Difference between revisions of "Part:BBa K1946007"

Revision as of 19:53, 26 October 2016

dCas9 E. Coli

dCas9 is a catalytically inactive Cas9 that cannot generate double strand cleavage. It recognises the sequence targeted by gRNA and binds to that sequence. It can be used for gene silencing and in an AND gate. Further information can be found here: http://2016.igem.org/Team:Bilkent-UNAMBG/Description

We cloned our sgRNA targeting lacI after pL(TetO) promoter which is operated by TetR and sfGFP operated by LacI. We verified our cloning with restriction digestion using EcoRI and BamHI. (Fig.1 lanes 3,4,5,6 and 7)

dCas9 construct from Addgene (44249) has pL(TetO) promoter followed by dCas9. dCas9 and sgRNA transcription is expected after induction with ATC. We observed an overexpression of a protein with the same mass with dCas9 after induction with ATC compared to uninduced samples (Fig.2 lane 7 and 9 ~160kDa).

Then we cotransformed our sgRNA containing construct (RC.2) with dCas9 containing construct. When they are present at same time, ATC induction which is expected to initiate transcription of sgRNA and dCas9, increases sfGFP signal by 3.5 fold while without induction their co-presence increases sfGFP signal by 2.5 fold. (Fig. 3) This data suggests that our lacI targeting sgRNA and dCas9 can inhibit lacI transcription only when they coexist so that repression of LacI on sfGFP can be diminished. Induction with ATC increased fold change but effects of leakage can be observed.

Sequence and Features