Engineering Transcription with a Novel Platform For Directed Evolution

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Adam Meyer, Jared W Ellefson, Andrew D Ellington

University of Texas, United States

The controlled transcription of genes and functional RNAs is important for the current generation of synthetic biology. In general, T7 RNA polymerase (T7 RNAP) has proven to be a workhorse enzyme for in vivo gene expression, and is also of great utility for in vitro RNA production in molecular biology and molecular diagnostics. We have endeavored to engineer T7 RNAP functionality using a novel platform for directed evolution termed Compartmentalized Partnered Replication (CPR). CPR is an emulsion-based selection system that couples the in vivo function of a gene to its in vitro amplification. In our initial efforts, a library of T7 RNAP variants was transformed into E. coli containing a Taq DNA polymerase (Taq DNAP) gene under the control of a T7 RNA polymerase promoter. After the induction of expression, cells containing the most active T7 RNAP genes also contained high levels of Taq DNAP protein. Cells were then compartmentalized in a water-in-oil emulsion containing buffer, dNTPs, and primers specific to the T7 RNAP gene. Emulsification was performed such that (on average) each cell was isolated in its own compartment. Upon thermal cycling, the most active T7 RNAP genes, which are compartmentalized with the most Taq DNAP protein, are preferentially amplified. Successfully amplified T7 RNAP genes were recovered and used to seed subsequent rounds of selection. Iterative rounds of selection and amplification were found to yield highly active T7 RNAP variants. When the promoter driving the Taq DNAP was altered, new promoter-specific variants of T7 RNAP were selected, including a variety of new orthogonal variants with high activity and specificity. Ongoing work includes expanding the nucleotide specificity of T7 RNAP as well as applying the CPR methodology to the evolution of other proteins and nucleic acids, in particular tRNA synthetase:tRNA pairs.