Difference between revisions of "Part:BBa K3017068"

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Construct for testing CRISPRi asRNA cross-talk with non-target sgRNA

Aim

This asRNA characterization construct is to characterize the cross talking by asRNA mismatch with the incorrect sgRNA.

Design

This asRNA characterization constructs all contain a constitutively expressed dCas9, a fluorescent protein RFP, asRNA (for sgRNA_GFP) under pBAD promoter, and sgRNA (RFP). Under the regulation of pBAD promoter, asRNA is transcripted when arabinose is added to the culture medium. The transcription start site of the pBAD promoter has been identified according to Brzozowska et al. (2018), in which the asRNA has been placed on the Transcription Start Site (TSS). Forl sgRNA transcription, weak Anderson promoter BBa_J23115 of strength 0.15 is used.

Since the sgRNA transcribed will be targeting for for RFP, suppression effect on the RFP should be observed. However, upon L-arabinose induction for the transcription of asRNA, no derepression effect should be observed as the asRNA’s extensor is not complementary to the sgRNA_RFP.

Reference

Characterizing Genetic Circuit Components in E. coli towards a Campylobacter jejuni Biosensor

Natalia Brzozowska, Jane Gourlay, Ailish O’Sullivan, Frazer Buchanan, Ross Hannah, Alison Stewart, Hannah Taylor, Reuben Docea, Greig McLay, Ambra Giuliano, James Provan, Katherine Baker, Jumai Abioye, Julien Reboud, Sean Colloms

bioRxiv 290155; doi: https://doi.org/10.1101/290155

Sequence and Features