. Although these parts are both entered in the registry, the sequences are not complete, so we are submitting new versions of them, constructed ourselves. Once we knew that these parts were functional, we went o to characterize them with our reporter constructs.<br />
</p><br />
<br />
<p><br />
<br />
<h2 style="color:#0066CC">Experiment 2: Characterization of the cpxR promoter's response to folding and misfolding proteins through co-transformation of MalE and MalE31 coupled to arabinose promoter in cpxR reporter competent cells</h2><br />
</p><br />
<br />
<h3><u>Protocol:</u></h3><br />
<br />
<p><br />
Arabinose inducible promoter (I0500) coupled with standard ribosome binding site (B0034) and the respective maltose binding protein were transformed into competent cells containing pCpxR coupled with RFP generator (I13507). These cells were plated and incubated overnight. Colonies from each of the plates were selected and overnight cultures were prepared at 37 C. These 5 ml overnight cultures were then sub-cultured in 20 ml broth. These were shaken for 6-8 hours and aliquoted into 5 ml cultures and induced with varying levels of arabinose(percent). This was incubated in the shaker for 12-14 hours and RFP output was measured using 555 excitation and 632 nm emission frequency.</p><br />
<br />
<h3><u>Results</u></h3><br />
<br />
<br />
</li><a href="http://s872.photobucket.com/albums/ab287/iGEMCalgary_2010/?action=view&current=Unititled-7.png" target="_blank"><img src="http://i872.photobucket.com/albums/ab287/iGEMCalgary_2010/Unititled-7.png" border="0" alt="CpxR"></a></li></td><br />
<br />
<br />
<p>Figure 2: RFP output produced by the CpxR-I13507 system when co-transfected with I0500-B0034-MalE (red) and I0500-B0034-MalE31 (blue) at different arabinose concentrations. RFP levels were measured at 555 nm excitation and 632 nm emission frequencies</p><br />
<br />
<br />
<br />
</li><a href="http://s872.photobucket.com/albums/ab287/iGEMCalgary_2010/?action=view&current=lineofbestfitCpxR.png" target="_blank"><img src="http://i872.photobucket.com/albums/ab287/iGEMCalgary_2010/lineofbestfitCpxR.png" border="0" alt="Photobucket"></a></li><br />
<br />
<p>Figure 3: RFP output produced by the CpxR-I13507 system when co-transfected with I0500-B0034-MalE (red) and I0500-B0034-MalE31 (blue) at different arabinose concentrations. RFP levels were measured at 555 nm excitation and 632 nm emission frequencies.</p><br />
<br />
<h3><u>Discussion of Results and Conclusion</u></h3><br />
<p><br />
Figure 2 and 3 indicate the RFP output normalized with growth ratio (OD) at different levels of arabinose. Figure 1 shows that CpxR-I13507 is activated at the highest level when MalE31, the periplasmic misfolder, is expressed. This occurs around 0.2% arabinose concentration. Similar trends are observed in the case of MalE which is a periplasmic folder. MalE and MalE31 activate the system at different levels. MalE31 has similar trends to MalE but has a higher level of RFP expression. These results prove that MalE and MalE31 can both activate the CpxR system however, MalE31, which misfolds, activates it more rapidly and at a lower level of arabinose concentration compared to MalE. If the line of best fit is studied, it is seen that MalE has very minimal level of Cpx activation. Whereas, malE31 has a linear regression which flattens out as the system reaches its upper threshold of detection. Biologically, this could mean that the MalE31 is activated at levels that saturate the cellular chaperones and cause the system to reach its threshold level of proteolytic and chaperone activities. Another interesting pattern observed is the fact that when MalE is constructed with CpxR-I13507 on the same plasmid (Green), the cell RFP output is much lower compared to cells co-transfected with CpxR-I13507 and I0500-B0034 –MalE. This indicates that insertion of high copy plasmid also induces stress in the periplasmic region of the cell consequently inducing the activation of CpxR system. <br />
</p><br />
<br />
<p><br />
<h2 style="color:#0066CC">Experiment 3: Characetrizing the cpxR promoter's response to varying temperatures over different time periods</h2></p><br />
<br />
<h3><u>Protocol:</u></h3><br />
<p><br />
Top 10 competent cells were transformed with CpxR-I13507 and plated. 5 ml overnight cultures were made from 5 different colonies using LB broth with appropriate antibiotics. Each of these cultures were aliquoted into six different tubes containing 600 µL of culture. These tubes were then placed in hot water baths at 30 C, 37C, 42C, 47C. Measurements were taken every hour for 5 hours after placing the tubes in different temperatures at 555 nm excitation and 632 nm emission.</p><br />
<br />
<br />
</li><a href="http://s872.photobucket.com/albums/ab287/iGEMCalgary_2010/?action=view&current=Untitled-6.png" target="_blank"><img src="http://i872.photobucket.com/albums/ab287/iGEMCalgary_2010/Untitled-6.png" border="0" alt="Temperature induction"></a> </li><br />
<br />
<p>Figure 3: RFP output produced by the CpxR-I13507 system when the system is heat shocked at different temperature for different lengths of time. The RFP output was measured at 555 nm excitation and 632 nm emission frequencies<br />
<br />
</p><br />
<br />
<h3><u>Discussion of Results and Conclusion</u></h3><br />
<p><br />
This graph shows that the CpxR system does respond to temperature activated stress. When the system is placed at 42 C the RFP output is much higher at t=0 compared to the system placed at 37 C or 30 C. This indicates that the system does get activated due to heat shock which matches the literature parameters. At 47 C, the system gets activated faster because the linear regression has a steeper slope. This indicates that the system is being stressed and it produces its downstream product which is RFP in this case and DegP and other chaperones in the genomic DNA much faster in order to cope with periplasmic protein denaturation. Also, it seems that the system gets activated dramatically after 3 hours regardless of the temperature, this could indicate that the system peaks after 3 hours and the genomic CpxR produces enough downstream chaperones and proteases in order for the system to be able to cope with stress which allows the RFP reading to decrease at 4 hours time because the cell reaches homeostasis. This allows the cell to get rid of misfolded protein and other factors that might be contributing to stressing it out and causing the Cpx regulon to be activated. The cell then shows a rapid rise again because it is still under heat shock stress. But, if the cell was placed at 37 degrees, the cell would show a flatline pattern rather than an oscillating pattern.<br />
</p><br />
<p><br />
<h2 style="color:#0066CC">Experiment 3: Measuring RFP output by co-transformation of MalE and MalE31 coupled to arabinose promoter in the DegP-RFP competent cells</h2></P><br />
<br />
<h3><u>Protocol</u></h3><br />
<br />
<br />
<p> Arabinose inducible promoter (I0500) coupled with standard ribosome binding site (B0034) and the respective maltose binding protein were transformed into competent cells containing pDegP coupled with RFP generator (I13507). These cells were plated and incubated overnight. Colonies from each of the plates were selected and overnight cultures were prepared at 37 C. These 5 ml overnight cultures were then subcultured in 20 ml LB broth. These were shaken for 6-8 hours and aliquoted into 5 ml cultures and induced with varying levels of arabinose. This was incubated in the shaker for 12-14 hours and RFP output was measured using 555/632 nm.</p><br />
<br />
<a href="http://s872.photobucket.com/albums/ab287/iGEMCalgary_2010/?action=view&current=DegPinduction.png" target="_blank"><img src="http://i872.photobucket.com/albums/ab287/iGEMCalgary_2010/DegPinduction.png" border="0" alt="Photobucket"></a><br />
<br />
<p> This figure demonstrates that the DegP promoter activated with 15 different concentrations of arabinose. This diagram shows that the DegP promoter (K239000) is not particularly sensitive to misfolding proteins.</p><br />
<br />
<h3><u>Discussion and conclusions</u></h3><br />
<p><br />
<br />
The figure shows that MalE and MalE31 express the DegP promoter in a similar fashion. This is slightly contradictory compared to the literature. The literature claims that the DegP promoter is upregulated in the case of protein misfolding, graph shown . Since MalE and MalE31 have been tested using other experiments described in this page, it is reasonable to conclude that K230009 is not very responsive to protein folding stress, that is , the DNA might not be consistent.<br />
<br />
</p><br />
<br />
<p><br />
<h2 style="color:#0066CC">Experiment 4: Measuring the GFP output through insertion of the mutant malE and malE31 with the transport signal sequence deleted into competent cells containing a fusion promoter (ibpAB-fsxA) coupled to a GFP reporter. </h2></P><br />
<br />
<h3><u>Purpose</u></h3><br />
<br />
<p><br />
The purpose of this assay is to test the output that the cytoplasmic acting fusion promoter (ibpAB-fsxA) will produce with proteins that are known to fold correctly (malEΔSS) and with proteins that are known to misfold (malE31ΔSS) in the cytoplasm. The plasmids containing malEΔSS and malE31ΔSS are coupled to an IPTG inducible promoter and were received from the lab of Jean-Michel Betton.<br />
</p><br />
</p><br />
<br />
</div><br />
<br />
</div><br />
<br />
</body><br />
</html></div>Emily Hickshttp://2010.igem.org/Team:Calgary/Parts/CharacterizationTeam:Calgary/Parts/Characterization2010-10-27T22:01:42Z<p>Emily Hicks: </p>
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<h1>Parts</h1><br />
<br />
<ul><br />
<li><a href="https://2010.igem.org/Team:Calgary/Parts/Parts_Submitted">Parts Submitted</a></li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Parts/Characterization">Characterization</a></li><br />
</ul><br />
<br />
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<br />
<div class="mainbody"><br />
<br />
<span id="bodytitle"><h1>Characterization</h1></span><br />
<br />
<p><br />
<h2 style="color:#0066CC">Experiment 1: Characterization of the cpxR promoter's response to folding and misfolding proteins through co-transformation of MalE and MalE31 coupled to arabinose promoter in cpxR reporter competent cells</h2><br />
</p><br />
<br />
<h3><u>Protocol:</u></h3><br />
<br />
<p><br />
Arabinose inducible promoter (I0500) coupled with standard ribosome binding site (B0034) and the respective maltose binding protein were transformed into competent cells containing pCpxR coupled with RFP generator (I13507). These cells were plated and incubated overnight. Colonies from each of the plates were selected and overnight cultures were prepared at 37 C. These 5 ml overnight cultures were then sub-cultured in 20 ml broth. These were shaken for 6-8 hours and aliquoted into 5 ml cultures and induced with varying levels of arabinose(percent). This was incubated in the shaker for 12-14 hours and RFP output was measured using 555 excitation and 632 nm emission frequency.</p><br />
<br />
<h3><u>Results</u></h3><br />
<br />
<br />
</li><a href="http://s872.photobucket.com/albums/ab287/iGEMCalgary_2010/?action=view&current=Unititled-7.png" target="_blank"><img src="http://i872.photobucket.com/albums/ab287/iGEMCalgary_2010/Unititled-7.png" border="0" alt="CpxR"></a></li></td><br />
<br />
<br />
<p>Figure 1: RFP output produced by the CpxR-I13507 system when co-transfected with I0500-B0034-MalE (red) and I0500-B0034-MalE31 (blue) at different arabinose concentrations. RFP levels were measured at 555 nm excitation and 632 nm emission frequencies</p><br />
<br />
<br />
<br />
</li><a href="http://s872.photobucket.com/albums/ab287/iGEMCalgary_2010/?action=view&current=lineofbestfitCpxR.png" target="_blank"><img src="http://i872.photobucket.com/albums/ab287/iGEMCalgary_2010/lineofbestfitCpxR.png" border="0" alt="Photobucket"></a></li><br />
<br />
<p>Figure 2: RFP output produced by the CpxR-I13507 system when co-transfected with I0500-B0034-MalE (red) and I0500-B0034-MalE31 (blue) at different arabinose concentrations. RFP levels were measured at 555 nm excitation and 632 nm emission frequencies.</p><br />
<br />
<h3><u>Discussion of Results and Conclusion</u></h3><br />
<p><br />
Figure 1 and 2 indicate the RFP output normalized with growth ratio (OD) at different levels of arabinose. Figure 1 shows that CpxR-I13507 is activated at the highest level when MalE31, the periplasmic misfolder, is expressed. This occurs around 0.2% arabinose concentration. Similar trends are observed in the case of MalE which is a periplasmic folder. MalE and MalE31 activate the system at different levels. MalE31 has similar trends to MalE but has a higher level of RFP expression. These results prove that MalE and MalE31 can both activate the CpxR system however, MalE31, which misfolds, activates it more rapidly and at a lower level of arabinose concentration compared to MalE. If the line of best fit is studied, it is seen that MalE has very minimal level of Cpx activation. Whereas, malE31 has a linear regression which flattens out as the system reaches its upper threshold of detection. Biologically, this could mean that the MalE31 is activated at levels that saturate the cellular chaperones and cause the system to reach its threshold level of proteolytic and chaperone activities. Another interesting pattern observed is the fact that when MalE is constructed with CpxR-I13507 on the same plasmid (Green), the cell RFP output is much lower compared to cells co-transfected with CpxR-I13507 and I0500-B0034 –MalE. This indicates that insertion of high copy plasmid also induces stress in the periplasmic region of the cell consequently inducing the activation of CpxR system. <br />
</p><br />
<br />
<p><br />
<h2 style="color:#0066CC">Experiment 2: Characetrizing the cpxR promoter's response to varying temperatures over different time periods</h2></p><br />
<br />
<h3><u>Protocol:</u></h3><br />
<p><br />
Top 10 competent cells were transformed with CpxR-I13507 and plated. 5 ml overnight cultures were made from 5 different colonies using LB broth with appropriate antibiotics. Each of these cultures were aliquoted into six different tubes containing 600 µL of culture. These tubes were then placed in hot water baths at 30 C, 37C, 42C, 47C. Measurements were taken every hour for 5 hours after placing the tubes in different temperatures at 555 nm excitation and 632 nm emission.</p><br />
<br />
<br />
</li><a href="http://s872.photobucket.com/albums/ab287/iGEMCalgary_2010/?action=view&current=Untitled-6.png" target="_blank"><img src="http://i872.photobucket.com/albums/ab287/iGEMCalgary_2010/Untitled-6.png" border="0" alt="Temperature induction"></a> </li><br />
<br />
<p>Figure 3: RFP output produced by the CpxR-I13507 system when the system is heat shocked at different temperature for different lengths of time. The RFP output was measured at 555 nm excitation and 632 nm emission frequencies<br />
<br />
</p><br />
<br />
<h3><u>Discussion of Results and Conclusion</u></h3><br />
<p><br />
This graph shows that the CpxR system does respond to temperature activated stress. When the system is placed at 42 C the RFP output is much higher at t=0 compared to the system placed at 37 C or 30 C. This indicates that the system does get activated due to heat shock which matches the literature parameters. At 47 C, the system gets activated faster because the linear regression has a steeper slope. This indicates that the system is being stressed and it produces its downstream product which is RFP in this case and DegP and other chaperones in the genomic DNA much faster in order to cope with periplasmic protein denaturation. Also, it seems that the system gets activated dramatically after 3 hours regardless of the temperature, this could indicate that the system peaks after 3 hours and the genomic CpxR produces enough downstream chaperones and proteases in order for the system to be able to cope with stress which allows the RFP reading to decrease at 4 hours time because the cell reaches homeostasis. This allows the cell to get rid of misfolded protein and other factors that might be contributing to stressing it out and causing the Cpx regulon to be activated. The cell then shows a rapid rise again because it is still under heat shock stress. But, if the cell was placed at 37 degrees, the cell would show a flatline pattern rather than an oscillating pattern.<br />
</p><br />
<br />
<p><br />
<h2 style="color:#0066CC">Experiment 4: Measuring the GFP output through insertion of the mutant malE and malE31 with the transport signal sequence deleted into competent cells containing a fusion promoter (ibpAB-fsxA) coupled to a GFP reporter. </h2></P><br />
<br />
<h3><u>Purpose</u></h3><br />
<p><br />
The purpose of this assay is to test the output that the cytoplasmic acting fusion promoter (ibpAB-fsxA) will produce with proteins that are known to fold correctly (malEΔSS) and with proteins that are known to misfold (malE31ΔSS) in the cytoplasm. The plasmids containing malEΔSS and malE31ΔSS are coupled to an IPTG inducible promoter and were received from the lab of Jean-Michel Betton.<br />
</p><br />
</p><br />
<br />
</div><br />
<br />
</div><br />
<br />
</body><br />
</html></div>Emily Hickshttp://2010.igem.org/Team:Calgary/Parts/CharacterizationTeam:Calgary/Parts/Characterization2010-10-27T21:59:08Z<p>Emily Hicks: </p>
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<h1>Parts</h1><br />
<br />
<ul><br />
<li><a href="https://2010.igem.org/Team:Calgary/Parts/Parts_Submitted">Parts Submitted</a></li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Parts/Characterization">Characterization</a></li><br />
</ul><br />
<br />
</div><br />
<br />
<div class="mainbody"><br />
<br />
<span id="bodytitle"><h1>Characterization</h1></span><br />
<br />
<p><br />
<h2 style="color:#0066CC">Experiment 1: Characterization of the cpxR promoter's response to folding and misfolding proteins through co-transformation of MalE and MalE31 coupled to arabinose promoter in cpxR reporter competent cells</h2><br />
</p><br />
<br />
<h3><u>Protocol:</u></h3><br />
<br />
