Part:BBa_K5179300
P4 CI RNA
This part is a minimal CI designed to induce transcription termination at the T1 terminator (BBa_K5179301). CI does this by binding to multiple regions on a nascent transcript containing transcribed T1, causing it to form a termination structure which stops further transcription by RNA polymerase. In order for the CI RNA to bind and form said termination structure it needs to have an 8bp region of complementarity with the relevant T1 terminator in its bulge loop secondary structure (Forti et al., 2002). For this part, the section of DNA corresponding to the wild type P4 CI bulge loop has been replaced with two outward facing BsaI cut sites so that said 8bp region can be easily inserted to create any one of the possible CI RNA varieties.
Usage
Both the CI RNA (BBa_K5179300) and T1 terminator (BBa_K5179301) are designed to be easily changed into any one of the possible CI/T1 variants through insertion of an 8bp fragment which defines the mature CI RNA’s bulge loop. In order for the CI RNA to bind to the nascent T1 transcript they must be complementary to each other in that 8bp region. To generate said complementarity each of a CI/T1 pair must be assembled in a golden gate reaction using BsaI along with a small double stranded DNA with appropriate fusion sites. Each of the CI and T1 have different fusion sites so two such inserts will be required, however each of the relevant fusion sites is not used by the iGEM type IIS/MoClo assembly standard so this step can be performed at the same time as another assembly with BsaI. These inserts can be created with inward facing BsaI cut sites, or synthesized as single stranded DNA oligos, which will then need to be annealed and phosphorylated on the 5’ ends before being used in an assembly.
Biology
The CI RNA is derived from the satellite bacteriophage P4. In P4 the CI RNA serves as an immunity factor, meaning that CI works to keep P4 integrated into its host bacteria’s chromosome and that its presence confers immunity to further infection by P4. The CI RNA does this by binding to regions on the nascent transcripts of P4’s chromosome which correspond to CI RNA dependent terminators, such as T1. When CI RNA binds to such a region, it causes the nascent transcript to take on a termination structure which halts further transcription by RNA polymerase (Sabbattini et al., 1995).
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 50
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 53
Illegal BsaI.rc site found at 47
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