RNA

Part:BBa_K4811018

Designed by: Kasper Krunderup Jakobsen   Group: iGEM23_DTU-Denmark   (2023-10-03)


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


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE 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
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


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