<p><br />
Arabinose inducible promoter (I0500) coupled with standard ribosome binding site (B0034) and the respective maltose binding protein were transformed into competent cells containing pCpxR coupled with RFP generator (I13507). These cells were plated and incubated overnight. Colonies from each of the plates were selected and overnight cultures were prepared at 37 C. These 5 ml overnight cultures were then sub-cultured in 20 ml broth. These were shaken for 6-8 hours and aliquoted into 5 ml cultures and induced with varying levels of arabinose(percent). This was incubated in the shaker for 12-14 hours and RFP output was measured using 555 excitation and 632 nm emission frequency.</p><br />
<br />
<h3><u>Results</u></h3><br />
<br />
<br />
</li><a href="http://s872.photobucket.com/albums/ab287/iGEMCalgary_2010/?action=view&current=Unititled-7.png" target="_blank"><img src="http://i872.photobucket.com/albums/ab287/iGEMCalgary_2010/Unititled-7.png" border="0" alt="CpxR"></a></li></td><br />
<br />
<br />
<p>Figure 1: RFP output produced by the CpxR-I13507 system when co-transfected with I0500-B0034-MalE (red) and I0500-B0034-MalE31 (blue) at different arabinose concentrations. RFP levels were measured at 555 nm excitation and 632 nm emission frequencies</p><br />
<br />
<br />
<br />
</li><a href="http://s872.photobucket.com/albums/ab287/iGEMCalgary_2010/?action=view&current=lineofbestfitCpxR.png" target="_blank"><img src="http://i872.photobucket.com/albums/ab287/iGEMCalgary_2010/lineofbestfitCpxR.png" border="0" alt="Photobucket"></a></li><br />
<br />
<p>Figure 2: RFP output produced by the CpxR-I13507 system when co-transfected with I0500-B0034-MalE (red) and I0500-B0034-MalE31 (blue) at different arabinose concentrations. RFP levels were measured at 555 nm excitation and 632 nm emission frequencies.</p><br />
<br />
<h3><u>Discussion of Results and Conclusion</u></h3><br />
<p><br />
Figure 1 and 2 indicate the RFP output normalized with growth ratio (OD) at different levels of arabinose. Figure 1 shows that CpxR-I13507 is activated at the highest level when MalE31, the periplasmic misfolder, is expressed. This occurs around 0.2% arabinose concentration. Similar trends are observed in the case of MalE which is a periplasmic folder. MalE and MalE31 activate the system at different levels. MalE31 has similar trends to MalE but has a higher level of RFP expression. These results prove that MalE and MalE31 can both activate the CpxR system however, MalE31, which misfolds, activates it more rapidly and at a lower level of arabinose concentration compared to MalE. If the line of best fit is studied, it is seen that MalE has very minimal level of Cpx activation. Whereas, malE31 has a linear regression which flattens out as the system reaches its upper threshold of detection. Biologically, this could mean that the MalE31 is activated at levels that saturate the cellular chaperones and cause the system to reach its threshold level of proteolytic and chaperone activities. Another interesting pattern observed is the fact that when MalE is constructed with CpxR-I13507 on the same plasmid (Green), the cell RFP output is much lower compared to cells co-transfected with CpxR-I13507 and I0500-B0034 –MalE. This indicates that insertion of high copy plasmid also induces stress in the periplasmic region of the cell consequently inducing the activation of CpxR system. <br />
</p><br />
<br />
<p><br />
<h2 style="color:#0066CC">Experiment 2: Measuring RFP output of the CpxR-I13507 cells after exposure to different temperature for different time periods</h2></p><br />
<br />
<h3><u>Protocol:</u></h3><br />
<p><br />
Top 10 competent cells were transformed with CpxR-I13507 and plated. 5 ml overnight cultures were made from 5 different colonies using LB broth with appropriate antibiotics. Each of these cultures were aliquoted into six different tubes containing 600 µL of culture. These tubes were then placed in hot water baths at 30 C, 37C, 42C, 47C. Measurements were taken every hour for 5 hours after placing the tubes in different temperatures at 555 nm excitation and 632 nm emission.</p><br />
<br />
<br />
</li><a href="http://s872.photobucket.com/albums/ab287/iGEMCalgary_2010/?action=view&current=Untitled-6.png" target="_blank"><img src="http://i872.photobucket.com/albums/ab287/iGEMCalgary_2010/Untitled-6.png" border="0" alt="Temperature induction"></a> </li><br />
<br />
<p>Figure 3: RFP output produced by the CpxR-I13507 system when the system is heat shocked at different temperature for different lengths of time. The RFP output was measured at 555 nm excitation and 632 nm emission frequencies<br />
<br />
</p><br />
<br />
<h3><u>Discussion of Results and Conclusion</u></h3><br />
<p><br />
This graph shows that the CpxR system does respond to temperature activated stress. When the system is placed at 42 C the RFP output is much higher at t=0 compared to the system placed at 37 C or 30 C. This indicates that the system does get activated due to heat shock which matches the literature parameters. At 47 C, the system gets activated faster because the linear regression has a steeper slope. This indicates that the system is being stressed and it produces its downstream product which is RFP in this case and DegP and other chaperones in the genomic DNA much faster in order to cope with periplasmic protein denaturation. Also, it seems that the system gets activated dramatically after 3 hours regardless of the temperature, this could indicate that the system peaks after 3 hours and the genomic CpxR produces enough downstream chaperones and proteases in order for the system to be able to cope with stress which allows the RFP reading to decrease at 4 hours time because the cell reaches homeostasis. This allows the cell to get rid of misfolded protein and other factors that might be contributing to stressing it out and causing the Cpx regulon to be activated. The cell then shows a rapid rise again because it is still under heat shock stress. But, if the cell was placed at 37 degrees, the cell would show a flatline pattern rather than an oscillating pattern.<br />
</p><br />
<br />
<p><br />
<h2 style="color:#0066CC">Experiment 4: Measuring the GFP output through insertion of the mutant malE and malE31 with the transport signal sequence deleted into competent cells containing a fusion promoter (ibpAB-fsxA) coupled to a GFP reporter. </h2></P><br />
<br />
<h3><u>Purpose</u></h3><br />
<p><br />
The purpose of this assay is to test the output that the cytoplasmic acting fusion promoter (ibpAB-fsxA) will produce with proteins that are known to fold correctly (malEΔSS) and with proteins that are known to misfold (malE31ΔSS) in the cytoplasm. The plasmids containing malEΔSS and malE31ΔSS are coupled to an IPTG inducible promoter and were received from the lab of Jean-Michel Betton.<br />
</p><br />
</p><br />
<br />
</div><br />
<br />
</div><br />
<br />
</body><br />
</html></div>Emily Hickshttp://2010.igem.org/Team:Calgary/Parts/CharacterizationTeam:Calgary/Parts/Characterization2010-10-27T21:58:37Z<p>Emily Hicks: </p>
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<h1>Parts</h1><br />
<br />
<ul><br />
<li><a href="https://2010.igem.org/Team:Calgary/Parts/Parts_Submitted">Parts Submitted</a></li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Parts/Characterization">Characterization</a></li><br />
</ul><br />
<br />
</div><br />
<br />
<div class="mainbody"><br />
<br />
<span id="bodytitle"><h1>Characterization</h1></span><br />
<br />
<p><br />
<h2 style="color:#0066CC">Experiment 1: Characterization of the cpxR promoter's response to folding and misfolding proteins through co-transformation of MalE and MalE31 coupled to arabinose promoter in CpxR reporter competent cells</h2><br />
</p><br />
<br />
<h3><u>Protocol:</u></h3><br />
<br />
<p><br />
Arabinose inducible promoter (I0500) coupled with standard ribosome binding site (B0034) and the respective maltose binding protein were transformed into competent cells containing pCpxR coupled with RFP generator (I13507). These cells were plated and incubated overnight. Colonies from each of the plates were selected and overnight cultures were prepared at 37 C. These 5 ml overnight cultures were then sub-cultured in 20 ml broth. These were shaken for 6-8 hours and aliquoted into 5 ml cultures and induced with varying levels of arabinose(percent). This was incubated in the shaker for 12-14 hours and RFP output was measured using 555 excitation and 632 nm emission frequency.</p><br />
<br />
<h3><u>Results</u></h3><br />
<br />
<br />
</li><a href="http://s872.photobucket.com/albums/ab287/iGEMCalgary_2010/?action=view&current=Unititled-7.png" target="_blank"><img src="http://i872.photobucket.com/albums/ab287/iGEMCalgary_2010/Unititled-7.png" border="0" alt="CpxR"></a></li></td><br />
<br />
<br />
<p>Figure 1: RFP output produced by the CpxR-I13507 system when co-transfected with I0500-B0034-MalE (red) and I0500-B0034-MalE31 (blue) at different arabinose concentrations. RFP levels were measured at 555 nm excitation and 632 nm emission frequencies</p><br />
<br />
<br />
<br />
</li><a href="http://s872.photobucket.com/albums/ab287/iGEMCalgary_2010/?action=view&current=lineofbestfitCpxR.png" target="_blank"><img src="http://i872.photobucket.com/albums/ab287/iGEMCalgary_2010/lineofbestfitCpxR.png" border="0" alt="Photobucket"></a></li><br />
<br />
<p>Figure 2: RFP output produced by the CpxR-I13507 system when co-transfected with I0500-B0034-MalE (red) and I0500-B0034-MalE31 (blue) at different arabinose concentrations. RFP levels were measured at 555 nm excitation and 632 nm emission frequencies.</p><br />
<br />
<h3><u>Discussion of Results and Conclusion</u></h3><br />
<p><br />
Figure 1 and 2 indicate the RFP output normalized with growth ratio (OD) at different levels of arabinose. Figure 1 shows that CpxR-I13507 is activated at the highest level when MalE31, the periplasmic misfolder, is expressed. This occurs around 0.2% arabinose concentration. Similar trends are observed in the case of MalE which is a periplasmic folder. MalE and MalE31 activate the system at different levels. MalE31 has similar trends to MalE but has a higher level of RFP expression. These results prove that MalE and MalE31 can both activate the CpxR system however, MalE31, which misfolds, activates it more rapidly and at a lower level of arabinose concentration compared to MalE. If the line of best fit is studied, it is seen that MalE has very minimal level of Cpx activation. Whereas, malE31 has a linear regression which flattens out as the system reaches its upper threshold of detection. Biologically, this could mean that the MalE31 is activated at levels that saturate the cellular chaperones and cause the system to reach its threshold level of proteolytic and chaperone activities. Another interesting pattern observed is the fact that when MalE is constructed with CpxR-I13507 on the same plasmid (Green), the cell RFP output is much lower compared to cells co-transfected with CpxR-I13507 and I0500-B0034 –MalE. This indicates that insertion of high copy plasmid also induces stress in the periplasmic region of the cell consequently inducing the activation of CpxR system. <br />
</p><br />
<br />
<p><br />
<h2 style="color:#0066CC">Experiment 2: Measuring RFP output of the CpxR-I13507 cells after exposure to different temperature for different time periods</h2></p><br />
<br />
<h3><u>Protocol:</u></h3><br />
<p><br />
Top 10 competent cells were transformed with CpxR-I13507 and plated. 5 ml overnight cultures were made from 5 different colonies using LB broth with appropriate antibiotics. Each of these cultures were aliquoted into six different tubes containing 600 µL of culture. These tubes were then placed in hot water baths at 30 C, 37C, 42C, 47C. Measurements were taken every hour for 5 hours after placing the tubes in different temperatures at 555 nm excitation and 632 nm emission.</p><br />
<br />
<br />
</li><a href="http://s872.photobucket.com/albums/ab287/iGEMCalgary_2010/?action=view&current=Untitled-6.png" target="_blank"><img src="http://i872.photobucket.com/albums/ab287/iGEMCalgary_2010/Untitled-6.png" border="0" alt="Temperature induction"></a> </li><br />
<br />
<p>Figure 3: RFP output produced by the CpxR-I13507 system when the system is heat shocked at different temperature for different lengths of time. The RFP output was measured at 555 nm excitation and 632 nm emission frequencies<br />
<br />
</p><br />
<br />
<h3><u>Discussion of Results and Conclusion</u></h3><br />
<p><br />
This graph shows that the CpxR system does respond to temperature activated stress. When the system is placed at 42 C the RFP output is much higher at t=0 compared to the system placed at 37 C or 30 C. This indicates that the system does get activated due to heat shock which matches the literature parameters. At 47 C, the system gets activated faster because the linear regression has a steeper slope. This indicates that the system is being stressed and it produces its downstream product which is RFP in this case and DegP and other chaperones in the genomic DNA much faster in order to cope with periplasmic protein denaturation. Also, it seems that the system gets activated dramatically after 3 hours regardless of the temperature, this could indicate that the system peaks after 3 hours and the genomic CpxR produces enough downstream chaperones and proteases in order for the system to be able to cope with stress which allows the RFP reading to decrease at 4 hours time because the cell reaches homeostasis. This allows the cell to get rid of misfolded protein and other factors that might be contributing to stressing it out and causing the Cpx regulon to be activated. The cell then shows a rapid rise again because it is still under heat shock stress. But, if the cell was placed at 37 degrees, the cell would show a flatline pattern rather than an oscillating pattern.<br />
</p><br />
<br />
<p><br />
<h2 style="color:#0066CC">Experiment 4: Measuring the GFP output through insertion of the mutant malE and malE31 with the transport signal sequence deleted into competent cells containing a fusion promoter (ibpAB-fsxA) coupled to a GFP reporter. </h2></P><br />
<br />
<h3><u>Purpose</u></h3><br />
<p><br />
The purpose of this assay is to test the output that the cytoplasmic acting fusion promoter (ibpAB-fsxA) will produce with proteins that are known to fold correctly (malEΔSS) and with proteins that are known to misfold (malE31ΔSS) in the cytoplasm. The plasmids containing malEΔSS and malE31ΔSS are coupled to an IPTG inducible promoter and were received from the lab of Jean-Michel Betton.<br />
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<p>Here is a summary of all the parts submitted to the iGEM Registry of Standard Biological Parts.</p><br />
<br />
<p> Characterization data for our parts can be found <a href="https://2010.igem.org/Team:Calgary/Parts/Characterization">here</a>.<br />
<Br><Br><br />
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<p>Here is a summary of all the parts submitted to the iGEM Registry of Standard Biological Parts.</p><br />
<br />
<p> Characterization data for our parts can be found <a href="https://2010.igem.org/Team:Calgary/Parts/Characterization">here</a>.<br />
<br />
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<span id="bodytitle"><h1>iGEM Safety Questionnaire</h1></span><br />
<p><br />
As a safety precaution, all Igem team members have been fully trained in WHIMIS as well as in introductory Biosfatey.<br />
</p><br /><br />
<p><br />
<em>Would any of your project ideas raise safety issues in terms of:</em><br /> <br />
<em>researcher safety</em><br />
<em>public safety, or</em> <br />
<em>environmental safety?</em> <br />
</p><br />
<p><br />
No, not directly. The goal of our project is to create a tool that can help solve protein expression problems in future projects both within the context of the iGEM competiion and beyond. For this reason, our project only really poses safety issues if it was to be used to aid in the expression of toxic or otherwise dangerous proteins. Part of our ethical analysis included a discussion of some of the specific ethical, social and economic issues raised by our project. Check out our ethics page for more information.<br />
</p><br />
<br /><br />
<p><em>Do any of the new BioBrick parts (or devices) that you made this year raise any safety issues?</em></p> <br />
<p><br />
No, none of the parts that we made alone raise any safety issues. Again, our project only has the possibility of posing safety issues in regards to the final product the user of our tool kit is trying to produce.<br />
</p><br />
<br /><br />
<p><br />
<em>Is there a local biosafety group, committee, or review board at your institution?</em><br />
</p> <br />
<p><br />
Yes, we have an office of medical bioethics at our Univeristy. Our project has never come up with this office. Due to the fact that we are using non-pathogenic bacteria and that our project poses no direct safety issues to researchers the public or the environment, they have no concerns with our project at present.</p><br />
<br /><br />
<p><br />
<em>Do you have any other ideas how to deal with safety issues that could be useful for future iGEM competitions? How could parts, devices and systems be made even safer through biosafety engineering?</em></p><br />
<br />
<p><br />
insert answer here!<br />
</p><br />
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<br />
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<br />
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<ul><br />
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<span id="bodytitle"><h1>Cytoplasmic Stress Detectors</h1></span><br />
<br />
<br />
<h2 style="color:#0066CC">How does a native <i>E. coli</i> cell combat protein related stress in the Cytoplasm?</h2><br />
<br />
<p><br />
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.<br />
</p><br />
<br />
<br />
<h2 style="color:#0066CC">iGEM Calgary cytoplasmic stress detection circuit</h2><br />
<br />
<br />
<table><br />
<tr><br />
<td><br />
<br />
<img src="http://i872.photobucket.com/albums/ab287/iGEMCalgary_2010/ibpab-1.png"></img> </td><br />
