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Want to know what the Berkeley iGEM is doing? Go to our page.

The Riboregulator Method of translational control of gene expression cis-repressive sequence (“lock”) upstream of a gene’s coding region forms a hairpin, sequestering the ribosome binding site trans-activating (“key”) mRNA strand binds and opens the hairpin thus allowing access to the RBS. Highly specific activation occurs. Very similar lock and key pair sequences do not exhibit crosstalk Isaacs et al., Nature Biotechnology, 2004

Results with lock3/key3 Strain Fluorescence no plasmids 31 lock3RFP 44 key3 + lock3RFP 78 OnRFP 6415 key3 + lock3-RFP

Improved locks and keys Frame of homology Presence of hairpin Position of promoter Degree of homology Position of terminator Transcriptional fusion Length of spacer Distance from RBS

Useful Specs? Gain = FL(lock + key) / FL(lock) Gain = FL(lock + key – BG) / FL(lock – BG) % Recovery = FL(lock + key) / FL(OnRFP)

New key architectures Increases effective key concentration; gene dosing 13x, δ=0.5 83x, δ=4 11%, δ=.5% Increases stability 8x, δ=1 30x, δ=5 9%, δ=1%

Unanswered questions Is 100% recovery possible? Limits to gain? Why is [mRNA] lower? Termination, RNAses, etc. QPCR How does FL vary over pop.? Actual FL levels? Flow Cytometry Is there a FL-OD relationship?  Time course