Team:BostonU/Composite Part
To create the Gemini parts library we made use of two “classes” of plasmid: guide RNA expressing vectors and guide RNA operator reporter vectors. When these two plasmids are co-transfected with a dCas9-VPR plasmid into a mammalian cell line, a gene of interest is expressed. Our work for iGEM dealt with fluorescent proteins; specifically, GFP for the parts submitted to the registry.
The guide RNA operator vector can be considered a composite part. It is constructed by combining the following basic parts in order: BBa_K1875009 (20 bp target sequence), BBa_K1875000 (minimal CMV promoter), BBa_K1875001 (Kozak sequence), BBa_K1875003 (GFP), BBa_K1875002 (Rabbit Beta Globin PolyA terminator). The sources of these parts can be found on their respective registry pages.
We have submitted BBa_K1875018 for best composite part due to both its high expression and consistency in experimentation. This particular composite part has a triple multimerized operator for gRNA 13 (g13), meaning that there are 3 20 bp target sequences for g13 (each separated by 24 random bases) upstream of the minimal CMV promoter. Through our experimentation, we identified that this part, when compared with a full-length CMV promoter, can actually express GFP at a higher level than the strong constitutive promoter. This means that this synthetic composite part enables even higher protein production compared to a natural promoter, and can be used usefully in many protein production applications. Additionally, because its activity is controlled by expression of a specific corresponding gRNA (g13), it can grant temporal control over protein expression.
