Part:BBa_K1946007
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)
Figure 1: Results of restriction enzyme digestion with EcoRI and BamHI to confirm cloning of sgRNA. Successful samples are seen on lanes 3-7
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).
Figure 2: SDS Page results of empty Dh5a uninduced, empty Dh5a ATC induced, empty Dh5a Pro uninduced, empty Dh5a Pro ATC induced, dCas9 Dh5a uninduced, dCas9 Dh5a ATC induced, dCas9 Dh5a Pro uninduced, dCas9 Dh5a Pro ATC induced in order. Bands around 160kDa (thought to be dCas9) are seen on only lanes 7 and 9 corresponding to samples transformed with dCas9 that are induced with ATC as expected
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.
Figure 3: GFP signal levels in the empty control, sample transformed with dCas9, transformed with sgRNA containing construct RC2, co-transformation of dCas9 and RC2 and positive control induced and uninduced with ATC. Y axis is fluorescence intensity (A.U.), X axis is constructs bacteria containing. Data normalized with respect to OD600 obsorbance. Positive control is dCas9 and sgRNA containing cells induced with IPTG.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 1340
- 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 1340
Illegal NheI site found at 1099 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 1340
Illegal BamHI site found at 3378 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 1340
- 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 1340
- 1000COMPATIBLE WITH RFC[1000]
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