Difference between revisions of "Part:BBa K3081058"

Revision as of 07:44, 21 October 2019

pBAD-dCas9-J23119-sgRNA

In the CRISPR replication inteference system, a 20-bp sgRNA is designed to be complementary to OriC, the genome replication origin. We use arabinose promoter to drive the expression of dCas9, while sgRNA is under the control of a J23119 promoter. Seven different targeting sites for dCas9 is designed to test the effect on cell growth. Binding box nomenclature used here is the conventional name of binding box (e.g. "M") followed by a "+" or "-" which stands for the direction of sgRNA from 5' to 3'. For instance, M box with its bottom strand bound by dCas9 is thus called M+ box.

This composite part is the principal design of the inducible CRISPRri system, but the intended sgRNAs are not assembled into the backbone, so this part contains a relatively meaningless sequence. Two BsaI sites flanking the meaningless sequence allow for easy assembly of intended sgRNA sequence by Golden Gate method. Length (412bp) of this meaningless sequence is far greater than that of normal sgRNAs (<20bp), facilitating easy detection of successfully assembled Golden Gate product when the colony PCR fragments are loaded onto gel.   After Golden Gate assembly, the ‘sgRNA’ sequence will be replaced by our carefully selected sequences (like M+, BBa_K3081107; R1+, BBa_K3081108; etc.). These newly generated pBAD-dCAS9-J23119-M+/R1/… parts each contains a meaningful sgRNA which guides the dCas9 protein to different boxes on E.coli genome replication initiation region, OriC, thus blocking the binding of relevant proteins essential for replication initiation to their boxes. Arrest and inhibition of genome replication initiation is achieved in this way.

polyA

M+

R1+

R3+

R4-

MR13

IHF

M-R2+

Sequence and Features