<br />
<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><br />
<br />
</tr><br />
<br />
</table><br />
<br />
<h3>The ibpAB Promoter</h3><br />
<br />
<br />
<p><br />
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 hydrofobicity, previngin firtyher 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).<br />
<br />
<p><br />
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.<br />
.</p><br />
<br />
<table><br />
<tr><br />
<td><br />
<br />
<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><br />
</tr><br />
</table><br />
<br />
<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><br />
<br />
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<br />
<br />
<ul><br />
<li><a href="https://2010.igem.org/Team:Calgary/Project/Transcription">Transcription/Translation Reporter Circuit</a></li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Project/misfolding_overview">Protein Misfolding Reporters</a><br />
<ul><br />
<li><a href="https://2010.igem.org/Team:Calgary/Project/IbpAB">Cytoplasmic Stress Detectors</a></li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Project/CpxP">Periplasmic Stress Detectors</a></li><br />
</ul><br />
</li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Project/Controls">Testing our system</a></li><br />
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</ul><br />
<br />
</div><br />
<br />
<div class="mainbody"><br />
<br />
<span id="bodytitle"><h1>Cytoplasmic Stress Detectors</h1></span><br />
<br />
<br />
<h2 style="color:#0066CC">How does a native <i>E. coli</i> cell combat protein related stress in the Cytoplasm?</h2><br />
<br />
<p><br />
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.<br />
</p><br />
<br />
<br />
<h2 style="color:#0066CC">iGEM Calgary cytoplasmic stress detection circuit</h2><br />
<br />
<br />
<table><br />
<tr><br />
<td><br />
<br />
<img src="http://i872.photobucket.com/albums/ab287/iGEMCalgary_2010/ibpab-1.png"></img> </td><br />
<br />
<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><br />
<br />
</tr><br />
<br />
</table><br />
<br />
<h3>The ibpAB Promoter</h3><br />
<br />
<br />
<p><br />
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 hydrofobicity, previngin firtyher 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).<br />
<br />
<p><br />
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.<br />
.</p><br />
<br />
<table><br />
<tr><br />
<td><br />
<br />
<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><br />
</tr><br />
</table><br />
<br />
<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><br />
<br />
<br />
<h3> How are we utilizing this promoter?</h3><br />
<p><br />
This fusion promoter will be connected to the registry part I13504 which<br />
is RBS-GFP-B0015. The ibpAB/fxsA circuit will be activated in the presence<br />
of aggregation in the cell. We will be using MalE31 with a signal sequence<br />
deletion (MalE31&#8710;SS) which was designed by Betton et al. The native<br />
E. coli protein MalE generally exported into the periplasmic space but<br />
this mutated protein does not get exported to the periplasmic space due to<br />
the signal sequence deletion. Also Betton et al designed MalE31such that<br />
there are two amino acid changes in the protein and it misfolds. The<br />
MalE31&#8710;SS protein coding region will be used in order to induce<br />
cytoplasmic protein stress in E. coli. </p><br />
<p>Ideally, this misfolded<br />
MalE31&#8710;SS should activate the plasmid system containing<br />
ibpAB/fxsA-I13504 which will produce GFP alerting the researcher that<br />
their protein is not being expressed in the cell because it is misfolding<br />
and as a result getting degraded. Our circuit should also be activated<br />
much faster than the native stress system because the ibpAB/fxsA promoter<br />
is much more sensitive to the presence of aggregate bodies in the cell.<br />
The promoter also gives a much higher output compared to the promoters<br />
individually, which is the case in the E. coli genome which should allow<br />
us to detect the fluorescence level much faster.<br />
</p><br />
<br />
</td><br />
</tr><br />
</table><br />
<br />
<br />
</div><br />
<br />
</div><br />
<br />
</body><br />
</html></div>Emily Hickshttp://2010.igem.org/Team:Calgary/Project/IbpABTeam:Calgary/Project/IbpAB2010-10-27T10:37:41Z<p>Emily Hicks: </p>
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<h1>Project Descriptions</h1><br />
<br />
<br />
<ul><br />
<li><a href="https://2010.igem.org/Team:Calgary/Project/Transcription">Transcription/Translation Reporter Circuit</a></li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Project/misfolding_overview">Protein Misfolding Reporters</a><br />
<ul><br />
<li><a href="https://2010.igem.org/Team:Calgary/Project/IbpAB">Cytoplasmic Stress Detectors</a></li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Project/CpxP">Periplasmic Stress Detectors</a></li><br />
</ul><br />
</li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Project/Controls">Testing our system</a></li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Project/Achievements">Achievements</a></li><br />
</ul><br />
<br />
</div><br />
<br />
<div class="mainbody"><br />
<br />
<span id="bodytitle"><h1>Cytoplasmic Stress Detectors</h1></span><br />
<br />
<br />
<h2 style="color:#0066CC">How does a native <i>E. coli</i> cell combat protein related stress in the Cytoplasm?</h2><br />
<br />
<p><br />
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.<br />
</p><br />
<br />
<br />
<h2 style="color:#0066CC">iGEM Calgary cytoplasmic stress detection circuit</h2><br />
<br />
<br />
<table><br />
<tr><br />
<td><br />
<br />
<img src="http://i872.photobucket.com/albums/ab287/iGEMCalgary_2010/ibpab-1.png"></img> </td><br />
<br />
<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><br />
<br />
</tr><br />
<br />
</table><br />
<br />
<h3>The ibpAB Promoter</h3><br />
<br />
<br />
<p><br />
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 hydrofobicity, previngin firtyher 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).<br />
. </p><br />
<br />
<table><br />
<tr><br />
<td><br />
<br />
<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><br />
</tr><br />
</table><br />
<br />
<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><br />
<br />
<br />
<h3> How are we utilizing this promoter?</h3><br />
<p><br />
This fusion promoter will be connected to the registry part I13504 which<br />
is RBS-GFP-B0015. The ibpAB/fxsA circuit will be activated in the presence<br />
of aggregation in the cell. We will be using MalE31 with a signal sequence<br />
deletion (MalE31&#8710;SS) which was designed by Betton et al. The native<br />
E. coli protein MalE generally exported into the periplasmic space but<br />
this mutated protein does not get exported to the periplasmic space due to<br />
the signal sequence deletion. Also Betton et al designed MalE31such that<br />
there are two amino acid changes in the protein and it misfolds. The<br />
MalE31&#8710;SS protein coding region will be used in order to induce<br />
cytoplasmic protein stress in E. coli. </p><br />
<p>Ideally, this misfolded<br />
MalE31&#8710;SS should activate the plasmid system containing<br />
ibpAB/fxsA-I13504 which will produce GFP alerting the researcher that<br />
their protein is not being expressed in the cell because it is misfolding<br />
and as a result getting degraded. Our circuit should also be activated<br />
much faster than the native stress system because the ibpAB/fxsA promoter<br />
is much more sensitive to the presence of aggregate bodies in the cell.<br />
The promoter also gives a much higher output compared to the promoters<br />
individually, which is the case in the E. coli genome which should allow<br />
us to detect the fluorescence level much faster.<br />
</p><br />
<br />
</td><br />
</tr><br />
</table><br />
<br />
<br />
</div><br />
<br />
</div><br />
<br />
</body><br />
</html></div>Emily Hickshttp://2010.igem.org/Team:Calgary/Project/IbpABTeam:Calgary/Project/IbpAB2010-10-27T10:32:48Z<p>Emily Hicks: </p>
<hr />
<div>{{CalgaryMenu}}<br />
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<br />
<h1>Project Descriptions</h1><br />
<br />
<br />
<ul><br />
<li><a href="https://2010.igem.org/Team:Calgary/Project/Transcription">Transcription/Translation Reporter Circuit</a></li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Project/misfolding_overview">Protein Misfolding Reporters</a><br />
<ul><br />
<li><a href="https://2010.igem.org/Team:Calgary/Project/IbpAB">Cytoplasmic Stress Detectors</a></li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Project/CpxP">Periplasmic Stress Detectors</a></li><br />
</ul><br />
</li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Project/Controls">Testing our system</a></li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Project/Achievements">Achievements</a></li><br />
</ul><br />
<br />
</div><br />
<br />
<div class="mainbody"><br />
<br />
<span id="bodytitle"><h1>Cytoplasmic Stress Detectors</h1></span><br />
<br />
<br />
<h2 style="color:#0066CC">How does a native <i>E. coli</i> cell combat protein related stress in the Cytoplasm?</h2><br />
<br />
<p><br />
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.<br />
</p><br />
<br />
<br />
<h2 style="color:#0066CC">iGEM Calgary cytoplasmic stress detection circuit</h2><br />
<br />
<br />
<table><br />
<tr><br />
<td><br />
<br />
<img src="http://i872.photobucket.com/albums/ab287/iGEMCalgary_2010/ibpab-1.png"></img> </td><br />
<br />
<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><br />
<br />
</tr><br />
<br />
</table><br />
<br />
<h3>The ibpAB Promoter</h3><br />
<br />
<br />
<p><br />
In our cytoplasmic stress detector circuit, we decided to fuse two<br />
different promoter regions from two heat shock proteins, which are ibpAB<br />
and fxsA. In a study done by Kraft et al, they demonstrate that a fusion<br />
of IbpAB/fxsA promoters combined along with T7 DNA has a significantly<br />
higher output as a result of heat shock compared to the promoters<br />
individually. </p><br />
<br />
<table><br />
<tr><br />
<td><br />
<br />
<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><br />
</tr><br />
</table><br />
<br />
<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><br />
<br />
<br />
<h3> How are we utilizing this promoter?</h3><br />
<p><br />
This fusion promoter will be connected to the registry part I13504 which<br />
is RBS-GFP-B0015. The ibpAB/fxsA circuit will be activated in the presence<br />
of aggregation in the cell. We will be using MalE31 with a signal sequence<br />
deletion (MalE31&#8710;SS) which was designed by Betton et al. The native<br />
E. coli protein MalE generally exported into the periplasmic space but<br />
this mutated protein does not get exported to the periplasmic space due to<br />
the signal sequence deletion. Also Betton et al designed MalE31such that<br />
there are two amino acid changes in the protein and it misfolds. The<br />
MalE31&#8710;SS protein coding region will be used in order to induce<br />
cytoplasmic protein stress in E. coli. </p><br />
<p>Ideally, this misfolded<br />
MalE31&#8710;SS should activate the plasmid system containing<br />
ibpAB/fxsA-I13504 which will produce GFP alerting the researcher that<br />
their protein is not being expressed in the cell because it is misfolding<br />
and as a result getting degraded. Our circuit should also be activated<br />
much faster than the native stress system because the ibpAB/fxsA promoter<br />
is much more sensitive to the presence of aggregate bodies in the cell.<br />
The promoter also gives a much higher output compared to the promoters<br />
individually, which is the case in the E. coli genome which should allow<br />
us to detect the fluorescence level much faster.<br />
</p><br />
<br />
</td><br />
</tr><br />
</table><br />
<br />
<br />
</div><br />
<br />
</div><br />
<br />
</body><br />
</html></div>Emily Hickshttp://2010.igem.org/Team:Calgary/Project/IbpABTeam:Calgary/Project/IbpAB2010-10-27T10:32:03Z<p>Emily Hicks: </p>
<hr />
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<body><br />
<br />
<div class="container"><br />
<br />
<br />
<div class="sidebar"><br />
<br />
<br />
<h1>Project Descriptions</h1><br />
<br />
<br />
<ul><br />
<li><a href="https://2010.igem.org/Team:Calgary/Project/Transcription">Transcription/Translation Reporter Circuit</a></li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Project/misfolding_overview">Protein Misfolding Reporters</a><br />
<ul><br />
<li><a href="https://2010.igem.org/Team:Calgary/Project/IbpAB">Cytoplasmic Stress Detectors</a></li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Project/CpxP">Periplasmic Stress Detectors</a></li><br />
</ul><br />
</li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Project/Controls">Testing our system</a></li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Project/Achievements">Achievements</a></li><br />
</ul><br />
<br />
</div><br />
<br />
<div class="mainbody"><br />
<br />
<span id="bodytitle"><h1>Cytoplasmic Stress Detectors</h1></span><br />
<br />
<br />
<h2 style="color:#0066CC">How does a native <i>E. coli</i> cell combat protein related stress in the Cytoplasm?</h2><br />
<br />
<p><br />
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.<br />
</p><br />
<br />
<br />
<h2 style="color:#0066CC">iGEM Calgary cytoplasmic stress detection circuit</h2><br />
<br />
<br />
<table><br />
<tr><br />
<td><br />
<br />
<img src="http://i872.photobucket.com/albums/ab287/iGEMCalgary_2010/ibpab-1.png"></img> </td><br />
<br />
<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><br />
<br />
</tr><br />
<br />
</table><br />
<br />
<h3>Rationale behind picking this promoter</h3><br />
<br />
<br />
<p><br />
In our cytoplasmic stress detector circuit, we decided to fuse two<br />
different promoter regions from two heat shock proteins, which are ibpAB<br />
and fxsA. In a study done by Kraft et al, they demonstrate that a fusion<br />
of IbpAB/fxsA promoters combined along with T7 DNA has a significantly<br />
higher output as a result of heat shock compared to the promoters<br />
individually. </p><br />
<br />
<table><br />
<tr><br />
<td><br />
<br />
<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><br />
</tr><br />
</table><br />
<br />
<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><br />
<br />
<br />
<h3> How are we utilizing this promoter?</h3><br />
<p><br />
This fusion promoter will be connected to the registry part I13504 which<br />
is RBS-GFP-B0015. The ibpAB/fxsA circuit will be activated in the presence<br />
of aggregation in the cell. We will be using MalE31 with a signal sequence<br />
deletion (MalE31&#8710;SS) which was designed by Betton et al. The native<br />
E. coli protein MalE generally exported into the periplasmic space but<br />
this mutated protein does not get exported to the periplasmic space due to<br />
the signal sequence deletion. Also Betton et al designed MalE31such that<br />
there are two amino acid changes in the protein and it misfolds. The<br />
MalE31&#8710;SS protein coding region will be used in order to induce<br />
cytoplasmic protein stress in E. coli. </p><br />
<p>Ideally, this misfolded<br />
MalE31&#8710;SS should activate the plasmid system containing<br />
ibpAB/fxsA-I13504 which will produce GFP alerting the researcher that<br />
their protein is not being expressed in the cell because it is misfolding<br />
and as a result getting degraded. Our circuit should also be activated<br />
much faster than the native stress system because the ibpAB/fxsA promoter<br />
is much more sensitive to the presence of aggregate bodies in the cell.<br />
The promoter also gives a much higher output compared to the promoters<br />
individually, which is the case in the E. coli genome which should allow<br />
us to detect the fluorescence level much faster.<br />
</p><br />
<br />
</td><br />
</tr><br />
</table><br />
<br />
<br />
</div><br />
<br />
</div><br />
<br />
</body><br />
</html></div>Emily Hickshttp://2010.igem.org/Team:Calgary/Notebook/SafetyTeam:Calgary/Notebook/Safety2010-10-27T10:29:58Z<p>Emily Hicks: </p>
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<h1>Sections</h1><br />
<br />
<ul><br />
<li><a href="https://2010.igem.org/Team:Calgary/Notebook/Calendar">Calendar</a></li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Notebook/Future_Directions">Future Directions</a></li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Notebook/Safety_And_Protocols">Protocols</a></li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Notebook/Safety">Safety</a></li><br />
</ul><br />
<br />
<br />
</div><br />
<br />
<div class="mainbody"><br />
As a safety precaution, all Igem team members have been fully trained in WHIMIS as well as in introductory Buosfatey.<br />
<br />
Would any of your project ideas raise safety issues in terms of: <br />
researcher safety<br />
public safety, or <br />
environmental safety? <br />
<br />
No, not directly. The goal of our project is to create a tool that can help solve protein expression problems in future projects both within the ontext of the Igem competiion and beyond. For this reason, our project nly really poses safety issues if it was to be used to aid in the expreession of toxic or otherwise dangerous proteins. help future projects both iGEM projectas as w<br />
Do any of the new BioBrick parts (or devices) that you made this year raise any safety issues? <br />
<br />
No, none of the parts that we made alone raise any safetry issuees. Again, our prject only has the possibility of posing safety issues if. In that sense, ot is very much dependent on the specific aopllication to which it is used. Pur parts alone rasuie no direct concerns that the registry needs to be aware about.<br />
<br />
Is there a local biosafety group, committee, or review board at your institution? <br />
<br />
Yes, we have an office of medical bioethics at our Univeristy. Our project has never come up with this office. Due to the fact that we are using non-pathogenic bacteria and that our project poses no direct safety issues to researchers the public or the environment, they have no concerns with our project at present.<br />
