Difference between revisions of "Part:BBa K537002:Design"

Latest revision as of 17:02, 15 September 2011

Theophylline Riboswitch 2-CheZ

Design Notes

The effectiveness of the theophylline riboswitch is determined by the ability of the RNA 'loop' to hide the RBS preventing translation. The sequence of bases within the riboswitch will determine the formation of the loop and its leakiness.

This biobrick part represents a composite of a theophylline riboswitch (type2/ clone 12.1) 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 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 could 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 12.1 is derived from Lynch and Gallivan NAR 2009.

References

1. Lynch S.A. and Gallivan J.P., A flow cytometry based screen for synthetic riboswitches. 2009, Nucleic Acids Res. 37(1)184-192

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