Thursday, August 16, 2012: 9:00 AM
Meeting Room 5, Columbia Hall, Terrace level (Washington Hilton)
Amin Espah Borujeni
1, Emily Dong
2 and
Howard Salis2, (1)Chemical engineering / Biological engineering, Pennsylvania State University, State College, PA, (2)Chemical engineering / Biological engineering, Pennsylvania State University, University Park, PA
Small RNAs (sRNA) are central regulators of metabolism, motility, and stress response in bacteria. We have developed a biophysical model of sRNA regulation that predicts the translation rate of a mRNA when regulated by a small RNA. The model calculates the kinetics of sRNA:mRNA complex formation, the thermodynamics of ribosome binding (Salis et. al., Nat. Biotech., 2009), and the resulting translation initiation rate, taking into account the sRNA and mRNA concentrations and the presence of RNA-RNA nucleation binding sites. By combining this biophysical model with an optimization algorithm, synthetic small RNAs are designed
de novo to activate or repress translation of one or more target genes. This design method for synthetic small RNAs, called the Small RNA Calculator, can create a limitless toolbox of versatile regulators for controlling enzyme expression for metabolic engineering applications.
We employ the Small RNA Calculator to design both repressing and activating synthetic small RNAs. Using flow cytometry, fluorescent protein reporters, and long-time cultures, we experimentally validate a collection of de novo designed synthetic small RNAs that are capable of decreasing protein expression by >100-fold (repression) and increasing protein expression by >10-fold (activation). We also use the model to highlight the trade-offs in small RNA design, showing how the sequence space is highly constrained with evolutionary consequences.
The Small RNA Calculator's ability to rationally design synthetic small RNAs to regulate targeted genes will improve our ability to rapidly tune, control, and optimize protein expression for metabolic engineering applications.
