Part:BBa_K1679039:Design
pT7-10 + RiboJ + GFP + LVA + 3 terminnators
- 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 1019
- 1000COMPATIBLE WITH RFC[1000]
Design Notes
This device is design for protein expression and promoter measurement. It constructed with a T7 RNA polymerase which has other 3 mutations, a RiboJ, a green fluorescent protein, a LVA degradation tag, a T7 terminator, a rrnB T1 terminator and a rrnB T2 terminator.
We added restriction sites flank these segments, so that users can change this segments into what they prefer. Users can replace T7 with other promoters through SalI and HindIII; replace RiboJ and GFP with other coding sequence through HindIII and StuI.
After transcription, RiboJ becomes a section of functional RNA which comprises the sTRSV- ribozyme which can excise its upstream fragment to reduce noise, and an additional 23-nt hairpin immediately downstream to help expose the RBS. We use it to buffer synthetic circuits from genetic context.
To adapt the protein expression device, three terminators are design to use cooperatively with strong promoters, the LVA degradation tag can accelerate protein degradation rate to exclude bacterial stress responses.
Source
Mutated from BBa_K1679026 by PCR.
References
Schaerli Y, Munteanu A, Gili M, et al. A unified design space of synthetic stripe-forming networks[J]. Nature Communications, 2014, 5:4905-4905.
Segall-Shapiro T H, Meyer A J, Ellington A D, et al. A‘resource allocator’ for transcription based on a highly fragmented T7 RNA polymerase[J]. Molecular Systems Biology, 2014, 10(7):742-742.