Part:BBa_K1946002
sgRNA targeting LacI
This part has a sgRNA targeting lacI. It also has 5' and 3' ribozyme which cleave the RNA molecule with self catalysis so that any sequence can be added to 5' or 3' to this part without preventing Cas9 binding and its activity.
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We cloned our sgRNA targeting LacI (K1946002) controlled by Tet operator into pGEX-6P-1 vector with sfGFP controlled by Lac operator and constitutively active LacI gene. We cotransformed the construct with Tet controlled dCas9 containing construct so that expression of dCas9 and sgRNA would inhibit LacI expression leading to increase in expression of sfGFP. In this figure: Cells without sgRNA or dCas9 or sfGFP (1,2), cells with only dCas9 (3,4) and cells with only sgRNA and sfGFP (5,6) showed less sfGFP activity than cells with sfGFP, sgRNA and dCas9 (7,8). Unfortunately, ATC induction had no effect on LacI repression which may suggest that even leakage of sgRNA and dCas9 is enough for suppressing LacI (7: not induced, 8: ATC induced). Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 43
- 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 43
- 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 43
- 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 43
- 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 43
- 1000COMPATIBLE WITH RFC[1000]
| target | LacI |