Difference between revisions of "Part:BBa K3365061"

Revision as of 13:10, 27 October 2020

Target transcription activating unit upstream of RFP

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

Fig.1: Schematic diagram of this part

We co-transformed plasmid contained this part and pWJ66 with dCas9-ωin △rpoZ-MG1655 to determine if the system can work well. The transformed △rpoZ-MG1655 without pWJ66 is used as control. We took isolated colonies to pre-inoculate in 5mL of Luria-Bertani (LB) , incubating overnight in a shaker at 37℃. After that we detect the fluorescence intensity of the bacterial solution by microplate reader every two hours.Ths result is shown as follow.

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 812
Illegal XhoI site found at 803
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal AgeI site found at 667
Illegal AgeI site found at 779
1000
COMPATIBLE WITH RFC[1000]

@@ Line 3: / Line 3: @@
 <partinfo>BBa_K3365061 short</partinfo>
-This part is a on-target detecting platform for dCas9-ω. We put this part in a plasmid and transfer it into E.coli, which was knocked out <i>rpoZ</i>. For convenience, we called it △<i>rpoZ</i>-MG1655. If dCas9-ω is binding to target 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 red fluorescence,which means dCas9-ωis on-target, else the weak promoter and the lack of <i>rpoZ</i> makes the reporter cannot express or just have a quite low expression level.
+This part is an on-target detecting platform for dCas9-ω. We put this part in a plasmid and transfer it into E.coli, which was knocked out <i>rpoZ</i>. For convenience, we called it △<i>rpoZ</i>-MG1655. If dCas9-ω is binding to target sequence, the subnit Omega fused to dCas9 C-terminal can activate the expression of reporter by recruiting the RNAP holoenzyme and the cell would emit red fluorescence,which means dCas9-ωis on-target, else the weak promoter and the lack of <i>rpoZ</i> makes the reporter cannot express or just have a quite low expression level.
 [[File:Target_binding_site_for_dCas9_and_subnit_Omega.png]]
 <b>Fig.1:</b> Schematic diagram of this part
+We co-transformed plasmid contained this part and  pWJ66 with dCas9-ωin △<i>rpoZ</i>-MG1655 to determine if the system can work well. The transformed △<i>rpoZ</i>-MG1655 without pWJ66 is used as control.
+We took isolated colonies to pre-inoculate in 5mL of Luria-Bertani (LB) , incubating overnight in a shaker at 37℃. After that we detect the fluorescence intensity of the bacterial solution by microplate reader every two hours.Ths result is shown as follow.
+[[File:The_result_of_microplate_reader.jpeg]]
 <!-- Add more about the biology of this part here