Difference between revisions of "Part:BBa K1689010"

Revision as of 15:58, 18 September 2015

N-luc-dCas9

Nluc-dCas9 fusion protein ORF

A catalytically dead Cas9 (dCas9), when coexpressed with a guide RNA, generates a DNA recognition complex which can binds to any targeted gene [1]. And luciferase, widely used as a reporter is split into two fragments, namely Nluc and Cluc [2]. Each protein fragment by itself is inactive, when two fragments are reassembled, the enzymatic activity of the original protein would be reconstituted, providing easily measurable read out. Thus, split luciferase is widely used as a reporter.

2015 Peking iGEM fused Nluc to N terminus of dCas9 (Figure 1). Guided by sgRNA, it binds to target DNA sequence. And together with Cluc-dCas9 (BBa_K1689008):sgRNA complex, our paired dCas9 (PC) reporter system was constructed (Figure 2) which converts invisible sequence information into measurable bioluminescence.

[[File: Sequence and Features