Team:UofC Calgary/Composite Part
Composite Parts
The backbone of the U of Calgary project are the composite parts. Our 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 modified Bowman-Birk Protease Inhibitor (mBBI) and the competency-inducing ComK part that was submitted to the registry by the 2014 UofC_Calgary team.
A variety of different parts were used across all of the mBBI composite constructs. We used Pveg, a constitutive E. coli and B. subtilis promoter, and double terminators to book-end internal genes upon chromosomal integration. To improve secretion and transdermal delivery of mBBI from B. subtilis, we added a secretion (sec) tag as well as a transdermal (TD1) tag. The secretion tag is a localization tag for the Sec pathway in B. subtilis (Fu et al., 2007). TD1 is a part previously developed by the USTC_China 2013 iGEM team, and it is designed to allow for the trafficking of peptides across the skin.
The second major composite part was the ComK construct. ComK is a transcription factor and master regulator for competency in B. subtilis. We modified the original ComK sequence, which was first submitted by the 2014 UofC_Calgary team, to remove unwanted restriction sites from within the ComK gene itself. The ComK gene and xylose-inducible promoter were then flanked with sequences homologous to the amyE locus in B. subtilis, a non-essential gene responsible for the degradation of starch. These 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 allows for the transformation of B. subtilis to be more time-efficient and less 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 mBBI and Comk constructs. Our cassette contains thrC integration sequences which flank the sec and TD1 tag. However, the mBBI sequence is replaced with the restriction sites for BamHI and XmaI restriction enzymes. Addition of the restriction sites allows for directional cloning of any desired insert, allowing B. subtilis to be used as a platform for future biotherapeutics, creating a portable mini-pharmacy.
Composite Parts
BBa_K2008001
BBI with N-terminal KSCI solubility tag fused to GFP for expression in E. coli
Sequence-confirmed
Submitted to the registry
BBa_K2008002
BBI with N-terminal sec and TD1 tags fused to GFP and under the pVeg promoter for expression in B. subtilis
Sequence-confirmed
Submitted to the registry
BBa_K2008003
BBI with N-terminal KSCI solubility, sec, and TD1 tags fused to GFP and under the pVeg promoter for expression in B. subtilis
Sequence-confirmed
Submitted to the registry
BBa_K2008004
BBI with N-terminal sec and TD1 tags under the pVeg promoter for expression in B. subtilis
Sequence-confirmed
Submitted to the registry
♥BBa_K2008005
BBI with KSCI solubility, sec, and TD1 tags under the pVeg promoter for expression in B. subtilis
Sequence-confirmed
Characterized
Submitted to the registry
BBa_K2008006
Improved version of comK gene with removal of internal restriction sites
Sequence-confirmed
Submitted to the registry
♥BBa_K2008007
Improved version of comK with addition of flanking regions of amyE homology
Sequence-confirmed
Submitted to the registry
♥BBa_K2008008
Modular secretion platform containing sec and TD1 tags with flanking regions of thrC homology for chromosomal integration in B. subtilis
Sequence-confirmed
Submitted to the registry
References
Fu, L.L., Xu, Z.R., Li, W.F., Shuai, J.B., Lu, P., and Hu, C.X. (2007). Protein secretion pathways in Bacillus subtilis: implication for optimization of heterlogous protein secretion. Biotechnology Advances 25(1): 1-12.
