Engineering Translation with a Novel Platform For Directed Evolution

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Jared Ellefson, Adam Meyer, Andrew Ellington

University of Texas at Austin, United States

An exciting avenue of synthetic biology is expanding the scope of life’s chemistry. Unnatural amino acids in proteins can drastically alter their functionality, enabling chemical reactions that would otherwise be impossible using the canonical genetic code. To achieve site directed incorporation of unnatural amino acids, aminoacyl tRNA synthetases are engineered that have a broadened substrate specificity and tRNAs are modified to read through amber (UAG) codons. Engineering these components can be challenging and improved methodologies are desirable. We developed a novel platform for the directed evolution of biomolecules called Compartmentalized Partnered Replication (CPR), which was used to evolve a tRNA synthetase pair. CPR is an emulsion-based selection system that couples the in vivo functioning of a gene to its in vitro amplification. Libraries of the Saccharomyces Cerevisiae Tryptophanyl tRNAs and cognate synthetases were generated and transformed into cells harboring a plasmid containing a Taq DNA polymerase gene with one or more amber codons in the open reading frame. The suppression of the amber codons in the Taq gene is essential for expressing full length Taq polymerase. The E. coli cells are emulsified along with buffers, dNTPs, and primers specific to the synthetase or tRNA genes. Upon thermal cycling, active synthetases are amplified due to their ability to make functional Taq polymerase. iterations of this process enrich for the most active variants in the population. By altering the binding pocket of the synthetase an unnatural amino acid, 5-HydroxyTryptophan can induce suppression of an amber codon. The cognate tRNA was also modified such that suppression of amber codons was increased by 6-fold over the starting construct. Since CPR is a generalizable platform for the evolution of biomolecules, future efforts will be directed towards expanding the repertoire of engineered biomolecules.