Part:BBa_K3081007
pBAD-dCas9-J23119-M+
This composite part is the principal design of the inducible CRISPR-based DNA replication interference system, with the 20 bp sgRNA targeting to the M+(top strand) DnaA box on E.coli genome replication initiation region, OriC. In natural situations, M+ is a low affinity box for DnaA binding. By blocking the binding of DnaA protein to M+ box, moderate arrest and inhibition of genome replication initiation is achieved. For more detailed information, see BBa_K3081058
Design
Based on CRISPR-interference method for transcription inhibition, we develop a novel approach for prokaryotic genome replication interference (CRISPRri). Hence, a 20-bp sgRNA is designed to be complementary to OriC, the genome replication origin (Figure 1). Instead of site-directed mutations one by one, CRISPRri allows for 20-bp scan each time. Although CRISPRri requires a PAM ("NGG") sequence to execute its function, we found a high occurrence frequency of PAM in the region of replication origin and all available sgRNAs can cover 76.2% (221 out of 290) of OriC. Seven different targeting sites for dCas9 is designed to test the effect on cell growth. We characterized M+ box in detail.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 1205
Illegal NheI site found at 5459
Illegal NheI site found at 5482 - 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 1470
Illegal BamHI site found at 1144 - 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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