Difference between revisions of "Featured Parts:RNA-Lock-and-Key"

Revision as of 08:17, 27 April 2006

This set of riboregulators are based upon the work of Isaacs et al 2004 (see reference #1). They work by repressing the expression of a target gene by introducing a complementary region to prevent transcription.
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 will fold to form a stem-loop secondary structure, thus blocking the RBS and preventing transcription. 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" f non-coding RNA are called "trans-activating" RNA (taRNA). They act to bind complementary regions within the lock to open the crRNA and allow proteins 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.

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

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 ====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.
 ===References===