Difference between revisions of "Team:UofC Calgary/Composite Part"
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<p>The backbone of the U of Calgary project are the composite parts. The composite parts are an amalgamation of different registry parts which act together to provide a novel function. The composite parts designed for this project are based around the Bowman-Birk Protease inhibitor (BBI) and the <a href="http://parts.igem.org/Part:BBa_K1444018">ComK part</a> that was submitted to the registry by the 2014 Calgary team.</p> | <p>The backbone of the U of Calgary project are the composite parts. The composite parts are an amalgamation of different registry parts which act together to provide a novel function. The composite parts designed for this project are based around the Bowman-Birk Protease inhibitor (BBI) and the <a href="http://parts.igem.org/Part:BBa_K1444018">ComK part</a> that was submitted to the registry by the 2014 Calgary team.</p> | ||
| − | <p>A variety of different parts were used across all of the BBI composite constructs. We used Pveg, a constitutive promoter, and double terminators to book end the rest of the internal genes. To improve secretion of BBI from B. subtilis, we added a secretion tag as well as a transdermal (TD1) tag. The secretion tag is a localization tag for the Sec pathway in B. subtilis | + | <p>A variety of different parts were used across all of the BBI composite constructs. We used Pveg, a constitutive promoter, and double terminators to book end the rest of the internal genes. To improve secretion of BBI from <i>B. subtilis</i>, we added a secretion tag as well as a transdermal (TD1) tag. The secretion tag is a localization tag for the Sec pathway in <i>B. subtilis</i> <Ruan et al., 2013). TD1 is a part developed by a previous iGEM team <a href="http://parts.igem.org/Part:BBa_E0040:Experience> (USTC_CHINA 2013)</a> which is designed to allow for the trafficking of peptides across the skin.</p> |
| − | <p>The second major composite part was the ComK construct. We modified the original ComK sequence, which was first submitted by the 2014 Calgary Team, to remove unwanted restriction sites from within Comk itself. The ComK gene and xylose inducible promoters were then flanked with sequences homologous to AmyE in B. subtilis. The flanking sequences were added to allow for the integration of a controllable ComK into the B. subtilis genome. The integration of a xylose inducible ComK is to make the transformation of B. subtilis less time and resource intensive. </p> | + | <p>The second major composite part was the ComK construct. We modified the original ComK sequence, which was first submitted by the 2014 Calgary Team, to remove unwanted restriction sites from within Comk itself. The ComK gene and xylose inducible promoters were then flanked with sequences homologous to AmyE in <i>B. subtilis</i>. The flanking sequences were added to allow for the integration of a controllable ComK into the <i>B. subtilis</i> genome. The integration of a xylose inducible ComK is to make the transformation of <i>B. subtilis</i> less time and resource intensive. </p> |
<p>Our last construct does not fall into either of the two previous categories. The construct is an integration cassette which is a mix of both the BBI and Comk constructs. The cassette contains Thrc integration sequences which flank the sec and TD1 tag. However the BBI sequence is replaced with the sequences for the BamHI and XmaI restriction enzymes. Addition of the restriction sites is to make a platform for future biotherapeutics that could be mass produced in bacteria, a mini-pharmacy. | <p>Our last construct does not fall into either of the two previous categories. The construct is an integration cassette which is a mix of both the BBI and Comk constructs. The cassette contains Thrc integration sequences which flank the sec and TD1 tag. However the BBI sequence is replaced with the sequences for the BamHI and XmaI restriction enzymes. Addition of the restriction sites is to make a platform for future biotherapeutics that could be mass produced in bacteria, a mini-pharmacy. | ||
</p> | </p> | ||
Revision as of 02:47, 18 October 2016
Composite Parts
The backbone of the U of Calgary project are the composite parts. The composite parts are an amalgamation of different registry parts which act together to provide a novel function. The composite parts designed for this project are based around the Bowman-Birk Protease inhibitor (BBI) and the ComK part that was submitted to the registry by the 2014 Calgary team.
A variety of different parts were used across all of the BBI composite constructs. We used Pveg, a constitutive promoter, and double terminators to book end the rest of the internal genes. To improve secretion of BBI from B. subtilis, we added a secretion tag as well as a transdermal (TD1) tag. The secretion tag is a localization tag for the Sec pathway in B. subtilis E. coli – GFP-BBI 5 B. subtilis– pVeg -> sec-> TD1-> BBI-GFP (BBI 1 GFP w/o) B. subtilis – pVeg->sec-TD1-KSCI-BBI-GFP (BBI 5 GFP w/o) B. subtilis – secretion tag, transdermal tag, and BBI without internal restriction sites (BBI 1 w/o) B. subtilis – secretion tag, transdermal tag, solubility tag and BBI without internal restriction sites (BBI 5 w/o) B. subtilis - Improved ComK: removal of internal restriction sites B. subtilis - Improved ComK: addition of AmyE homology B. subtilis - Bacillus subtilis modular secretion platform with ThrC homology for chromosomal integration Composite Parts
BBa_K2008001
BBI with N-terminal KSCI solubility tag fused to GFP
Registry Link
BBa_K2008002
Registry Link
BBa_K2008003
Registry Link
BBa_K2008004
Registry Link
BBa_K2008005
Registry Link
BBa_K2008006
Registry Link
BBa_K2008007
Registry Link
BBa_K2008008
Registry Link
