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SYNTHETIC BIOLOGY GAMES WITH RNA IN BACTERIA ROEE AMIT DEPARTMENT OF BIOTECHNOLOGY AND FOOD ENGINEERING TECHNION-ISRAEL INSTITUTE OF TECHNOLOGY 7TH ANNUAL WINTER Q-BIO MEETING IN HAWAII FEB. 24TH, 2017 WHY RNA? HOW IS IT DIFFERENT FROM DNA? • RNA makes secondary and tertiary structure. • RNA degrades. • RNA-based or translational regulation is not well understood. • RNA regulation is faster than transcriptional regulation. WHY SYNTHETIC BIOLOGY? SYSTEMATIC LARGE SCALE VARIATIONS OF DIFFERENT REGULATORY ARCHITECTURES. • RBP-RNA regulation • RNA-based insulation in bacteria SYNTHETIC BIOLOGY FORCES US TO TEST WHAT WE THINK WE UNDERSTAND ABOUT BIOLOGY! Lior Levy Leon Anavy Noa Katz SYNTHETIC RBP-BASED REGULATION RNA VERSION OF BUILDING SYNTHETIC PROMOTERS IN VIVO RBP BINDING ASSAY CAN CHARACTERIZE RBP BINDING AFFINITIES CURRENTLY EXPANDING TO A LARGE SCALE SCREEN. BASAL LEVEL PERIODICITY - WHY? RIBOSOME CAN UNWIND HAIRPIN STRUCTURES • Rate of unwinding is position dependent with 3nt periodicity. • Every binding site tested exhibited this behavior. • Kinetic hopping model can explain this behavior. SYNTHETIC TRANSLATION REPRESSION • RBPs can repress translation only when the binding site is encoded within the initiation region. • Mechanism is likely due to blocking of 70S formation • No evidence for scanning 70S initiation. SUMMARY • Showed that Ribosome can unwind hairpin of various sizes inside CDS. • Hairpins encoded within initiation region do not affect translation. This goes against current understanding. • RBP can repress expression in the initiation region or 5’UTR (data not shown). • Multiple RBP binding sites alter binding affinity - stabilize one another (data not shown). WHAT IS CONTEXT? • Sequence level effects that may be embedded in the core “element”. • Secondary regulatory functions that only emerge when the elements is taken out of “context”. • Encoded in sparse or not easily identifiable sequence elements. EXAMPLE: BACTERIAL INSULATION • We engineered this synthetic configuration and found silencing - how can one promoter silence another? IT’S NOT IN THE DNA…. LOOKS LIKE IT’S IN THE RNA SO HOW IS IT ENCODED? USE A SYNTHETIC LIBRARY APPROACH… 135 MUTATIONS OF GLNKP IN PARALLEL THERE IS IT? - IN THE BLUE REGION! WHAT ABOUT OTHER SIGMA54 PROMOTERS? • 3-5 nt PSSM. Impossible to identify by looking at a single or handful of variants! HOW IS INSULATION ENCODED? • Strength of insulation is proportional to how many CT-rich motifs are encoded within the core region, and how close they are to RBS. • Phenomenon is enriched for σ54 promoter sub-class in E.coli WHAT ABOUT SYNTHETIC BIOLOGY? NEW DESIGN RULE FOR CIRCUIT DESIGN • Model that takes into account the new insulation phenomenon outperforms RBS calculators. SUMMARY • Synthetic OLs can help unravel “context” dependent effects in vivo. • Bacterial insulators predominantly encoded in sig54 promotes. • Insulators are encode by multiple sparse 3-5 nt CT-(purine-rich) motif. • Mechanism is anti-Shine Dalgarno:Shine Dalgarno binding. ACKNOWLEDGEMENTS Insulators RBP-RNA Interactions • Lior Levy • Noa Katz • Leon Anavy • Ronni Cohen • Oz Solomon • Beate Kauffman • Roni Cohen • Sarah Goldberg • Orna Atar • Shilo Ohayon • Sarah Goldberg • Orna Atar • Zohar Yakhini Positions Available: Ph.D Students Post Docs