Part:BBa_K1689009

C-luc-dCas9

C-luc-dCas9 fusion protein ORF

A catalytically dead Cas9 (dCas9), when co-expressed with a guide RNA, forms a DNA recognition complex which can bind any sequence [1]. Firefly luciferase, widely used as a reporter, is split into two fragments, namely N-luc and C-luc [2]. Each fragment by itself is inactive; when two fragments are reassembled, the enzymatic activity of the original protein would be reconstituted, providing easily measurable readout.

Peking iGEM 2015 fused C-luc to N terminus of dCas9 (Figure 1) and purified it (Figure 2). Guided by sgRNA, it binds to target DNA sequence. Together with another part, N-luc-dCas9 (BBa_K1689010):sgRNA complex, our paired dCas9 (PC) reporter system would work to (Figure 3) to convert the sequence-specific information of pathogenic bacteria's genome (in our case, M. tuberculosis) into easily readable bioluminescence signal.

Figure 1. Schematic cartoon of C-luc-dCas9 fusion protein.

Figure 2. SDS-PAGE of purified Cluc-dCas9 fusion protein