Difference between revisions of "Part:BBa K3081058"

Revision as of 18:23, 16 October 2019

pBAD-dCas9-J23119-sgRNA

This composite part is the principal design of the inducible CRISPR-based DNA replication interference 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.

Sequence and Features