Generator
thRS1-ChZ

Part:BBa_K537001:Design

Designed by: Natasia Kruger   Group: iGEM11_WITS-CSIR_SA   (2011-07-19)

Theophylline Riboswitch 1-CheZ


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


Design Notes

The effectiveness of the theophylline riboswitch is determined by the ability of the RNA 'loop' to hide the RBS preventing translation.

This biobrick part represents a composite of a theophylline riboswitch (type1/ clone 8.1) directly upstream of an E.coli CheZ motility factor. Here the riboswitch and its adjacent CheZ coding region are considered together as a single biobrick. Previous teams: Lethbridge 2007, Lethbridge 2009, and NYMU Taipei 2010 all considered strategies for making the riboswitch a separate biobrick, however standard assembly techniques are inadequate since the sequence distance between the ATG, RBS and aptamer domain of the riboswitch are critical. We have explored this further in a more elaborate discussion (see here).

This part represents an N-terminal fusion part. The part contains a standard prefix and a “assembly standard 25” suffix (Freiburg Fusions/ RFC 25). Because of multiple AgeI sites within CheZ, full compatibility with RFC 25 is not maintained. Cloning to a C-terminal part occurs by cutting with NgoMIV. The CheZ CDS therefore lacks a TAATAA double stop codon (TAG stop provided by RFC 25 suffix).

Source

The cheZ sequence was PCR amplified from E.coli strain DH5α, where the forward primers in the reaction contained the sequence for the theophylline riboswitch.

The riboswitch clone 8.1 is derived from Topp and Gallivan JACS 2007, as well as previous biobricks BBa_K249026 and BBa_K411001

References

Topp S, Gallivan JP. Guiding bacteria with small molecules and RNA. J Am Chem Soc 2007;129:6807-11

Lynch S.A. Desai S.K.,Sajja,H.K., and Gallivan J.P.A high-trhoughput screen for synthetic riboswitches reveals mechanistic insight into their function. 2007, Chem Biol 14:173-184

Topp S. and Gallivan J.P. Random walks to synthetic riboswitches – a high throughput selection based on cell motility. 2008, ChemBiochem 9:210-213