Team:UofC Calgary/Composite Part

iGEM Calgary 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 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.

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.