Do you have any other ideas how to deal with safety issues that could be useful for future iGEM competitions? How could parts, devices and systems be made even safer through biosafety engineering?<br />
<br />
<br />
<br />
<br />
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</html></div>Emily Hickshttp://2010.igem.org/Team:Calgary/Project/IbpABTeam:Calgary/Project/IbpAB2010-10-27T10:27:23Z<p>Emily Hicks: </p>
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<br />
<br />
<ul><br />
<li><a href="https://2010.igem.org/Team:Calgary/Project/Transcription">Transcription/Translation Reporter Circuit</a></li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Project/misfolding_overview">Protein Misfolding Reporters</a><br />
<ul><br />
<li><a href="https://2010.igem.org/Team:Calgary/Project/IbpAB">Cytoplasmic Stress Detectors</a></li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Project/CpxP">Periplasmic Stress Detectors</a></li><br />
</ul><br />
</li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Project/Controls">Testing our system</a></li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Project/Achievements">Achievements</a></li><br />
</ul><br />
<br />
</div><br />
<br />
<div class="mainbody"><br />
<br />
<span id="bodytitle"><h1>Cytoplasmic Stress Detectors</h1></span><br />
<br />
<br />
<h2 style="color:#0066CC">How does a native <i>E. coli</i> cell combat protein related stress in the Cytoplasm?</h2><br />
<br />
<p><br />
There are several heat shock pathways in E. coli which are actively<br />
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. Sigma32 is a factor that is crucial for maintaining and monitoring heat shock responses in the cytoplasm of <i>E. coli</i>. Sigma 32 and other house keeping factors act as transcription factors for small heat shock proteins (sHsps). sHsps consist of proteins such<br />
as ibpA, ibpB, DnaK, DnaJ, GroEL and GroES. Amongst these, IbpA (inclusion body binding proteins) and ibpB<br />
are two different proteins that are activated as a result of cytoplasmic<br />
stress response. IbpA and ibpB proteins are chaperones that are<br />
responsible for refolding aggregated bodies and inclusion bodies into<br />
their native conformation.</p><br />
<br />
<br />
<h2 style="color:#0066CC">iGEM Calgary cytoplasmic stress detection circuit</h2><br />
<br />
<br />
<table><br />
<tr><br />
<td><br />
<br />
<img src="http://i872.photobucket.com/albums/ab287/iGEMCalgary_2010/ibpab-1.png"></img> </td><br />
<br />
<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><br />
<br />
</tr><br />
<br />
</table><br />
<br />
<h3>Rationale behind picking this promoter</h3><br />
<br />
<br />
<p><br />
In our cytoplasmic stress detector circuit, we decided to fuse two<br />
different promoter regions from two heat shock proteins, which are ibpAB<br />
and fxsA. In a study done by Kraft et al, they demonstrate that a fusion<br />
of IbpAB/fxsA promoters combined along with T7 DNA has a significantly<br />
higher output as a result of heat shock compared to the promoters<br />
individually. </p><br />
<br />
<table><br />
<tr><br />
<td><br />
<br />
<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><br />
</tr><br />
</table><br />
<br />
<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><br />
<br />
<br />
<h3> How are we utilizing this promoter?</h3><br />
<p><br />
This fusion promoter will be connected to the registry part I13504 which<br />
is RBS-GFP-B0015. The ibpAB/fxsA circuit will be activated in the presence<br />
of aggregation in the cell. We will be using MalE31 with a signal sequence<br />
deletion (MalE31&#8710;SS) which was designed by Betton et al. The native<br />
E. coli protein MalE generally exported into the periplasmic space but<br />
this mutated protein does not get exported to the periplasmic space due to<br />
the signal sequence deletion. Also Betton et al designed MalE31such that<br />
there are two amino acid changes in the protein and it misfolds. The<br />
MalE31&#8710;SS protein coding region will be used in order to induce<br />
cytoplasmic protein stress in E. coli. </p><br />
<p>Ideally, this misfolded<br />
MalE31&#8710;SS should activate the plasmid system containing<br />
ibpAB/fxsA-I13504 which will produce GFP alerting the researcher that<br />
their protein is not being expressed in the cell because it is misfolding<br />
and as a result getting degraded. Our circuit should also be activated<br />
much faster than the native stress system because the ibpAB/fxsA promoter<br />
is much more sensitive to the presence of aggregate bodies in the cell.<br />
The promoter also gives a much higher output compared to the promoters<br />
individually, which is the case in the E. coli genome which should allow<br />
us to detect the fluorescence level much faster.<br />
</p><br />
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</html></div>Emily Hickshttp://2010.igem.org/Team:Calgary/Project/misfolding_overviewTeam:Calgary/Project/misfolding overview2010-10-27T10:24:48Z<p>Emily Hicks: </p>
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<br />
<br />
<ul><br />
<li><a href="https://2010.igem.org/Team:Calgary/Project/Transcription">Transcription/Translation Reporter Circuit</a></li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Project/misfolding_overview">Protein Misfolding Reporters</a><br />
<ul><br />
<li><a href="https://2010.igem.org/Team:Calgary/Project/IbpAB">Cytoplasmic Stress Detectors</a></li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Project/CpxP">Periplasmic Stress Detectors</a></li><br />
</ul><br />
</li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Project/Controls">Testing our system</a></li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Project/Achievements">Achievements</a></li><br />
</ul><br />
<br />
</div><br />
<br />
<div class="mainbody"><br />
<br />
<span id="bodytitle"><h1>Misfolding detection circuit overview</h1></span><br />
<br />
<h2 style="color:#0066CC">How Does Protein Misfolding Occur?</h2><br />
<p><br />
Protein misfolding can occur as a result of a variety of factors. Overproduction of proteins in the cell is a good example. When proteins are overproduced, the cell can become overwhelmed and lack the necessary resources such as chaperones in order to deal with the large amount of protein. Proteins can also misfold due to mutations that occur in the coding region of the protein that can alter the amino acid sequence thereby interrupting the native structure of the protein. This can cause it to misfold into a non-functional state. Proteins can also misfold due to cellular stress such as changes in pH, temperature and changes in media. Localization can also be an issue. If a periplasmic protein lacks a signal sequence for example, it could misfold in the cytoplasm because the conditions are different in the two cellular compartments.<br />
</p><br />
<br /><br />
<br />
<h2 style="color:#0066CC">Why do we care?</h2><br />
<p><br />
Protein misfolding is an important topic in mnay regards. Many diseases, particularly neurodegenerative disorders such as Alzheimer’s Disease and __ result from misfolding proteins. The production recmonbaint proteins in prokaryoes such as E. Coli can also pose a problem. Non-native proteins are more susceptible to misfolding. This can compliacte many lab projectas such as the deisgn of protein drugs.<br />
</p><br />
<br /><br />
<br />
<h2 style="color:#0066CC">How does our system detect protein misfolding?</h2><br />
<h3>Current methods</h3><br />
<p><br />
GFP fusions are a method commonly used to detect protein misfolding. Targeted proteins can be fused to the C-Terminal of reporter genes such as GFP or Luciferase. If the target gene folds correctly, it would permit the reporter gene to also fold correctly, thus giving a measurable output. If the target gene was not able to fold however, the thought is that the reporter gene would not be able to fold correctly either, Arguments have been made however, that the fusion may affect the solubility of the target protein, thus resulting in an ineffective testing system. A more recent system has been the use of a split GFP system. Cabantous et al (2005) describe a system using two fractions of GFP. The smaller part is fused to the target protein. The small size of the fraction of GFP fused to the target protein is thought to not affect the solubility of the protein of interest. Nevertheless, many heterologous proteins often are not suitable for fusion with such reporters due to inaccessible C terminus of the target protein. <br />
</p><br />
<br />
<h3>Our System</h3><br />
<p><br />
Another method of protein misfolding detecton is thus to look at transcription levels of different heat shock promoters. By monitoring the activity levels of native stress promoters, you cab look more to the cell to report in its own stress levels. Because the reporter itself is decoupled from the stress, there is a minimized chance of the reporter having a stabilizing effect on the misfolding protein.Because transcription from these promoters is drastically increased during times of stress in the cell, these promoters, when coupled with different reporter genes such as GFP or lacZ, can be used as indicators of protein misfolding, as this is a stress for the cell. <br />
</p><br />
<br />
<h3>Our stress promoters</h3><br />
<p><br />
We chose four stress promoters to look at: three that monitor stress in periplasm of E Coli: <a href="https://2010.igem.org/Team:Calgary/Project/CpxP">Periplasmic Stress Detectors</a>, and one that monitors stress in the cytoplasm of E. Coli: <a href="https://2010.igem.org/Team:Calgary/Project/IbpAB">Cytoplasmic Stress Detectors</a><br />
</p><br />
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<li><a href="https://2010.igem.org/Team:Calgary/Project/Transcription">Transcription/Translation Reporter Circuit</a></li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Project/misfolding_overview">Protein Misfolding Reporters</a><br />
<ul><br />
<li><a href="https://2010.igem.org/Team:Calgary/Project/IbpAB">Cytoplasmic Stress Detectors</a></li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Project/CpxP">Periplasmic Stress Detectors</a></li><br />
</ul><br />
</li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Project/Controls">Testing our system</a></li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Project/Achievements">Achievements</a></li><br />
</ul><br />
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<br />
<span id="bodytitle"><h1>Achievements</h1></span><br />
<br />
<p><br />
<br />
Bronze Medal Requirements<br />
<br />
Register the team<br />
<br />
Successfully complete and submit a project summary form<br />
<br />
Create a Wiki<br />
<br />
Present a presenattiona nd a potsre at the Igem Jamboree<br />
<br />
Enter information detailing at least one new standard BioBrick Part or Device in the Registry of Parts <br />
<br />
-Data was entered for 11 new biobrick parts<br />
<br />
Submit DNA for at least one new BioBrick Part or Device to the Registry of Parts<br />
<br />
<br />
We designed and submitted DNA for three new biobrick parts<br />
--cytoplasm maltose binding protein (malESS)<br />
-I bpAb/ fxsA fusion promoter<br />
- cpxP Promoter<br />
<br />
We also built the following new constructs:<br />
<br />
-ibpAB reporter<br />
CpxR reporter<br />
DegP reporter<br />
MalE generator<br />
MalE31 generator<br />
K13500-I13507-I0500-B0034-malE31<br />
<br />
All of these parts were verified via resriction digests and PCR, and then finally sequenced.<br />
<br />
Silver medal Requirments<br />
<br />
Demonstrate that one of your parts works as expected<br />
<br />
Through our characeroization data, we showed that several of our parts worked as expected. <br />
<br />
The cpxR reporter was found to report on misfolding protein. We characterized this promoter with varying concentrations of folding and midfolding proteins produced. We also tested this promoter with NLPE, an outer membrane lipoprotein that literature has found activates the cpx pathway. Finally, we tested this promoter in varying heat shock conditions.<br />
<br />
Both MalE and malE31 also worked as expected, malE folding in the periplams and malE31 showing a misfolding reponse. This was indictaed through tetsing with a reporter from another lab (Raivio) (for more info click here) as well as tetsing with our cpxR reporter click here.<br />
<br />
We characetrized the cpxR reporter, malE31 ibpAB and . See characterization data here.<br />
<br />
Characterize or improve an existing BioBrick Part or Device and enter this information back on the Registry. <br />
<br />
-we entered new DNA as well as new sequences for malE and malE31.<br />
- we entered characterization data for the cpxR promoter<br />
<br />
Help another iGEM team by, for example, characterizing a part, debugging a construct, or modeling or simulating their system. <br />
<br />
-We tetsed out a part for Lethbridge. We characterized it for possible misfodling in the cytoplasm. Resuylts can be found here.<br />
<br />
- We also attended a regional workship as well as a mock Igem event with the Univeristy of Salberta as well as the Univeristy of Lethbridge. Here we helped each their out with our projects by practicing our presentaitons for each other, giving suggestions, future directions as well as touble shooting<br />
<br />
-Finally our team filled out surveys for the following teams:<br />
<br />
<br />
- This year we chose to approach ethjcs by making a pods acts covering a few different ethical issues pertaining to synthetic biology. We felt tat this would be a novel way to increase awareness about the field of syntyehtic biology while having a discussion of important issues. We felt that this would be a much more accessible way for the public to gain a better understanding of what the real fears in sythetic biology is as well as to dispell some of the misleading iompressions given by media sources.<br />
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</html></div>Emily Hickshttp://2010.igem.org/Team:Calgary/Community/EthicsTeam:Calgary/Community/Ethics2010-10-27T10:18:25Z<p>Emily Hicks: </p>
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<h1>Community</h1><br />
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<ul><br />
<li><a href="https://2010.igem.org/Team:Calgary/Community/Ethics">Human Practices</a></li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Community/Gallery">Photo Gallery</a></li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Community/Conferences">Conferences</a></li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Community/Podcasts">Podcasts</a></li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Community/Blog">Visit Our Blog!</a></li><br />
</ul><br />
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<div class="mainbody"><br />
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<span id="bodytitle"><h1>Ethics and Human Practices</h1></span><br />
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<h3>Ethics Podcast and Paper</h3><br />
<table><br />
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<td><p>Synthetic Biology is a new and emerging field with many potential benefits. Because the public perception of synthetic biology is still very limited, knowledge and understanding of projects relating to synthetic biology continues to remain limited, as well. Therefore, the iGEM Calgary team believes that it is essential to recognize the ethical implications of our project. We hope to improve the public's understanding of Synthetic Biology and alleviate common concerns with the field. With this aim, our team began working on two major projects for the Ethics and Human Practices component. </p><br />
</td><br />
<td><a href="http://s872.photobucket.com/albums/ab287/iGEMCalgary_2010/?action=view&current=Untitled-8-1.png" target="_blank"><img src="http://i872.photobucket.com/albums/ab287/iGEMCalgary_2010/Untitled-8-1.png" border="0" alt="Photobucket"></a><br />
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<p><br />
One project examines Synthetic Biology from a general perspective, while the other discusses the ethical implications of our project specifically.<br />
In the first project, we have created a series of <a href="https://2010.igem.org/Team:Calgary/Community/Podcasts">podcasts</a> regarding various issues relating to Synthetic Biology. In the podcasts, members of our group have an open discussion about issues such as the concept of synthetic life and the nature of open-source science. This allowed us to reflect on the potentials of Synthetic Biology, and hence, enrich our understanding about this field. We also wrote a paper where we discuss the ethical, economical and social implications of our project. This allowed us to understand our project from different perspectives. The significance of this paper is that it allowed us, as a team, to reflect on the potential benefits and risks that our project can entail. <br />
Apart from working on these two projects over the summer, we have also incorporated an outreach program into our Ethics portion. We have presented to several high schools, with the aim of increasing awareness of Synthetic Biology to high-school students. As a team, we believe that high school students will be the generation that will be increasingly exposed to this field.</p><br />
<br />
<br />
<h3>High School Presentations</h3><br />
<br />
<p> Synthetic Biology is a new emerging field of Science and the general public are still unfamiliar about its potentials. High school students are an important generation who should become aware of this fascinating approach to science. This is not just to increase public awareness but also because high school students are the ones who will be most exposed to Synthetic Biology in the upcoming years. With that intention, we went to several high schools to introduce Synthetic Biology and its potentials. We discussed with the students about the three components of projects related to Synthetic Biology in iGEM: Wetlab, Modelling and Human Practice. We emphasized on the interdisciplinary characteristic of projects in iGEM that are related to Synthetic Biology. With this, we hope that students of next generations are more interested in Synthetic Biology. In addition, we hope to empower the students with the growing techniques and perspectives of looking at biological functions. </p><br />
