NanoLuc-aTcdB Delivery System: BBa_K2087000

This gene codes for a prototype of a fusion protein which is designed to deliver the luminescent protein NanoLuc. The NanoLuc protein is connected to the aTcdB domain via a glycine/serine linker. The protein is HIS tagged at the C terminus with a 6x HIS tag. The part is designed without a promoter, allowing it to be placed under the control of any promoter desired. However, given the length of the transcript, E. coli would not be the best organism. Instead, a strain of bacteria which can handle large proteins would be ideally for expression.

S. pyogenes Cas9 sgRNA DD96 gene Target Locus 618: BBa_K2087000

This is the transcript for the sgRNA that hybridizes with S. pyogenes Cas9 in order to guide the endonuclease to make a double stranded break at a sequence complementary to the RNA. The transcription is under a the control of a T7 promoter, and targets the 618 bp locus on the human DD96 gene. DD96 was intended to be the target of the Cas9-aTcdB protein, and is found in HeLa cells. The DD96 gene is found in humans, and is upregulated in ulcerative colitis. The specific gene was chosen as it is a gene of interest in a separate project being undertaken by Tufts Synthetic Biology. This particular sgRNA is created from the BBa_K1818000 biobrick, which is an empty sgRNA cassette with two adjacent Bbs1 cut sites where the target spacer should go. This part was created by digesting the empty cassette with Bbs1 and ligating the 20bp spacer to reform the plasmid. This target has been confirmed to work with S. pyogenes Cas9 in vitro.

S. pyogenes Cas9 sgRNA DD96 gene Target Locus 401: BBa_K2087002

This biobrick is very similar to BBa_K2087000, in that it is a transcript for S. pyogenes Cas9 sgRNA targeting the DD96 gene. This sgRNA targets a different locus on the DD96 gene, the 401 bp position. We designed and tested both targets in vitro in anticipation of testing our Cas9-aTcdB protein in HeLa cells. This target has been confirmed to work with S. pyogenes Cas9 in vitro.