Part:BBa_K3365006
Target sequence downstream of pBAD/araC
We add target sequence downstream of BBa_K1321333 created by Group iGEM14_Imperial and create 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.
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 product and the sequencing result reveal the successful construction of the fragment.
F1: 5’-CGAGCTCTTATGACAACTTGACGGCTACATCATTCAC-3’
R1: 5’-TTCTTAAAGGCAGTTGTGTGACACGGAAGCGGATGGAGAAACAGTAGAGAGTTGCG-3’
This part 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.
To verify the inhibition effect of dCas9 on pIn-RTr, we set up six culturing system (as shown in the table below) and use microplate spectrophotometer to examine fluorescence intensity. L-arabinose is 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 does work.
To know about the inhibition of the expression of RFP by dCas9 in a single cell as well as verify our mutant library, the obtained dCas9 mutant library and the wild-type dCas9 are transformed into MG1655 carrying pIN-RTr. Then, we analyze the red fluorescence by flow cytometry. The results are shown below. 77.12% cells have positive red fluorescence in the absence of dCas9, while the proportion of positive cells decreases to 0.08% in the presence of dCas9, showing a quite efficient inhibition of wild-type dCas9. And the positive rate rises to 78.37% when the cells are transformed into dCas9 mutant library, which means the lack of function in most of mutant dCas9 and the successful construction of our mutant library.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 979
- 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI site found at 961
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