<br />
<h3>Research Symposiums</h3><br />
<br />
<p> <br />
This year our iGEM team participated in a few research sympoisums. This was a great way to raise more awareness for iGEM at our own Univeristy while getting to practice presenting our work to varied audiences.<br />
<br><br><br />
''Bhsc Research Symposium''<Br><br />
On October 7th we presented an oral presentation as well as a poster rpesentation at the Bachelor of Health Scinecs research symposium. This was a great way to get more people from the faculty of medicine interested in iGEM. There was a mix of undergradutae students, researchers and professors in attendance, so this was a great place to show pff what we did this Summer.<br />
<Br><Br><br />
'''USRP Research Symposium'''<Br><br />
On October 7th we also had the opportunity to present a poster at the USRP Markin Research Symposium at our university. This was a great opportunity to get the word out about Igem and our project to a wider audience at our university. There were students and faculty mambers from across all faculties ad it was good practice trying to explain our project to people with very little biological background.<br />
<Br><Br><br />
'''Student’s Union Research Symposium'''<Br><br />
Still to come we have the Univeristy of Calgary’s Student’s Union Research Symposium in late November. This will be a good platform to start recruitmnent for next year’s team as this symposium attracts students and faculty from across the university. This will also be a great wrap-up to our iGEM season a ocuple of weeks after the actual Igem Jamboree.</p><br />
<h3>Bake Sale</h3><br />
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<p> This year, the purpose of the bake sale included two aspects: firstly, it was to raise awareness about Synthetic Biology in our Health Sciences department and secondly, it was a fundraising activity to have the financial support to complete the Wetlab portion of our project.</p><br />
<br />
<p><br />
The Bake Sale was a great way to interact with the undergraduate and graduate students in the Health Sciences department of the University of Calgary. Many researchers were interested in finding out more about Synthetic Biology in the small time of interaction during the fundraising. <br />
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</html></div>Emily Hickshttp://2010.igem.org/Team:Calgary/Project/ControlsTeam:Calgary/Project/Controls2010-10-27T09:20:30Z<p>Emily Hicks: </p>
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<h1>Project Descriptions</h1><br />
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<ul><br />
<li><a href="https://2010.igem.org/Team:Calgary/Project/Transcription">Transcription/Translation Reporter Circuit</a></li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Project/misfolding_overview">Protein Misfolding Reporters</a><br />
<ul><br />
<li><a href="https://2010.igem.org/Team:Calgary/Project/IbpAB">Cytoplasmic Stress Detectors</a></li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Project/CpxP">Periplasmic Stress Detectors</a></li><br />
</ul><br />
</li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Project/Controls">Testing our system</a></li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Project/Achievements">Achievements</a></li><br />
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<span id="bodytitle"><h1>System Controls</h1></span><br />
<br />
<p>Testing our System<br />
<br />
Once constructed, we needed a way to test the cytoplasm and periplasmic stress promoters in order to characterize them. We did this in three different ways.<br />
<br />
1) tetsing with known folding and misfolding proteins<br />
<br />
2) tetsing with NLPE, an outer membrane lipoprotein known to activate the cpx pathway<br />
<br />
2) tetsing with varying temperature conditions.<br />
<br />
<br />
Tetsing with known folding and misfolding proteins<br />
<br />
We first needed to identify proteins that we know fold and don’t fold well inE. coili. For this we chose the maltose binding protein. This is a protein known to fold extremely well in the periplasm of E. Coli. MalE31, a mutant with two amino acid substitutions at postion 33 and 34, does not fold and is classified as a non-folder. MalE with the signal sequence removed, does not move in to the periplas, but remains in the cytoplasm where it folds extremely well. Male31 with the signal sequence removed, is a non folder in the cytoplasm. Thus we have four proteins coverning folding and non-folding in botht he periplas and the cytoplasm.<br />
<br />
(Show the chart here of the maltose binding proteins)<br />
<br />
We received these genes from the Betton labs in France. We biobricked these parts, but before testing our stress reporters with them, we wanted to first test these parts to sohow that they work as expected.. To do this, we transformed them into strains of cells containing cpxR and degP promoters up stream of a lacZ rpeorter (Raivio labs). We would expect malE31, if it misfolded, to activate the cpxR and degP stress promoters, thsus providing a blue output from lacZ. MalE on the other hand would not misfod, and therefore would not activate these promoters, and we would not expect to see any lacZ activity. This allowed us to conlcude that malE and malE31 work the way that we expected them to. See results on our characterization page.<br />
<br />
Once malE and malE31 were shown to be functional, we then used them to test out the stress promoters. We did this by making competent cells containing our reporeter circuits. We then transofmed in exprressio constructs for our malE and mutant malE proteins. We then measured fluorescence output from our reporter constructs. See resuts for this on our characterization opage.<br />
<br />
Testing with NLPE<br />
<br />
NLPE is an outer membrane lipoprotein that literature has shown actibates the cpX pathway. We transformed expression costructs for this protien (obtained from the Rvaio lab) into competent cells containing our cpxR reporter and looked for fluoresecnt output. Results for this experiment can be viewed on our characyerization page.<br />
<br />
Testing with Vraying Temperature Conditions<br />
<br />
Finally we tested the cpxR promoter</p><br />
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</html></div>Emily Hickshttp://2010.igem.org/Team:Calgary/Project/misfolding_overviewTeam:Calgary/Project/misfolding overview2010-10-27T08:39:16Z<p>Emily Hicks: </p>
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<h1>Project Descriptions</h1><br />
<br />
<br />
<ul><br />
<li><a href="https://2010.igem.org/Team:Calgary/Project/Transcription">Transcription/Translation Reporter Circuit</a></li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Project/misfolding_overview">Protein Misfolding Reporters</a><br />
<ul><br />
<li><a href="https://2010.igem.org/Team:Calgary/Project/IbpAB">Cytoplasmic Stress Detectors</a></li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Project/CpxP">Periplasmic Stress Detectors</a></li><br />
</ul><br />
</li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Project/Controls">System Controls</a></li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Project/Achievements">Achievements</a></li><br />
</ul><br />
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<span id="bodytitle"><h1>Misfolding detection circuit overview</h1></span><br />
<p> Protein misfolding<br />
<br />
Protein misfolding can occur as a result of a variety of factors. Overproduction of proteins in the cell is a good example. When proteins are overproduced, the cell can become overwhelmed and lack the necessary resources such as chaperones in order to deal with the large amount of protein. Proteins can also misfold due to mutations that occur in the coding region of the protein that can alter the amino acid sequence thereby interrupting the native structure of the protein. This can cause it to misfold into a non-functional state. Proteins can also misfold due to cellular stress such as changes in pH, temperature and changes in media. Localization can also be an issue. If a periplasmic protein lacks a signal sequence for example, it could misfold in the cytoplasm because the conditions are different in the two cellular compartments.<br />
<br />
Why do we care?<br />
<br />
Protein misfolding is an important topic in mnay regards. Many diseases, particularly neurodegenerative disorders such as Alzheimer’s Disease and __ result from misfolding proteins. The production recmonbaint proteins in prokaryoes such as E. Coli can also pose a problem. Non-native proteins are more susceptible to misfolding. This can compliacte many lab projectas such as the deisgn of protein drugs.<br />
<br />
How does our system detect protein misfolding?<br />
<br />
Current methods<br />
<br />
GFP fusions are a method commonly used to detect protein misfolding. Targeted proteins can be fused to the C-Terminal of reporter genes such as GFP or Luciferase. If the target gene folds correctly, it would permit the reporter gene to also fold correctly, thus giving a measurable output. If the target gene was not able to fold however, the thought is that the reporter gene would not be able to fold correctly either, Arguments have been made however, that the fusion may affect the solubility of the target protein, thus resulting in an ineffective testing system. A more recent system has been the use of a split GFP system. Cabantous et al (2005) describe a system using two fractions of GFP. The smaller part is fused to the target protein. The small size of the fraction of GFP fused to the target protein is thought to not affect the solubility of the protein of interest. Nevertheless, many heterologous proteins often are not suitable for fusion with such reporters due to inaccessible C terminus of the target protein. <br />
<br />
Our System<br />
<br />
Another method of protein misfolding detecton is thus to look at transcription levels of different heat shock promoters. By monitoring the activity levels of native stress promoters, you cab look more to the cell to report in its own stress levels. Because the reporter itself is decoupled from the stress, there is a minimized chance of the reporter having a stabilizing effect on the misfolding protein.Because transcription from these promoters is drastically increased during times of stress in the cell, these promoters, when coupled with different reporter genes such as GFP or lacZ, can be used as indicators of protein misfolding, as this is a stress for the cell. <br />
<br />
Our stress promoters<br />
<br />
We chose four stress promoters to look at: three that monitor stress in periplasm of E Coli (click here) and one that monitirs stress in the cytoplasm of E. Coli (click here) <br />
<br />
Cytoplamsic Stress<br />
<br />
The Cytplasmic Stress Pathway<br />
<br />
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. <br />
<br />
The ibpAB Promoter<br />
<br />
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 hydrofobicity, previngin firtyher 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).<br />
<br />
The ibpAB fusion promoter<br />
<br />
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.<br />
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</html></div>Emily Hickshttp://2010.igem.org/Team:Calgary/Parts/CharacterizationTeam:Calgary/Parts/Characterization2010-10-27T06:27:47Z<p>Emily Hicks: </p>
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<h1>Parts</h1><br />
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<ul><br />
<li><a href="https://2010.igem.org/Team:Calgary/Parts/Parts_Submitted">Parts Submitted</a></li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Parts/Favourites">Group Favourites</a></li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Parts/Characterization">Characterization</a></li><br />
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<span id="bodytitle"><h1>Characterization</h1></span><br />
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<p><br />
<h3>Experiment 1: Measuring RFP output by co-transformation of MalE and MalE31 coupled to arabinose promoter in the CpxR-RFP competent cells</h3><br />
</p><br />
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<p><u>Protocol:</u></p><br />
<br />
<p><br />
Arabinose inducible promoter (I0500) coupled with standard ribosome binding site (B0034) and the respective maltose binding protein were transformed into competent cells containing pCpxR coupled with RFP generator (I13507). These cells were plated and incubated overnight. Colonies from each of the plates were selected and overnight cultures were prepared at 37 C. These 5 ml overnight cultures were then sub-cultured in 20 ml broth. These were shaken for 6-8 hours and aliquoted into 5 ml cultures and induced with varying levels of arabinose(percent). This was incubated in the shaker for 12-14 hours and RFP output was measured using 555 excitation and 632 nm emission frequency.</p><br />
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<p><u>Results</u></p><br />
<br />
</li><a href="http://s872.photobucket.com/albums/ab287/iGEMCalgary_2010/?action=view&current=Unititled-7.png" target="_blank"><img src="http://i872.photobucket.com/albums/ab287/iGEMCalgary_2010/Unititled-7.png" border="0" alt="CpxR"></a></li><br />
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<p>RFP output produced by the CpxR-I13507 system when co-transfected with I0500-B0034-MalE (red) and I0500-B0034-MalE31 (blue) at different arabinose concentrations. RFP levels were measured at 555 nm excitation and 632 nm emission frequencies</p><br />
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</li><a href="http://s872.photobucket.com/albums/ab287/iGEMCalgary_2010/?action=view&current=lineofbestfitCpxR.png" target="_blank"><img src="http://i872.photobucket.com/albums/ab287/iGEMCalgary_2010/lineofbestfitCpxR.png" border="0" alt="Photobucket"></a></li><br />
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<p>RFP output produced by the CpxR-I13507 system when co-transfected with I0500-B0034-MalE (red) and I0500-B0034-MalE31 (blue) at different arabinose concentrations. RFP levels were measured at 555 nm excitation and 632 nm emission frequencies.</p><br />
<br />
<p><u>Discussion of Results and Conclusion</u></p><br />
<p><br />
Figure 1 and 2 indicate the RFP output normalized with growth ratio (OD) at different levels of arabinose. Figure 1 shows that CpxR-I13507 is activated at the highest level when MalE31, the periplasmic misfolder, is expressed. This occurs around 0.2% arabinose concentration. Similar trends are observed in the case of MalE which is a periplasmic folder. MalE and MalE31 activate the system at different levels. MalE31 has similar trends to MalE but has a higher level of RFP expression. These results prove that MalE and MalE31 can both activate the CpxR system however, MalE31, which misfolds, activates it more rapidly and at a lower level of arabinose concentration compared to MalE. If the line of best fit is studied, it is seen that MalE has very minimal level of Cpx activation. Whereas, malE31 has a linear regression which flattens out as the system reaches its upper threshold of detection. Biologically, this could mean that the MalE31 is activated at levels that saturate the cellular chaperones and cause the system to reach its threshold level of proteolytic and chaperone activities. Another interesting pattern observed is the fact that when MalE is constructed with CpxR-I13507 on the same plasmid (Green), the cell RFP output is much lower compared to cells co-transfected with CpxR-I13507 and I0500-B0034 –MalE. This indicates that insertion of high copy plasmid also induces stress in the periplasmic region of the cell consequently inducing the activation of CpxR system. <br />
</p><br />
<br />
<p><br />
<h3>Experiment 2: Measuring RFP output of the CpxR-I13507 cells after exposure to different temperature for different time periods</h3></p><br />
<br />
<p><u>Protocol:</u></p><br />
<p><br />
Top 10 competent cells were transformed with CpxR-I13507 and plated. 5 ml overnight cultures were made from 5 different colonies using LB broth with appropriate antibiotics. Each of these cultures were aliquoted into six different tubes containing 600 µL of culture. These tubes were then placed in hot water baths at 30 C, 37C, 42C, 47C. Measurements were taken every hour for 5 hours after placing the tubes in different temperatures at 555 nm excitation and 632 nm emission.</p><br />
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</li><a href="http://s872.photobucket.com/albums/ab287/iGEMCalgary_2010/?action=view&current=Untitled-6.png" target="_blank"><img src="http://i872.photobucket.com/albums/ab287/iGEMCalgary_2010/Untitled-6.png" border="0" alt="Temperature induction"></a> </li><br />
<br />
<p>Figure 2: RFP output produced by the CpxR-I13507 system when the system is heat shocked at different temperature for different lengths of time. The RFP output was measured at 555 nm excitation and 632 nm emission frequencies<br />
<br />
</p><br />
<br />
<p><u>Discussion of Results and Conclusion</u></p><br />
<p><br />
