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 three 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.