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<h2> Overview </h2>
<h2> Overview </h2>
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Inspired from the previous Stanford-Brown-Spielman 2014 IGEM team, we wanted to create a universal cellulose binding construct that can attach any proteins onto a cellulose binding domain. Our design used a cellulose binding domain called the cellulosomal-scaffolding protein A (cipA) of <i> Clostridium thermocellum </i>. We chose cipA because of the high binding affinity tested by the 2014 IGEM team. We also modified the cipA to remove the illegal ecoRI site within the gene to make it BioBrick compatible. We attached the cipA protein onto a monomeric streptavidin (mSA) to prevent protein aggregation because the wild-type streptavidin is a tetramer. The overall goal is to create this universal CBD that can attach to any biotinylated protein of interest. This involves molecular cloning any protein of interest onto the bifunctional ligase/repressor (birA) protein that is attached to an acceptor peptide. BirA is found in <i> E. coli </i> and it catalyzes attachment of biotin onto the biotin acceptor peptide such as the Avitag [5]. Because the acceptor peptide is connected to the protein of interest, this protein can be extracted and purified from the cells and can then attach to Part I via streptavidin-biotin interaction. The overall second construct would be called protein of interest-birA (POI-birA).
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We used the cellulose binding domain cellulosomal-scaffolding protein A (cipA) of <i> Clostridium thermocellum </i> for construction of our cipA-mSA construct ([https://parts.igem.org/Part:BBa_K1692035 BBa_K1692035]) and our spore coat construct ([https://parts.igem.org/Part:BBa_K1692028 BBa_K1692028]). The cipA used is modified to get rid of the illegal ecoRI site found in the 2014 Imperial College IGEM team construct.
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We had successfully sequenced cipA-mSA construct and attempted to extract protein by Flag-tag purification. Our attempt at protein extraction, however, failed because of the concern that the beads attached to the flag antibody might not have functioned properly. We also believed that our failed extraction attempt was because we didn't transform our construct into a T7 competent cells. Thus, our future goal is to transform the cipA-mSA construct into T7 competent cells and extracted proteins by flag-tag purification. We can then do cellulose binding assay to test the binding affinity of the cipA onto a cellulose scaffold.
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Latest revision as of 09:48, 24 September 2015
CIPA-MSA
Overview
We used the cellulose binding domain cellulosomal-scaffolding protein A (cipA) of Clostridium thermocellum for construction of our cipA-mSA construct (BBa_K1692035) and our spore coat construct (BBa_K1692028). The cipA used is modified to get rid of the illegal ecoRI site found in the 2014 Imperial College IGEM team construct.