This graph shows that the CpxR system does respond to temperature activated stress. When the system is placed at 42 C the RFP output is much higher at t=0 compared to the system placed at 37 C or 30 C. This indicates that the system does get activated due to heat shock which matches the literature parameters. At 47 C, the system gets activated faster because the linear regression has a steeper slope. This indicates that the system is being stressed and it produces its downstream product which is RFP in this case and DegP and other chaperones in the genomic DNA much faster in order to cope with periplasmic protein denaturation. Also, it seems that the system gets activated dramatically after 3 hours regardless of the temperature, this could indicate that the system peaks after 3 hours and the genomic CpxR produces enough downstream chaperones and proteases in order for the system to be able to cope with stress which allows the RFP reading to decrease at 4 hours time because the cell reaches homeostasis. This allows the cell to get rid of misfolded protein and other factors that might be contributing to stressing it out and causing the Cpx regulon to be activated. The cell then shows a rapid rise again because it is still under heat shock stress. But, if the cell was placed at 37 degrees, the cell would show a flatline pattern rather than an oscillating pattern.<br />
</p><br />
<br />
<p><br />
<h3>Experiment 4: Measuring the GFP output through insertion of the mutant malE and malE31 with the transport signal sequence deleted into competent cells containing a fusion promoter (ibpAB-fsxA) coupled to a GFP reporter. </h3></P><br />
<br />
<p><u>Purpose</u></p><br />
<p><br />
The purpose of this assay is to test the output that the cytoplasmic acting fusion promoter (ibpAB-fsxA) will produce with proteins that are known to fold correctly (malEΔSS) and with proteins that are known to misfold (malE31ΔSS) in the cytoplasm. The plasmids containing malEΔSS and malE31ΔSS are coupled to an IPTG inducible promoter and were received from the lab of Jean-Michel Betton.<br />
</p><br />
</p><br />
<br />
</div><br />
<br />
</div><br />
<br />
</body><br />
</html></div>Emily Hickshttp://2010.igem.org/Team:Calgary/Parts/CharacterizationTeam:Calgary/Parts/Characterization2010-10-27T06:27:05Z<p>Emily Hicks: </p>
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<h1>Parts</h1><br />
<br />
<ul><br />
<li><a href="https://2010.igem.org/Team:Calgary/Parts/Parts_Submitted">Parts Submitted</a></li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Parts/Favourites">Group Favourites</a></li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Parts/Characterization">Characterization</a></li><br />
</ul><br />
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<div class="mainbody"><br />
<br />
<span id="bodytitle"><h1>Characterization</h1></span><br />
<br />
<p><br />
<h3>Experiment 1: Measuring RFP output by co-transformation of MalE and MalE31 coupled to arabinose promoter in the CpxR-RFP competent cells</h3><br />
</p><br />
<br />
<p><u>Protocol:</u></p><br />
<br />
<p><br />
Arabinose inducible promoter (I0500) coupled with standard ribosome binding site (B0034) and the respective maltose binding protein were transformed into competent cells containing pCpxR coupled with RFP generator (I13507). These cells were plated and incubated overnight. Colonies from each of the plates were selected and overnight cultures were prepared at 37 C. These 5 ml overnight cultures were then sub-cultured in 20 ml broth. These were shaken for 6-8 hours and aliquoted into 5 ml cultures and induced with varying levels of arabinose(percent). This was incubated in the shaker for 12-14 hours and RFP output was measured using 555 excitation and 632 nm emission frequency.</p><br />
<br />
<p><u>Results</u></p><br />
<br />
</li><a href="http://s872.photobucket.com/albums/ab287/iGEMCalgary_2010/?action=view&current=Unititled-7.png" target="_blank"><img src="http://i872.photobucket.com/albums/ab287/iGEMCalgary_2010/Unititled-7.png" border="0" alt="CpxR"></a></li><br />
<br />
<p>RFP output produced by the CpxR-I13507 system when co-transfected with I0500-B0034-MalE (red) and I0500-B0034-MalE31 (blue) at different arabinose concentrations. RFP levels were measured at 555 nm excitation and 632 nm emission frequencies</p><br />
<br />
<br />
<br />
</li><a href="http://s872.photobucket.com/albums/ab287/iGEMCalgary_2010/?action=view&current=lineofbestfitCpxR.png" target="_blank"><img src="http://i872.photobucket.com/albums/ab287/iGEMCalgary_2010/lineofbestfitCpxR.png" border="0" alt="Photobucket"></a></li><br />
<br />
<p>RFP output produced by the CpxR-I13507 system when co-transfected with I0500-B0034-MalE (red) and I0500-B0034-MalE31 (blue) at different arabinose concentrations. RFP levels were measured at 555 nm excitation and 632 nm emission frequencies.</p><br />
<br />
<p><u>Discussion of Results and Conclusion</u></p><br />
<p><br />
Figure 1 and 2 indicate the RFP output normalized with growth ratio (OD) at different levels of arabinose. Figure 1 shows that CpxR-I13507 is activated at the highest level when MalE31, the periplasmic misfolder, is expressed. This occurs around 0.2% arabinose concentration. Similar trends are observed in the case of MalE which is a periplasmic folder. MalE and MalE31 activate the system at different levels. MalE31 has similar trends to MalE but has a higher level of RFP expression. These results prove that MalE and MalE31 can both activate the CpxR system however, MalE31, which misfolds, activates it more rapidly and at a lower level of arabinose concentration compared to MalE. If the line of best fit is studied, it is seen that MalE has very minimal level of Cpx activation. Whereas, malE31 has a linear regression which flattens out as the system reaches its upper threshold of detection. Biologically, this could mean that the MalE31 is activated at levels that saturate the cellular chaperones and cause the system to reach its threshold level of proteolytic and chaperone activities. Another interesting pattern observed is the fact that when MalE is constructed with CpxR-I13507 on the same plasmid (Green), the cell RFP output is much lower compared to cells co-transfected with CpxR-I13507 and I0500-B0034 –MalE. This indicates that insertion of high copy plasmid also induces stress in the periplasmic region of the cell consequently inducing the activation of CpxR system. <br />
</p><br />
<br />
<p><br />
<h3>Experiment 2: Measuring RFP output of the CpxR-I13507 cells after exposure to different temperature for different time periods</h3></p><br />
<br />
<p><u>Protocol:</u></p><br />
<p><br />
Top 10 competent cells were transformed with CpxR-I13507 and plated. 5 ml overnight cultures were made from 5 different colonies using LB broth with appropriate antibiotics. Each of these cultures were aliquoted into six different tubes containing 600 µL of culture. These tubes were then placed in hot water baths at 30 C, 37C, 42C, 47C. Measurements were taken every hour for 5 hours after placing the tubes in different temperatures at 555 nm excitation and 632 nm emission.</p><br />
<br />
<br />
</li><a href="http://s872.photobucket.com/albums/ab287/iGEMCalgary_2010/?action=view&current=Untitled-6.png" target="_blank"><img src="http://i872.photobucket.com/albums/ab287/iGEMCalgary_2010/Untitled-6.png" border="0" alt="Temperature induction"></a> </li><br />
<br />
<p>Figure 2: RFP output produced by the CpxR-I13507 system when the system is heat shocked at different temperature for different lengths of time. The RFP output was measured at 555 nm excitation and 632 nm emission frequencies<br />
<br />
</p><br />
<br />
<p><u>Discussion of Results and Conclusion</u></p><br />
<p><br />
This graph shows that the CpxR system does respond to temperature activated stress. When the system is placed at 42 C the RFP output is much higher at t=0 compared to the system placed at 37 C or 30 C. This indicates that the system does get activated due to heat shock which matches the literature parameters. At 47 C, the system gets activated faster because the linear regression has a steeper slope. This indicates that the system is being stressed and it produces its downstream product which is RFP in this case and DegP and other chaperones in the genomic DNA much faster in order to cope with periplasmic protein denaturation. Also, it seems that the system gets activated dramatically after 3 hours regardless of the temperature, this could indicate that the system peaks after 3 hours and the genomic CpxR produces enough downstream chaperones and proteases in order for the system to be able to cope with stress which allows the RFP reading to decrease at 4 hours time because the cell reaches homeostasis. This allows the cell to get rid of misfolded protein and other factors that might be contributing to stressing it out and causing the Cpx regulon to be activated. The cell then shows a rapid rise again because it is still under heat shock stress. But, if the cell was placed at 37 degrees, the cell would show a flatline pattern rather than an oscillating pattern.<br />
</p><br />
<br />
<p><br />
<h3>Experiment 4: Measuring the GFP output through insertion of the mutant malE and malE31 with the transport signal sequence deleted into competent cells containing a fusion promoter (ibpAB-fsxA) coupled to a GFP reporter. </h4><br />
<br />
<p><u>Purpose</u></p><br />
<p><br />
The purpose of this assay is to test the output that the cytoplasmic acting fusion promoter (ibpAB-fsxA) will produce with proteins that are known to fold correctly (malEΔSS) and with proteins that are known to misfold (malE31ΔSS) in the cytoplasm. The plasmids containing malEΔSS and malE31ΔSS are coupled to an IPTG inducible promoter and were received from the lab of Jean-Michel Betton.<br />
</p><br />
</p><br />
<br />
</div><br />
<br />
</div><br />
<br />
</body><br />
</html></div>Emily Hickshttp://2010.igem.org/Team:Calgary/Project/TranscriptionTeam:Calgary/Project/Transcription2010-10-27T06:24:32Z<p>Emily Hicks: </p>
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<h1>Project Descriptions</h1><br />
<br />
<br />
<ul><br />
<li><a href="https://2010.igem.org/Team:Calgary/Project/Transcription">Transcription/Translation Reporter Circuit</a></li><br />
<li>Protein Misfolding Reporters<br />
<ul><br />
<li><a href="https://2010.igem.org/Team:Calgary/Project/IbpAB">Cytoplasmic Stress Detectors</a></li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Project/CpxP">Periplasmic Stress Detectors</a></li><br />
</ul><br />
</li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Project/Controls">System Controls</a></li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Project/Achievements">Achievements</a></li><br />
</ul><br />
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<div class="mainbody"><br />
<br />
<span id="bodytitle"><h1>Transcription/Translation Circuit</h1></span><br />
<br />
<p><b>Background:</b></p><br />
<p><br />
Transcription and translation are essential processes for protein expression. Problems that arise during these processes could lead to improper protein formation. Issues that can occur include shortage in length, folding problems, low or no expression, etc. These issues are accentuated in synthetic biology as foreign genes are implemented into prokaryotes such as ''Escherichia coli''. The transcription translation detector circuit was developed in order to test whether or not a gene of interest is being correctly transcribed and translated. <br />
<br/><br/><br />
<u> How the Circuit Works </u><br />
<br />
PICTURE OF CIRCUIT HERE<br />
<br />
The gene of interest is fused to a mutant RFP. Downstream of this is GFP with its own ribosomal binding site. If trabnscription is occuring, the transcript would include the gene of interest, RFP as well as GFP. Because GFP has its own ribosomal biding site, it should be translated if transcription is happening. <br />
<br/><br />
PICTURE GOES HERE: Green plate --> Transcription!!<br />
<br />
If the gene of interest is also being translated, then RFP should also be translated because it is fused to the GOI.the RFP was specially selected from Dr. Lewenza’s lab. This RFP (nicknamed sRFP or special red fluorescent protein) can fold in the cytoplasm, periplasm and the cellular membrane.<br />
<br />
PICTURE GOES HERE; RED PLATE<br />
</p><br />
<br />
<p><b>Assumptions:</b></p><br />
<p><br />
For this circuit to work, there are several assumptions that must be made. The first of which is a result of a limitation within the design which is that sRFP will not affect the stability of the protein of interest. Both positives and negatives are not ideal because the circuit functions as an indicator any assistance could lead to false positives or vice versa. Second, AraC is the right promoter for this circuit. Although there are many benefits for using an arabinose inducible promoter, however evolutionary conditions have established optimal expression in natural promoters. Third is folding properties in the periplasm and cytoplasm (Lewenza, et al., 2006) had to be the same such that a sRFP in the cytoplasm will give the same absorbance as one in the periplasm. Fourth would be that the GOI does not contain a “rut” site (Rho utilization site) which would prematurely stop transcription using Rho dependent termination. Fifth would be that E.coli would be the most compatible cell available for protein expression. Much like the second assumption, genes are optimally expressed in its natural host. Transferring these genes into E.coli might decrease the efficiency of protein expression. These are considerations that must be made in order to ensure the success of this circuit towards its utilization within our testing kit. It is definitely more “artificial” compared to the other two mostly because it overrides the necessity for the natural systems within. However if all limitations are accounted for, this could be a very useful tool if coupled with our other systems. </p><br />
<br />
<p>Helpful tips with understanding the circuit: With the way the circuit is developed, a failure of transcription will lead to a failure of translation. Therefore it is impossible to see only red cells, but possible to see green cells. If a brownish color is expressed (a mixture of red and green), this is the best. Also if only green cells are noticed, then to definitively test whether or not there is something wrong with translation, a user must employ the other two circuits. Meaning positive in the folding circuits indicates the translation mechanism works however due to the design of attaching sRFP with the GOI, the GOI misfolding will affect the stability of sRFP.</p><br />
<br />
<p><b>Problems that can arise during transcription/translation:</b></p><br />
<p>There are numerous problems that can arise in the transcription and translation especially when trying to turn E.coli into a factory for foreign proteins. Each category of transcription and translation can be broken down into pre, during and post. Although some aspects between post-transcription and pre-translation are slightly grey, there are parts of it that are quite clear. For example the attachment of the 30S subunit from rRNA would be considered pre-translational but not post-transcriptional. This section describes the possible transcription/translation issues and the following responses by the system. </p><br />
<br />
<p>Note: this by no means is an indication of a complete list of problems. In fact many of the problems surrounding transcription and translation are still being researched today because aspects of the mechanism are still not fully understood.</p> <br />
<br />
<p><u>Transcription</u></p><br />
<p>Pre-transcription</p><br />
<p>-transcription factors</p><br />
<p>One of the main ideas of synthetic biology is the expression of proteins from foreign enzymes, for example GFP comes from Aequorea Victoria (Andersen, et al., 1998) . One of the considerations is whether or not foreign circuits have the corresponding transcriptions within E.coli. If these transcription factors have a profound effect on whether or not transcription can occur (Kleinert, et al., 2003) , then natural promoters might be hindered or lack the necessary transcription factors for expression. Therefore it is necessary to include an arabinose promoter (pBad/araC), a well characterized and working promoter in E.coli. (iGEM registry,2003) </p> <br />
<br />
<p>If the problem of the foreign circuit lies in the promoter, the circuit can be used to detect this simply through inserting the RBS+GOI into the circuit and compare this with inserting the foreign promoter + RBS + GOI. If there is expression without the foreign promoter, and no expression with it, then there could be a repressor bounded to the operon of the circuit. If there is expression in both then a third circuit can be constructed with just the foreign promoter + RBS + GOI without the arabinose promoter. If there is no expression in the third, then the foreign promoter lacks the necessary transcription factors to operate in the host E.coli.</p> <br />
<br />
<p>-promoter strength</p><br />
<p>This is not a problem with natural promoters however this is an issue faced by many synthetic biologist when matching a promoter with a GOI. More is not always better, over expression of protein could lead to higher amounts of aggregation and longer folding time.(Brock, 2010) Choosing the pBAD/araC promoter is beneficial because induction varies with arabinose concentrations. Therefore it is possible to use a 96 well plate with varying levels of arabinose to promote induction at various strengths. A plate reader can then be used to read absorbance levels to find the optimal amount of indicator expressed.</p><br />
<br />
<p>Transcription</p><br />
<p>-Repressor/amount of inducer</p> <br />
<p>The ratio of inducer to plasmid copy number would be a problem when trying to express a foreign protein in E.coli. Much like issue with transcription factors, the circuit was designed to include an arabinose promoter that way it is possible to control the concentration of the inducer arabinose. In that case there will be no shortage in the concentration of inducer because the promoter is well characterized meaning its induction is known.</p><br />
<br />
<p>-Hair pin loop/rho dependent termination</p><br />
<p>The formation of premature hair pin loops and rho utilizations sites formed from within the gene are potential methods of premature stops to transcription. Hair pin loops are typically 7 to 20 amino acids long (Lewin, 2007) and ruts sites are 22-116 base pairs.(Banerjee, et al., 2007) The more likely of the two when forming an accidental termination site would be a hair pin loop. This relies on the palindrome formation with high concentrations of guanine and cysteine which results in a RNA pulling from the DNA. Our system would detect premature termination of RNA would result in no signal with our GFP signal.</p> <br />
