Difference between revisions of "Part:BBa K3365007"

Latest revision as of 19:02, 27 October 2020

Lure1 sequence downstream of pBAD

The PAM and potential off-target sequence (lure1) are located directly downstream of the promoter (BBa_K3365003), where dCas9 might bind wrongly and block RNAP. In our part, the “lure1” is the potential off-target sequence for PDCD1 CRISPR gene editing mentioned in the literature, which might be identified and bound by the complex of dCas9 and sgRNA. According to the reference, the absolute off-target rate is 0.08286%.

Usage and Biology

This part is used to combine the signal of arabinose and the off-target or not of dCas9. The uninduced transcriptional level downstream the signaling is very low. In the presence of arabinose, transcription from the pBAD promoter is turned on. In the presence of both arabinose and the complex of dCas9 and sgRNA, the complex might bind to the lure1 sequence and the transcription might be partially inhibited because of the potential block of RNAP. It is characterized in combination with BBa_K3365054, and the resulting part is submitted as BBa_K3365015. BBa_K3365007 and BBa_K3365054 are ligated through overlap PCR.

Results

The lure1 sequence is added to the promoter of the L-arabinose operon of E. coli (pBAD) (BBa_K3365003) by PCR. The eletrophoretic profile of the PCR product and the sequencing result reveal the successful construction of the fragment.

Sequence and Features