Difference between revisions of "Part:BBa K3365062"

Revision as of 01:57, 25 October 2020

First lure sequence upstream of eGFP

This part is a off-target detecting platform for dCas9-ω. We put this part in a plasmid and transfer it into △rpoZ-MG1655, a strain of E.coli that was koncked out rpoZ. If dCas9-ω is binding to this lure sequence, the submit Omega fused to dCas9 C-terminal can activate the expression of reporter by recruiting the RNAP holoenzyme and the cell would emit green fluorescence,which means dCas9-ωis off-target, else the weak promoter and the lack of rpoZ makes the reporter cannot express or just have a quite low expression level.

According to the refrence, the off-target rate of the lure sequence in this part is 0.08286%.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
INCOMPATIBLE WITH RFC[12]
Illegal NheI site found at 66
Illegal NheI site found at 89
21
INCOMPATIBLE WITH RFC[21]
Illegal BamHI site found at 834
Illegal XhoI site found at 843
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]

Revision as of 01:54, 25 October 2020 (view source) Gyaxxxxxx (Talk \| contribs) ← Older edit		Revision as of 01:57, 25 October 2020 (view source) Gyaxxxxxx (Talk \| contribs) Newer edit →
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	This part is a off-target detecting platform for dCas9-ω. We put this part in a plasmid and transfer it into △rpoZ-MG1655, a strain of E.coli that was koncked out rpoZ. If dCas9-ω is binding to this lure sequence, the submit Omega fused to dCas9 C-terminal can activate the expression of reporter by recruiting the RNAP holoenzyme and the cell would emit green fluorescence,which means dCas9-ωis off-target, else the weak promoter and the lack of rpoZ makes the reporter cannot express or just have a quite low expression level.		This part is a off-target detecting platform for dCas9-ω. We put this part in a plasmid and transfer it into △rpoZ-MG1655, a strain of E.coli that was koncked out rpoZ. If dCas9-ω is binding to this lure sequence, the submit Omega fused to dCas9 C-terminal can activate the expression of reporter by recruiting the RNAP holoenzyme and the cell would emit green fluorescence,which means dCas9-ωis off-target, else the weak promoter and the lack of rpoZ makes the reporter cannot express or just have a quite low expression level.
		+
	According to the refrence, the off-target rate of the lure sequence in this part is 0.08286%.		According to the refrence, the off-target rate of the lure sequence in this part is 0.08286%.