Part:BBa_K3365006
Target sequence downstream of pBAD/araC
We added target sequence downstream of Part:BBa_K1321333(view:[1]) created by Group iGEM14_Imperial(view:[2]) and created this new part The PAM and target sequence are located directly downstream of the promoter, where dCas9 could bind and block RNAP. In our part, the “target” is the target sequence for PDCD1 CRISPR gene editing in one clinical trial, which can be identified and bound by the complex of dCas9 and corresponding sgRNA.
Usage and Biology
This part is used to combine the signal of arabinose and the on-target or not of dCas9. The pBAD is regulated by the AraC protein, which is both a positive and a negative regulator. 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 target sequence and the transcription is partially inhibited because of the block of RNAP. It is characterized in combination with BBa_K3365053, and the resulting part is submitted as BBa_K3365014. BBa_K3365006 and BBa_K3365053 are ligated through overlap PCR.
Results
The target sequence is added to the inducible pBAD/araC promoter (BBa_K3365013) by PCR with primer F1/R1. The eletrophoretic profile of the PCR (F1/R1) product and the sequencing result reveal the successful construction of the fragment. F1: 5’-CGAGCTCttatgacaacttgacggctacatcattcac-3’ R1: 5’-ttcttaaaGGCAGTTGTGTGACACGGAAGCGGatggagaaacagtagagagttgcg-3’
To verify the inhibition effect of dCas9 on pIn-RTr, we set up six culturing system (as shown in the table below) and used microplate spectrophotometer to examine fluorescence intensity. L-arabinose was added to induced the expression of RFP.
The results are shown below, from which we could see 64.39 times of fluorescence/OD600 difference between transform group (without the expression of dCas9) and co-transform group (with the expression of dCas9), indicating our transcription inhibition system did work.
Sequence and Features BBa_K3365006 Se Not understood
| None |