Part:BBa_K4811018
PT7-TMS(GFP1)-TT7
This is a transcriptional unit for a tRNA mimicking structure (TMS), using the T7 promoter and terminator. In E. coli BL21(DE3), the T7 polymerase is under LacI control, meaning it will be expressed by IPTG, leading to expression of the TMS RNA. The TMS is a novel piece of synthetically designed RNA, which folds in the same manner as tRNA, developed by Paul et al. It is a trans-encoded genetic switch, which binds to the Ribosome Binding Site (RBS) BBa_K4811001, repressing translation. The binding regions are flanking the RBS, and no binding happens in the RBS consensus sequence. This means that there are very few limitations on both the possible TMSs and repressible RBSs which could be engineered. Also, the D-loop of the TMS can be exchanged for additional functionality, such as an aptamer, making the TMS responsive to a ligand.
In this particular part, the D-loop has been exchanged for a GFP-sensitive aptamer, with the sequence: GGACAGATCTGGGAGCACGATGGCGTGGCGAATTGGGTGGGGAAAGTCCTTAAAAGAGGGCCACCACAGAAGCAATGGGCTTCTGGACTCGGTAGATCTGTCC
This particular TMS was tested by A. Paul during their PhD, and showed that the TMS represses translation, but the repression is relieved in the presence of GFP.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 33
Illegal BglII site found at 122
Illegal XhoI site found at 131
Illegal XhoI site found at 167 - 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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