Part:BBa_K4897016
sgRNA-R
This is a unique sgRNA contained by the Cas9 protein artificially added in our engineering E. coli, to ensure the blue light suicide system could successfully identify the target DNA region for gene editing and eventually kill the E. coli. The sgRNA-R in CAS9 is designed by similar methods as BBa_K4897015, except for the fact that it is used to target DNA-directed RNA polymerase subunit beta which serves as the building block of RNA polymerase in E. coli. Without the enzyme catalysing the transcription of DNA into RNA, E. coli would not be able to produce the proteins needed for its survival and would thus die. The specific sgRNA-R sequence is CGAGAAAAAACGTATTCGTAAGG. The CAS9 protein targeting the parts involved in essential bacterial metabolism serve as a strong double-layered protection to the environment, as the engineering E. coli will be deactivated as soon as CAS9 is activated.
Usage and Biology
In this way, once blue light is undetectable by EL222, the formation of EL222 dimers is terminated and the RNAP is allowed to bind back to the -35 to -10 region of the luxI promoter. This thereby activates the expression of CAS9 and its sgRNA-R sequence. The gene editing process takes place after that, disrupting the function of RNA polymerase of the bacterial cell and its protein production, which eventually causes the death of bacteria. By integrating the blue light suicide system containing this part into our engineering bacteria, precautions against potential leakage risks are established. Our engineering E. coli will not be able to survive in the external environment for more than a day, as when the night falls, it is forced to “commit suicide”. The detailed designs and procedures are illustrated in the figure below.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
| None |