Difference between revisions of "Part:BBa K1189033:Experience"

 
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===Applications of BBa_K1189033===
 
===Applications of BBa_K1189033===
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<html>
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<h1>Purpose</h1>
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<p>The <a href="2013.igem.org/Team:Calgary">Calgary iGEM 2013</a> team sought to detect pathogenic enterohemorrhagic <i>E. coli</i> (EHEC) DNA using pairs of TALEs designed to pathogenic <i>E. coli</i> markers. This TALE was designed based on a sequence alignment of the <i>stx2</i> gene found in EHEC organisms. It binds to a 19 bp region of the toxin, will detect EHEC strains including <i>E. coli O157:H7</i>, as well as several other EHEC strains. This TALE, along with the complementary <a href="https://parts.igem.org/wiki/index.php?title=Part:BBa_K1189032">BBa_K1189032</a>, will eventually be integrated into the final <a href="http://2013.igem.org/Team:Calgary/Project/OurSensor/Prototype">lateral flow strip FerriTALE prototype</a>.</p>
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<h1>Design details</h1>
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<p>The sequence includes a KasI restriction enzyme cut site so that other TALEs may be substituted with the C-terminal linker and K coil. This is one element in which the 2013 Calgary team attempted to make their DNA detection system modular and useful for detection of other DNA sequences based on the selection of an appropriate TALE. The target site was specifically selected to increase binding strength of the TALE to DNA as per Meckler et al. (2013). Please see the <a href="http://2013.igem.org/Team:Calgary/Project/OurSensor/Detector#Engineered TALEs">iGEM Calgary 2013 Wiki</a> for more details about how these TALEs were engineered for optimal binding.</a>
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<figure>
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<img src="https://static.igem.org/mediawiki/2013/5/50/UCalgary2013TRTwotargetTALE.png" alt="EHEC TALE 2 SOE PCR Product" width="600" height="300">
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<figcaption>
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<p><b>Figure 1.</b> Final size of the EHEC TALE 2 generated from IDT Gene Blocks using SOE PCR.</p>
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</figcaption>
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</figure>
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<h1>Progress</h1>
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<p>The Calgary team has tested their <a href="http://2013.igem.org/Team:Calgary/Project/OurSensor">DNA detection system </a>using proof of concept TALEs designed by the <a href="http://2012.igem.org/Team:Slovenia">2012 Slovenia iGEM team</a>. They have assembled BBa_K1189033 as shown in Figure 2 and are working on expressing, testing, and integrating these protein components into the final system.</p>
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<figure>
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<img src="https://static.igem.org/mediawiki/2013/b/ba/ET2_gel_finalSOEPCR.png" alt="EHEC TALE 2 SOE PCR Product" width="275" height="350">
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<figcaption>
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<p><b>Figure 2.</b> Final size of the EHEC TALE 2 generated from IDT Gene Blocks using SOE PCR.</p>
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</figcaption>
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</figure>
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<br></br>
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<h1>References</h1>
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<p>Meckler, J.F., Bhakta, M.S., Kim, M.S., Ovadia, R., Habrian, C.H., Zykovich, A., ... Baldwin, E.P. (2013). Quantitative analysis of TALE-DNA interactions suggests polarity effects. Nucleic Acids Research, 41(7), 4118–28. doi:10.1093/nar/gkt085</p>
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<p>Heckman, K. L., & Pease, L. R. (2007). Gene splicing and mutagenesis by PCR-driven overlap extension. Nature protocols, 2(4), 924-932.</p>
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<br></br>
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</html>
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===User Reviews===
 
===User Reviews===

Revision as of 10:37, 28 October 2013

This experience page is provided so that any user may enter their experience using this part.
Please enter how you used this part and how it worked out.

Applications of BBa_K1189033

Purpose

The Calgary iGEM 2013 team sought to detect pathogenic enterohemorrhagic E. coli (EHEC) DNA using pairs of TALEs designed to pathogenic E. coli markers. This TALE was designed based on a sequence alignment of the stx2 gene found in EHEC organisms. It binds to a 19 bp region of the toxin, will detect EHEC strains including E. coli O157:H7, as well as several other EHEC strains. This TALE, along with the complementary BBa_K1189032, will eventually be integrated into the final lateral flow strip FerriTALE prototype.

Design details

The sequence includes a KasI restriction enzyme cut site so that other TALEs may be substituted with the C-terminal linker and K coil. This is one element in which the 2013 Calgary team attempted to make their DNA detection system modular and useful for detection of other DNA sequences based on the selection of an appropriate TALE. The target site was specifically selected to increase binding strength of the TALE to DNA as per Meckler et al. (2013). Please see the iGEM Calgary 2013 Wiki for more details about how these TALEs were engineered for optimal binding.

EHEC TALE 2 SOE PCR Product

Figure 1. Final size of the EHEC TALE 2 generated from IDT Gene Blocks using SOE PCR.

Progress

The Calgary team has tested their DNA detection system using proof of concept TALEs designed by the 2012 Slovenia iGEM team. They have assembled BBa_K1189033 as shown in Figure 2 and are working on expressing, testing, and integrating these protein components into the final system.

EHEC TALE 2 SOE PCR Product

Figure 2. Final size of the EHEC TALE 2 generated from IDT Gene Blocks using SOE PCR.



References

Meckler, J.F., Bhakta, M.S., Kim, M.S., Ovadia, R., Habrian, C.H., Zykovich, A., ... Baldwin, E.P. (2013). Quantitative analysis of TALE-DNA interactions suggests polarity effects. Nucleic Acids Research, 41(7), 4118–28. doi:10.1093/nar/gkt085

Heckman, K. L., & Pease, L. R. (2007). Gene splicing and mutagenesis by PCR-driven overlap extension. Nature protocols, 2(4), 924-932.




User Reviews

UNIQc279b10cc1c4c496-partinfo-00000001-QINU UNIQc279b10cc1c4c496-partinfo-00000002-QINU