RNA Synthetic Biology. Farren J Isaacs, Daniel J Dwyer, & James J Collins Nature Biotechnology May 2006. iGEM 2010 Journal Club 7/7/2010. Any sequence diverse 2° structure and function Interact with proteins, metabolites, other nucleic acids Levels of modulation: Transcription
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Farren J Isaacs, Daniel J Dwyer, & James J Collins
iGEM 2010 Journal Club
Small regulatory RNAs (sRNAs) repress andactivate(unlike antisense RNAs) bacterial gene expression in trans by base pairing with target RNAs
Highly specific pockets in the 5′ UTR of the mRNAs that bind ligands conformational change in RNA structure change in gene expression.
Unlike ribozymes, use only changes in DNA conformation, no catalytic activity.
Isaac et al 2004
See next slide…
Unfolds hairpin to expose RBS
(non-coding RNA [ncRNA])
Images from Isaac 2004, Engineered riboregulators enable post-transcriptional control of gene expression
Blue – normal GFP
Green – with taRNA and crRNA
Red – with crRNA only
Black – no GFP gene
Image from Isaac 2004
Black and white bars – GFP fluorescence
Dark and light grey – taRNA concentrations
(pBAD promoter for taRNA)
Blue = original ribosome; purple = second ribosome.
Green = original mRNA; orange= duplicate.
Evolution until pairs do not interact anymore.
Image from Rackham and Chin 2005
1. Select for mRNA sequences that are not substrates for endogenous ribosomes
2. Transformed with library of mutant ribosomes and grown in chlor+ media
Negative selection: uracilphosphoribosyltransferase (UPRT).
Fused CAT (cat) and UPRT (upp) downstream of a constitutive promoter and RBS so the single transcript can be either positively or negatively selected.
And now for more cell logic…
Thanks for listening!