Design and Characterization of a Translation Initiation Region Library for Efficient Translation in E. coli.

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Justin Vigar, Hans-Joachim Wieden

Alberta RNA Research and Training Institute, Canada

Manipulation and fine-tuning of gene expression is critical for a large number of bioengineering applications aiming at altering a wide range of cellular behaviours. Regulation can be achieved on the transcriptional and the translational level. Transcriptional promoters and regulatory factors can be manipulated and changed, allowing control over how fast (or slow) RNA is synthesized or degraded. Alternatively, at the translational level the copy number of a protein, e.g. influencing the flux through metabolic pathways, can be fine-tuned by altering ribosomal binding sites, initiation factors, and optimizing codon usage. Translation initiation is the most regulated phase during protein biosynthesis and is also the rate-limiting step. It is therefore a promising target for expanding our toolbox of cellular control devices. Viruses and eukaryotic cells have very successfully evolved mechanisms using internal ribosomal entry sites (IRES) to bypass the rate-limiting and highly regulated classical eukaryotic translation initiation machinery. IRESs are able to drive translation with a reduced (or no) set of initiation factors and initiate translation through direct interaction with the ribosome. It would be of significant value for bioengineers to have a tool analogous to the virus IRESs for use in prokaryotes, enabling rational fine-tuning of translation initiation. To this end we have designed standardized translation initiation region (TIR) constructs based on an existing Escherichia coli (E.coli) IRES-like TIR. We have generated a randomized library of TIRs using error-prone PCR and directed evolution. Using an in vitro reconstituted translation system we have assessed the ability of our TIRs to drive protein synthesis in the presence and absence of specific translation factors. The aim of this study is to provide the Registry of Standard Biological Parts with a well characterized TIR library able to efficiently drive protein synthesis in a predictable way using a reduced number of translational factors.