Part:BBa_K2019002
mRFP + sgRNA targeting mRFP Coding Region (saCas9)
Part BBa_K2019002 consists of constitutively expressed mRFP gene (BBa_J04550) along with a constitutively expressed sgRNA that targets the mRFP gene.
Usage and Biology
Usage and Biology
The aim of this part is test the functionality of saCas9. This composite part has mRFP constitutively expressed along with a constitutively expressed sgRNA that targets the mRFP gene. Therefore, if saCas9 is functional, it will successfully cut and inactivate the mRFP gene, resulting in the cells to longer be red.
This part was used in the functional test for part BBa_K2019000, as BBa_K2019000 and BBa_K2019002 were cotransformed into the same E. Coli cell line and were compared to the cotransfomed BBa_K2019000 and BBa_K2019001 E. Coli cell line. BBa_K2019001 is very similar to BBa_K2019002, but it does not have a guide sequence that targets the mRFP gene; thus mRFP is constitutively expressed in the cell line. The results included below demonstrate that the designed guide sequence effectively cuts the mRFP and significantly reduces the relative fluorescence of the cell line.
Figure 1: Relative fluorescence of BB_K2019000+BB_K2019001 ("template"), BB_K2019000+BB_K2019002("guide"), and BB_K2019000 ("Cas9 only")
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal SpeI site found at 1099
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 1076
Illegal SpeI site found at 1099 - 21COMPATIBLE WITH RFC[21]
- 23INCOMPATIBLE WITH RFC[23]Illegal SpeI site found at 1099
- 25INCOMPATIBLE WITH RFC[25]Illegal SpeI site found at 1099
Illegal AgeI site found at 781
Illegal AgeI site found at 893 - 1000COMPATIBLE WITH RFC[1000]
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