<br />
<br />
<p>Post-transcription</p><br />
<p>-mRNA shape degradation</p><br />
<p>Although transcription occurs, mRNA instability results in the degradation of the mRNA. The circuit would suggest that the GFP report was not expressed. Despite transcription occurring completely, the most logical approach would be to group this under issues with transcription, also because pre-translational steps have not occurred yet.</p> <br />
<br />
<p>Translation</p><br />
<p>Pre-translation</p><br />
<p>No current issues arise from this step.</p> <br />
<p>Translation</p><br />
<p>-Multi codon usage</p> <br />
<p>When inserting foreign genes into E.coli, the ratios of tRNAs in E.coli in comparison to the foreign source can vary. Shortages in tRNA can lead to problems with rate and accuracy of translation. (Ran and Higgs, 2010) Kinetics is a factor of rate of protein formation, decreased concentrations of necessary tRNAs results in slower formation of proteins. Based on the research by Drummond and Wilke, lack of accuracy is caused by mistranslation causing higher amount of misfolding.(Drummond and Wilke, 2008) If the GOI’s multi codon usage disagrees with the host E.coli, there would be aggregation which will inhibit protein expression. The circuit can detect that there are problems with translation because sRFP would be form aggregate bodies with the protein of interest (POI). </p><br />
<br />
<br />
<p>-Premature stop codon<p><br />
<p>Stop codons inhibit translation. The circuit would indicate the presence of a premature stop codon because the sRFP would not be translated therefore no signal would be present. </p><br />
<br />
<p>-RBS compatibility</p><br />
<p>The ribosome binding site allows the attachment of the ribosome. Differences in ribosome strength could change the translation frequencies. This leaves room for protein misfolding. Because of the specificity of the RBS to the expression of the gene, as well as the potential of affecting the triple nucleotide site which could shift the reading frame. The circuit was designed in such a way that the user is capable of attaching their own RBS.</p><br />
<br />
<p>-Copy number</p><br />
<p>Copy number refers to the number of plasmids that can exist within on E.coli cell.(iGEM Registry, 2009) Although this doesn’t change the rate of transcription (polymerase per second, PoPs) like promoter strength, the effects are similar. Increasing the concentration of slower folding proteins could result in aggregation due to exposed hydrophobic segments. . (Ran and Higgs, 2010) The circuit will detect this as an issue with translation as this could affect the protein.</p><br />
<br />
<p>Post-translation</p><br />
<p>-Lack of chaperones</p><br />
<p>The lack of essential chaperones could result in protein misfolding. E.coli may not have the necessary chaperones to correct the conformation of the POI. The formation of misfolded protein will cause the aggregation of pRFP, therefore indicating an error in translation.</p> <br />
<br />
<p><b>Design of the Circuit:</b></p><br />
<p>pBad/araC Promoter- This promoter was chosen because it allow for variable strength without replacing the promoter (if the circuit had a promoter library). Because more isn’t always better, the user can customize optimal levels of promoter strength in protein expression. This part is also highly characterized (iGEM Registry, 2003).</p><br />
<br />
<p>Multiple Cloning Sites- We are using modified biobrick prefix and suffix. What this means is that these sites are not separating the biobrick parts from the sequences, rather they are located between the arabinose promoter and the sRFP.</p><br />
<p>ccdB- A place holder that was added for selection in addition to antibiotic selection. The circuit will contain a suicide ccdB gene as a placeholder for the GOI. If this is not removed, the cell which has this transformed plasmid will die. This will ensure that the only cells present on the plate will only express the genes intended to be there.</p> <br />
<p>RBS- We have decided not include a RBS within this sequence to allow customizability. Natural RBS are known to indicate optimal PoPs plus issues with this would indicate compatibility problems on the part of the RBS and GOI. This would also eliminate any issues regard reading frame shifts of the RBS to the GOI for those that are biobricking new parts.</p> <br />
<p>sRFP (special red fluorescent protein)- This is part of the translation portion of the circuit. This indicator was chosen because it can fold in the cytoplasm, periplasm and membranes.(Lewenza, et al., 2006) One of the limitations of this circuit is that the GOI must be fused to sRFP in order for translation detection to occur. This means additional time on the part of the user to rebiobrick the end portion of the GOI such that the stop codons are removed. Current studies by Lewenza, et al. reveals that RFP can be localized in the cytoplasm as well as the outer membrane.</p> <br />
<br />
<br />
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<br />
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<br />
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</html></div>Emily Hickshttp://2010.igem.org/Team:Calgary/Project/TranscriptionTeam:Calgary/Project/Transcription2010-10-27T06:22:16Z<p>Emily Hicks: </p>
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<h1>Project Descriptions</h1><br />
<br />
<br />
<ul><br />
<li><a href="https://2010.igem.org/Team:Calgary/Project/Transcription">Transcription/Translation Reporter Circuit</a></li><br />
<li>Protein Misfolding Reporters<br />
<ul><br />
<li><a href="https://2010.igem.org/Team:Calgary/Project/IbpAB">Cytoplasmic Stress Detectors</a></li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Project/CpxP">Periplasmic Stress Detectors</a></li><br />
</ul><br />
</li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Project/Controls">System Controls</a></li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Project/Achievements">Achievements</a></li><br />
</ul><br />
<br />
</div><br />
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<div class="mainbody"><br />
<br />
<span id="bodytitle"><h1>Transcription/Translation Circuit</h1></span><br />
<br />
<p><b>Background:</b></p><br />
<p><br />
Transcription and translation are essential processes for protein expression. Problems that arise during these processes could lead to improper protein formation. Issues that can occur include shortage in length, folding problems, low or no expression, etc. These issues are accentuated in synthetic biology as foreign genes are implemented into prokaryotes such as ''Escherichia coli''. The transcription translation detector circuit was developed in order to test whether or not a gene of interest is being correctly transcribed and translated. <br />
<br/><br/><br />
<u> How the Circuit Works </u><br />
<br />
PICTURE OF CIRCUIT HERE<br />
<br />
The gene of interest is fused to a mutant RFP. Downstream of this is GFP with its own ribosomal binding site. If trabnscription is occuring, the transcript would include the gene of interest, RFP as well as GFP. Because GFP has its own ribosomal biding site, it should be translated if transcription is happening. <br />
<br/><br />
PICTURE GOES HERE: Green plate --> Transcription!!<br />
<br />
If the gene of interest is also being translated, then RFP should also be translated because it is fused to the GOI.the RFP was specially selected from Dr. Lewenza’s lab. This RFP (nicknamed sRFP or special red fluorescent protein) can fold in the cytoplasm, periplasm and the cellular membrane.<br />
<br />
PICTURE GOES HERE; RED PLATE<br />
</p><br />
<br />
<p><b>Assumptions:</b></p><br />
<p><br />
For this circuit to work, there are several assumptions that must be made. The first of which is a result of a limitation within the design which is that sRFP will not affect the stability of the protein of interest. Both positives and negatives are not ideal because the circuit functions as an indicator any assistance could lead to false positives or vice versa. Second, AraC is the right promoter for this circuit. Although there are many benefits for using an arabinose inducible promoter, however evolutionary conditions have established optimal expression in natural promoters. Third is folding properties in the periplasm and cytoplasm (Lewenza, et al., 2006) had to be the same such that a sRFP in the cytoplasm will give the same absorbance as one in the periplasm. Fourth would be that the GOI does not contain a “rut” site (Rho utilization site) which would prematurely stop transcription using Rho dependent termination. Fifth would be that E.coli would be the most compatible cell available for protein expression. Much like the second assumption, genes are optimally expressed in its natural host. Transferring these genes into E.coli might decrease the efficiency of protein expression. These are considerations that must be made in order to ensure the success of this circuit towards its utilization within our testing kit. It is definitely more “artificial” compared to the other two mostly because it overrides the necessity for the natural systems within. However if all limitations are accounted for, this could be a very useful tool if coupled with our other systems. </p><br />
<br />
<p>Helpful tips with understanding the circuit: With the way the circuit is developed, a failure of transcription will lead to a failure of translation. Therefore it is impossible to see only red cells, but possible to see green cells. If a brownish color is expressed (a mixture of red and green), this is the best. Also if only green cells are noticed, then to definitively test whether or not there is something wrong with translation, a user must employ the other two circuits. Meaning positive in the folding circuits indicates the translation mechanism works however due to the design of attaching sRFP with the GOI, the GOI misfolding will affect the stability of sRFP.</p><br />
<br />
<p><b>Problems that can arise during transcription/translation:</b></p><br />
<p>There are numerous problems that can arise in the transcription and translation especially when trying to turn E.coli into a factory for foreign proteins. Each category of transcription and translation can be broken down into pre, during and post. Although some aspects between post-transcription and pre-translation are slightly grey, there are parts of it that are quite clear. For example the attachment of the 30S subunit from rRNA would be considered pre-translational but not post-transcriptional. This section describes the possible transcription/translation issues and the following responses by the system. </p><br />
<br />
<p>Note: this by no means is an indication of a complete list of problems. In fact many of the problems surrounding transcription and translation are still being researched today because aspects of the mechanism are still not fully understood.</p> <br />
<br />
<p><u>Transcription</u></p><br />
<p>Pre-transcription</p><br />
<p>-transcription factors</p><br />
<p>One of the main ideas of synthetic biology is the expression of proteins from foreign enzymes, for example GFP comes from Aequorea Victoria (Andersen, et al., 1998) . One of the considerations is whether or not foreign circuits have the corresponding transcriptions within E.coli. If these transcription factors have a profound effect on whether or not transcription can occur (Kleinert, et al., 2003) , then natural promoters might be hindered or lack the necessary transcription factors for expression. Therefore it is necessary to include an arabinose promoter (pBad/araC), a well characterized and working promoter in E.coli. (iGEM registry,2003) </p> <br />
<br />
<p>If the problem of the foreign circuit lies in the promoter, the circuit can be used to detect this simply through inserting the RBS+GOI into the circuit and compare this with inserting the foreign promoter + RBS + GOI. If there is expression without the foreign promoter, and no expression with it, then there could be a repressor bounded to the operon of the circuit. If there is expression in both then a third circuit can be constructed with just the foreign promoter + RBS + GOI without the arabinose promoter. If there is no expression in the third, then the foreign promoter lacks the necessary transcription factors to operate in the host E.coli.</p> <br />
<br />
<p>-promoter strength</p><br />
<p>This is not a problem with natural promoters however this is an issue faced by many synthetic biologist when matching a promoter with a GOI. More is not always better, over expression of protein could lead to higher amounts of aggregation and longer folding time.(Brock, 2010) Choosing the pBAD/araC promoter is beneficial because induction varies with arabinose concentrations. Therefore it is possible to use a 96 well plate with varying levels of arabinose to promote induction at various strengths. A plate reader can then be used to read absorbance levels to find the optimal amount of indicator expressed.</p><br />
<br />
<p>Transcription</p><br />
<p>-Repressor/amount of inducer</p> <br />
<p>The ratio of inducer to plasmid copy number would be a problem when trying to express a foreign protein in E.coli. Much like issue with transcription factors, the circuit was designed to include an arabinose promoter that way it is possible to control the concentration of the inducer arabinose. In that case there will be no shortage in the concentration of inducer because the promoter is well characterized meaning its induction is known.</p><br />
<br />
<p>-Hair pin loop/rho dependent termination</p><br />
<p>The formation of premature hair pin loops and rho utilizations sites formed from within the gene are potential methods of premature stops to transcription. Hair pin loops are typically 7 to 20 amino acids long (Lewin, 2007) and ruts sites are 22-116 base pairs.(Banerjee, et al., 2007) The more likely of the two when forming an accidental termination site would be a hair pin loop. This relies on the palindrome formation with high concentrations of guanine and cysteine which results in a RNA pulling from the DNA. Our system would detect premature termination of RNA would result in no signal with our GFP signal.</p> <br />
<br />
<br />
<p>Post-transcription</p><br />
<p>-mRNA shape degradation</p><br />
<p>Although transcription occurs, mRNA instability results in the degradation of the mRNA. The circuit would suggest that the GFP report was not expressed. Despite transcription occurring completely, the most logical approach would be to group this under issues with transcription, also because pre-translational steps have not occurred yet.</p> <br />
<br />
<p>Translation</p><br />
<p>Pre-translation</p><br />
<p>No current issues arise from this step.</p> <br />
<p>Translation</p><br />
<p>-Multi codon usage</p> <br />
<p>When inserting foreign genes into E.coli, the ratios of tRNAs in E.coli in comparison to the foreign source can vary. Shortages in tRNA can lead to problems with rate and accuracy of translation. (Ran and Higgs, 2010) Kinetics is a factor of rate of protein formation, decreased concentrations of necessary tRNAs results in slower formation of proteins. Based on the research by Drummond and Wilke, lack of accuracy is caused by mistranslation causing higher amount of misfolding.(Drummond and Wilke, 2008) If the GOI’s multi codon usage disagrees with the host E.coli, there would be aggregation which will inhibit protein expression. The circuit can detect that there are problems with translation because sRFP would be form aggregate bodies with the protein of interest (POI). </p><br />
<br />
<br />
<p>-Premature stop codon<p><br />
<p>Stop codons inhibit translation. The circuit would indicate the presence of a premature stop codon because the sRFP would not be translated therefore no signal would be present. </p><br />
<br />
<p>-RBS compatibility</p><br />
<p>The ribosome binding site allows the attachment of the ribosome. Differences in ribosome strength could change the translation frequencies. This leaves room for protein misfolding. Because of the specificity of the RBS to the expression of the gene, as well as the potential of affecting the triple nucleotide site which could shift the reading frame. The circuit was designed in such a way that the user is capable of attaching their own RBS.</p><br />
<br />
<p>-Copy number</p><br />
<p>Copy number refers to the number of plasmids that can exist within on E.coli cell.(iGEM Registry, 2009) Although this doesn’t change the rate of transcription (polymerase per second, PoPs) like promoter strength, the effects are similar. Increasing the concentration of slower folding proteins could result in aggregation due to exposed hydrophobic segments. . (Ran and Higgs, 2010) The circuit will detect this as an issue with translation as this could affect the protein.</p><br />
<br />
<p>Post-translation</p><br />
<p>-Lack of chaperones</p><br />
<p>The lack of essential chaperones could result in protein misfolding. E.coli may not have the necessary chaperones to correct the conformation of the POI. The formation of misfolded protein will cause the aggregation of pRFP, therefore indicating an error in translation.</p> <br />
<br />
<p><b>Design of the Circuit:</b></p><br />
<p>The final circuit that a user will receive, in its respective order would include: pBad/araC, ccdB, RFP, RBS, GFP, Terminator.<p> <br />
<p>pBad/araC Promoter- This promoter was chosen because it allow for variable strength without replacing the promoter (if the circuit had a promoter library). Because more isn’t always better, the user can customize optimal levels of promoter strength in protein expression. This part is also highly characterized (iGEM Registry, 2003) and many teams know how to use this promoter. </p><br />
<br />
<p>Multiple Cloning Sites- We are using modified biobrick prefix and suffix. What this means is that these sites are not separating the biobrick parts from the sequences, rather they are located between the arabinose promoter and the sRFP.</p><br />
