Part:BBa_K3081022
pBAD-dCas9-J23119-NT1
This composite part is the principal design of the inducible CRISPRi systemBBa_K3081024, with assistance of constantly expressed sgRNA (NT1) that is complementary to the corresponding sequence of mRFP coding region. So it needs to cotransform with a plasmid coding mRFP.Fluorescence is decreased as the concentration of arabinose increases.
To investigate CRISPR-dCas9 binding specificity and affinity with DNA, we constructed arabinose-induced expression of dCas9 targeted to mRFP coding region, with assistance of constantly-expressed single guide RNA that is complementary to the corresponding sequence. Since normal mRNA elongation is interrupted by occurrence of dCas9, fluorescence is greatly decreased as the arabinose concentration increases, comparing to a single guide RNA that has no binding specificity to DNA. This proves the basic concept that a dCas9 protein is able to bind to DNA with sequence specificity and interferes with the physiological process. All plasmids we use to interfere with DNA replication subsequently are derived from this.
Figure 1. CRISPR-interference causes a rapid decrease in mRFP as the arabinose concentration increases. Target sequence of CRISPRi, NT1, is located on the coding region of mRFP. The control group, of which the sgRNA is composed of a Poly-adenine that has no binding affinity to DNA strand.
Reference:
[1]Wolański M, Donczew R, Zawilakpawlik A, et al. oriC-encoded instructions for the initiation of bacterial chromosome replication.[J]. Frontiers in Microbiology, 2015, 5(735):735.
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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