Featured Parts:RNA-Lock-and-Key

The Parts

BBa_J01008 - ("key 1") Biobricked version of Isaacs' riboregulator trans activating key, taR12
BBa_J01010 - ("lock 1") Biobricked version of Isaacs' riboregulator cis repressed lock, crR12.
BBa_J01080 - ("lock 3") Modified version of J01010
BBa_J01086 - ("key 3") Modified version of J01008

Background: How the Riboregulator Works

These riboregulators are based on the work of Isaacs et al. 2004 (see reference #1). They supress the expression of a target gene by introducing a complementary region that prevents translation initiation. The DNA sequences of the "locks" are modified directly upsteam of the target gene's ribosome binding site (RBS) such that the 5' untranslated region folds to form a stem-loop secondary structure, blocking the RBS and preventing translation. This stem-loop structure is known as the 'cis-repressed' mRNA (crRNA). These "locked" stem-loops are 'unlocked' via what we call "keys". These "keys" for non-coding RNA are called "trans-activating" RNA (taRNA). They bind complementary regions within the lock to open the crRNA and allow the translation machinery to bind at the RBS.

Suggested Uses for these Biobricks

Experimental and Technical

Technical

  • crR12 ("lock1"):
    • Isaacs documentation (see reference #1): Sequence secondary structure forms a stem-loop with two dispersed "bulge" sites (formed by intentionally mismatched base pairings) which destabilize the stem-loop enough to allow the corresponding taR12 "key1" to interject itself and open the loop.
  • taR12 ("key1"):
    • Isaacs documentation (see reference #1): Under the induction of the araC promoter (using 0.25% arabinose solution), the taR12 key activated the corresponding crR12 lock to express the downstream indicator, GFP, to levels of flourescence 19-fold greater than without taR12 expression. Maximal expression was noted at 70 minutes after addition of arabinose solution, and was maintained after this point.

Previous Experiments

  • taR12/crR12 (key1/lock1): For the gene sequence [araC][taR12][crR12][GFP], Activation of taR12 transcription induced by araC promoter unlocked the crR12 stem-loop enough to cause a 19-fold increase in GFP flourescence. For more experiments see reference #1 Isaacs et al 2004.
  • cross-talk: The cross talk between the two different pairs of locks and keys has not yet been tested. Putatively, they should not interact but this has not yet been proven experimentally.

References

  • Publications on the Isaacs Riboregulator:
    • Isaacs, Dwyer DJ, Ding C, Pervouchine DD, Cantor CR, Collins JJ. 2004. "Engineered riboregulators enable post-transcriptional control of gene expression". Nature Biotechnology 2004. [http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=15208640&query_hl=7&itool=pubmed_docsum Pubmed] [http://www.nature.com/nbt/journal/v22/n7/abs/nbt986.html;jsessionid=ABB92F041E994E3B8E4A5213699C3CB0 Journal]
  • More parts and resources
    • A listing of more parts made by the Berkeley 2005 iGEM team including constituitively 'on' promoter-controlled riboregulators and other experimental constructs can be found here
    • The second generation of riboregulators designed to optimize their specificity and efficiency are available from the Berkeley iGEM 2006 page.