Part:BBa_K5179301

P4 T1 P4 T1 terminator is a terminator from the satellite bacteriophage P4, it is reliant on the P4 CI RNA (BBa_K5179300) to efficiently stop transcription. The CI RNA 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 which must be complementary to the CI bulge loop for efficient termination 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 T1 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 RNA and T1 terminator 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

P4 T1 plays a role in P4 immunity, meaning that it works to keep P4 integrated into its host bacteria’s chromosome in the presence of the CI RNA. T1, and other CI RNA dependent terminators do this by prematurely stopping transcription of P4’s alpha operon before P4 late genes, including its excisionase, can be transcribed. However, T1 only terminates transcription efficiently in the presence of the CI RNA (Sabbattini et al., 1995).