Part:BBa_K3365007
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%.
Figure1. Gene circuit
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.
Figure2. Eletrophoretic profile of the PCR result
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 74
- 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI site found at 56
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