<p>ccdB- A place holder that was added for selection in addition to antibiotic selection. The circuit will contain a suicide ccdB gene as a placeholder for the GOI. If this is not removed, the cell which has this transformed plasmid will die. This will ensure that the only cells present on the plate will only express the genes intended to be there.</p> <br />
<p>RBS- We have decided not include a RBS within this sequence to allow customizability. Natural RBS are known to indicate optimal PoPs plus issues with this would indicate compatibility problems on the part of the RBS and GOI. This would also eliminate any issues regard reading frame shifts of the RBS to the GOI for those that are biobricking new parts.</p> <br />
<p>sRFP (special red fluorescent protein)- This is part of the translation portion of the circuit. This indicator was chosen because it can fold in the cytoplasm, periplasm and membranes.(Lewenza, et al., 2006) One of the limitations of this circuit is that the GOI must be fused to sRFP in order for translation detection to occur. This means additional time on the part of the user to rebiobrick the end portion of the GOI such that the stop codons are removed. Current studies by Lewenza, et al. reveals that RFP can be localized in the cytoplasm as well as the outer membrane.</p> <br />
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</div><br />
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</html></div>Emily Hickshttp://2010.igem.org/Team:Calgary/Community/BlogTeam:Calgary/Community/Blog2010-10-27T00:26:53Z<p>Emily Hicks: </p>
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<h1>Community</h1><br />
<br />
<ul><br />
<li><a href="https://2010.igem.org/Team:Calgary/Community/Ethics">Human Practices</a></li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Community/Gallery">Photo Gallery</a></li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Community/Conferences">Conferences</a></li><br />
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<li><a href="https://2010.igem.org/Team:Calgary/Community/Blog">Visit Our Blog!</a></li><br />
</ul><br />
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<br />
<div class="mainbody"><br />
<span id="bodytitle"><h1>Our Blog</h1></span><br />
<br />
<p>This year, iGEM Calgary created a blog that updated with information regarding synthetic biology news, features of other iGEM projects as well as updates of our own team and our project. Our goal was to improve the public image of synthetic biology and showcase the many cool and interesting things that the field can bring. In this way, we hope to reach out to the community and hopefully alleviate some skepticism behind synthetic biology.</p><br />
<br />
<a href="http://igemcalgary2010.blogspot.com" target="_blank">Check Out Our Blog!</a><br />
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</html></div>Emily Hickshttp://2010.igem.org/Team:Calgary/18_August_2010Team:Calgary/18 August 20102010-10-26T23:43:52Z<p>Emily Hicks: </p>
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<div>{{CalgaryNotebookTemplate|<br />
Wednesday August 18, 2010|<br />
[[Image:08.18.2010.Chris - Various-1-.jpg|thumb|400px|Chris's first gel with a variety of CpxP, ibpAB, and construction of I0500-I13507]]<br />
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[[Image:08.18.2010.Chris - Various-2-.jpg|thumb|400px|Chris's second gel with a variety of CpxP and ibpAB]]<br />
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[[Image:08.18.2010.Chris, Emily - VariousRD.jpg|thumb|400px|Chris and Emily's gel with a mixture of digested parts including I0500, CpxP, ibpAB, and I0500-I13507]]<br />
<br />
<u>Himika</u><br />
<br />
Today I spoke to Paul, Dave and Patrick about my ideas on the modeling project that I decided. We had a 3 hour debate on the topic and we came up with a different plan. We plan to make our model such that it predicts the concentration of inclusion body formation based on the free energy and temperature of the cellular microenvironment. We are still haveing issues as to how to pull of this model but we are gradually progressing towards building a concrete one.<br />
<br />
<br />
<u>Chris</u><br />
<br />
Today, I ran several gels. The first gel that was run had CpxP, ibpAB and the constructions of I0500-I13507 and was run with a Fermentas 1 kb Ladder. The lanes were CpxP (Lane 2, 5, 6, 12, 13 on Gel 1 and Lane 4, 5, 6-10 on Gel 2), ibpAB (Lane 3, 4, 7-11, 14, and 19 in Gel 1 and Lanes 2, 3, 6, 11-19 on Gel 2) and I0500-I13507 (Lanes 15-18 on Gel 1). The gel results are shown on the right. After this, restriction digests were run of CpxP, ibpAB and I0500-I13507 along with a control of I0500. The digests were then run on a 1.0% gel which turned up empty. We suspect that the DNA was fried due to a high exposure time that was not noticed until too late. Then, we ran digests of the parts of I0500, I13507, CpxP, and ibpAB before running them on a gel. Finally, I transformed the parts of R0011 (lacI-repressible promoter/IPTG-inducible promoter), ipbAB (stress-activated promoter), pSB2K3 (for cloning purposes) and constructed I0500-I13507. Emily and I also made kanamycin-chloramphenicol and chloramphenicol plates.<br />
<br />
<u>Emily</u><br />
<br />
Today I made kanmycin and chloramphenicol plates. I also ran a gel of digests of I0500, I13507, CpxP and ibpAB in order to verify that we have the parts that we think we have. Today I also diluted biobrick and malE BBK primers.<br />
<u> Raida </u> <br />
<br />
Today, I countinued working on the Ethics paper.<br />
}}</div>Emily Hickshttp://2010.igem.org/Team:Calgary/23_October_2010Team:Calgary/23 October 20102010-10-26T23:33:06Z<p>Emily Hicks: </p>
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Saturday October 23, 2010|<br />
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<u>Emily</u><br />
<br />
Today I set up a massive colony PCR with the BBK-CP primers in order to veirfy that we were able to get all of our parts into the psB1C3 vector. Today I also made more LB broth. I also did transformations of a few other parts into the psB1C3 vector as well as the construction of I0500-B0034 with the Lethbridge part.<br />
<br />
}}</div>Emily Hickshttp://2010.igem.org/Team:Calgary/23_October_2010Team:Calgary/23 October 20102010-10-26T23:32:55Z<p>Emily Hicks: </p>
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Saturday October 23, 2010|<br />
<br />
<u>Emily<u><br />
<br />
Today I set up a massive colony PCR with the BBK-CP primers in order to veirfy that we were able to get all of our parts into the psB1C3 vector. Today I also made more LB broth. I also did transformations of a few other parts into the psB1C3 vector as well as the construction of I0500-B0034 with the Lethbridge part.<br />
<br />
}}</div>Emily Hickshttp://2010.igem.org/Team:Calgary/22_October_2010Team:Calgary/22 October 20102010-10-26T23:29:39Z<p>Emily Hicks: </p>
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Friday October 22, 2010|<br />
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<u>Emily</u><br />
<br />
This morning Chris and I went to Churchill High School where we did a presentation at their Science Cafe. We talked a bit about Synthetic Biology, iGEM and a bit about our project in general. The students seemed quite interested and we had a ton of questions about iGEM and various iGEM projects. This afternoon I made competant cells with the ibpAB-I13504 plasmid in them. We made both electrocompetant cells as well as chemically competant Calcium chloride cells. Today we also constructed all of the registry parts that we want to submit into the psB1C3 vector. After digestion and Antarctic Phosphotase treatment of the vector, I ligated, transformed into TOP10 E. Coli cells and then plated on Chloramphenicol plates. Today we also spent a fair bit of time looking into what functional testing has been done on our system up to date and deciding what more needs to be accomplished over the next few days and how we are ging to do that.<br />
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}}</div>Emily Hickshttp://2010.igem.org/Team:Calgary/24_October_2010Team:Calgary/24 October 20102010-10-26T23:18:53Z<p>Emily Hicks: </p>
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Sunday October 24, 2010|<br />
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<u>Patrick</u><br />
<br />
Final touches being added to the wiki over the next few days. Team pages and new header put into place.<br />
<br />
<u>Emily</u><br />
<br />
Today I trabsformed and plated our remianing plasmid switches (getting our final parts and constructs into the Regitsry accepted psB1C3 vector). I also prepared competant cells containing the I0500-I13504 plasmid in the psB1C3 vector. These cells were then used to transform malESSDel and malE31SSDel expression constructs from the Betton labs so that we can see if malE31SSDel and malESSDel work the way that we expect them to. I also transformed the Lethbridge team's construct into these cells in order to assay cytoplasmic protein msfolding. I also ran gels of my PCRs from yesterday in order to confirm that my construction of I0500-B0034 with the part that we got from Lethbridge was successful as well as to confirm that we were able to get ibpAB into the psBA1C3 vector.<br />
<br />
}}</div>Emily Hickshttp://2010.igem.org/Team:Calgary/25_October_2010Team:Calgary/25 October 20102010-10-26T23:13:09Z<p>Emily Hicks: </p>
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Monday October 25, 2010|<br />
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<u>Patrick</u><br />
<br />
Assigned and obtained various paragraphs from other team members. Uploaded many on the Wiki.<br />
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<u>Emily</u><br />
<br />
Set up digests of cpxP, malE, malESSdel, I0500-I13507, K135000-I13507-I0500-B0034-malE31 and the psB1AC3 vector. This is to plasmid switch all of the remianing registry parts into the accepted shipping plasmid. After construction and Antarctic Phosphotase treatment, I ligated them and then transformed them into TOP10 Competant E. Coli cells and then innocultaed LB broth culture and left the cultures to grow overnight. Today I also preparted ovenright cultures for an arabinose induction in order to get some more characterizatuon data of the I0500 (arabinose inducible) promoter. I also entered our parts into the Registry of Standard Biological Prats in preparation for shipping tomorrow.<br />
}}</div>Emily Hickshttp://2010.igem.org/Team:Calgary/26_October_2010Team:Calgary/26 October 20102010-10-26T23:06:12Z<p>Emily Hicks: </p>
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Tuesday October 26, 2010|<br />
<br />
<u>Patrick</u><br />
<br />
Final touches of the wiki being done by every member of the team. Uploaded the Maya animation.<br />
<br />
<u>Emily</u><br />
<br />
Today I miniprepped our overnight cultures of our parts for the registry in the psB1AC3 vector and I shipped this off to the Registry. Today I also set up ovenright cultures for three final inductions: I0500-I13504, ibpAB-I13504 and I0500-B0034-mns6.<br />
}}</div>Emily Hickshttp://2010.igem.org/Team:Calgary/13_September_2010Team:Calgary/13 September 20102010-09-14T16:26:34Z<p>Emily Hicks: </p>
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'''Monday September 13, 2010'''|<br />
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[[Image:09.13.2010-Himika-male31+degp+i13507 rd.jpg|400px|thumb| Himika's restriction digest of DegP system with EcoRI/ SpeI]]<br />
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[[Image:09.13.2010-Himika-male31+degp+i13504 rd.jpg|400px|thumb| Himika's restriction digest of DegP system with EcoRI/ SpeI]]<br />
<br />
<u>Himika</u><br />
<br />
Today I did a restriction digest of all the miniprepped colonies for DegP systems with EcoRI and SpeI. It looked like the construction worked but I will do a PCR of the plasmids before sending them off to sequencing.<br />
<br />
<br />
<u>Emily</u><br />
<br />
Today I made overnight cultures of my malE31 colonies that I think might be biobricked and in the AK Vector. I will miniprep these tomorrow and then proceed with a verification restriction digest. Tonight I also helped Chris and Jeremy clean up some things in the lab.<br />
<br />
<br />
}}</div>Emily Hickshttp://2010.igem.org/Team:Calgary/10_September_2010Team:Calgary/10 September 20102010-09-14T16:24:43Z<p>Emily Hicks: </p>
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'''Friday September 10, 2010'''|<br />
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<br />
<u>Emily</u><br />
Worked on some finishing up details for the presentation for aGEM and did a mock question period. Off to aGEM, hope that it's an excellent weekend!<br />
<br />
<br />
<u> Himika</u><br />
Came in for a few hours today and presented to Dr. Logan to get ready for aGEM. I also did the miniprep for MalE31-DegP system. Off to aGEM!<br />
}}</div>Emily Hickshttp://2010.igem.org/Team:Calgary/4_September_2010Team:Calgary/4 September 20102010-09-07T18:05:43Z<p>Emily Hicks: </p>
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'''Saturday September 4, 2010'''|<br />
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[[Image:09.04.2010.CpxPRDVerjpg.jpg|thumb|400px|Chris's restriction digest of random CpxP that Emily miniprepped/ Results are inconclusive]]<br />
<br />
Weekends are awesome!! 1 week to aGEM!<br />
<br />
<u>Chris</u><br />
<br />
Today, I ran a restriction digest of a bunch of the CpxP promoters that Emily miniprepped and ran them on a 1.0% gel. The results can be seen on the right. There are no real conclusive results so they were redigested and the gel will be run tomorrow. I also did plasmid preparations of a bunch of overnight cultures of ibpAB and CpxP that were set up yesterday.<br />
<br />
<u>Emily</u><br />
<br />
This evening I helped Chris with miniprpeps of various cultues from yesterday. I also set up a colony PCR of new malESSdel colonies as well as malE and malE31 colonies.<br />
}}</div>Emily Hickshttp://2010.igem.org/Team:Calgary/5_September_2010Team:Calgary/5 September 20102010-09-07T18:00:14Z<p>Emily Hicks: </p>
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'''Sunday September 5, 2010'''|<br />
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[[Image:09.05.2010.CpxPibpABAttempt1RD.jpg|thumb|400px|Chris's gel of the restriction digest of all old CpxP and ibpAB stuff. Lane 10 and Lane 4 show possible cuts but all other lanes show nothing. Lane 10 is ibpAB and Lane 4 is CpxP3 but those two tubes have no more DNA and must be redone.]]<br />
<br />
Who doesn't love weekends?<br />
<br />
<u>Chris</u><br />
<br />
Today, I ran a gel of the restriction digest of all the CpxP and ibpAB stuff from yesterday which was done with NEB enzymes. The gel results can be seen on the right. ibpAB1 (Lane 10) showed digestion at about the right size, but there was no more in the tube to construct with. As a result, overnight cultures were made of it. Another digest was set up of all the CpxP and ibpAB stuff that was ligated into Biobrick plasmids and a positive control of a Lux circuit from last year was also digested to affirm the enzyme activity. Finally, I helped Emily set up her multi-salt concentration PCR of MalE once again.<br />
<br />
<u>Emily</u><br />
<br />
Today I ran a gel of my colony PCR's from yesterday of malE and malESSdel. Unfortunately, I didn't get any amplification. So I proceeded to set up three PCR reactions of malE and malE31, varying the concentration of mgcl2 from 1.5 to 2.5 as well as running a temperature gradient from 51 to 59 degrees celcius.<br />
}}</div>Emily Hickshttp://2010.igem.org/Team:Calgary/7_September_2010Team:Calgary/7 September 20102010-09-07T17:52:17Z<p>Emily Hicks: </p>
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<div>{{CalgaryNotebookTemplate|<br />
'''Tuesday September 7, 2010'''|<br />
<br />
<u>Emily</u><br />
<br />
Today I PCR Purified malE and malE31. I then digested these as well as the psB1AK3 vector with a combination of restriction enzymes to try to get it into an AK BBK construction vector. I ligated these and transformed them into TOP10 competant cells and plated on K plates, leaving for overnight growth. I also made more K plates as well as more AK broth. Today we also all worked on the presentation that we will be giving at the 3rd annual aGEM Jamboree this coming weekend in Edmonton.<br />
}}</div>Emily Hickshttp://2010.igem.org/Team:Calgary/6_September_2010Team:Calgary/6 September 20102010-09-07T17:51:44Z<p>Emily Hicks: </p>
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<div>{{CalgaryNotebookTemplate|<br />
'''Monday September 6, 2010'''|<br />
<br />
<u>Emily</u><br />
<br />
Today I ran PCR reactions of malE, malE31, malESSdel and malE31SSdel using the appropriate primers and an mgcl2 concentration of 2.5 as well as an annealing temperature of 55.5-57 degrees C. I got amplification of the expected bands (~1.2 KB) for the malE and malE31, however I once again got no bands for any of the SSdel stuff. These reactions will have to be further optimized tomorrow. Today I also induced my I0500-I13504 cultures as well as Himika's cpxR reporter with malE31 circuit construct cultures with varying concentrations of Arabinose. We will be looking at GFP and RFP output for these tomorrow. Today a lot of work was also put into the presentation for aGEM this weekend in Edmonton.<br />
}}</div>Emily Hickshttp://2010.igem.org/Team:Calgary/6_September_2010Team:Calgary/6 September 20102010-09-07T17:50:25Z<p>Emily Hicks: </p>
<hr />
<div>{{CalgaryNotebookTemplate|<br />
'''Monday September 6, 2010'''|<br />
<br />
<u>Emily</u><br />
<br />
Today I ran PCR reactions of malE, malE31, malESSdel and malE31SSdel using the appropriate primers and an mgcl2 concentration of 2.5 as well as an annealing temperature of 55.5-57 degrees C. I got amplification of the expected bands (~1.2 KB) for the malE and malE31, however I once again got no bands for any of the SSdel stuff. These reactions will have to be further optimized tomorrow. Today a lot of work was also put into the presentation for aGEM this weekend in Edmonton.<br />
}}</div>Emily Hicks