Difference between revisions of "Part:BBa K1946002"

Revision as of 14:17, 14 October 2016

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.

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

Assembly Compatibility:

10
INCOMPATIBLE WITH RFC[10]
Illegal EcoRI site found at 43
12
INCOMPATIBLE WITH RFC[12]
Illegal EcoRI site found at 43
21
INCOMPATIBLE WITH RFC[21]
Illegal EcoRI site found at 43
23
INCOMPATIBLE WITH RFC[23]
Illegal EcoRI site found at 43
25
INCOMPATIBLE WITH RFC[25]
Illegal EcoRI site found at 43
1000
COMPATIBLE WITH RFC[1000]

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 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.
+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).
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 ===Usage and Biology===