Display of Functional Nucleic Acid Polymerase on Escherichia coli Surface and Its Application in Directed Polymerase Evolution

DNA polymerase-based techniques such as PCR and DNA sequencing have become indispensable for modern molecular biology. These techniques foster an expanding field of applications, including clinical diagnostics, drug screening, forensic research and archaeology. To tailor the properties of a polymerase for the desired application, various directed evolution strategies have been employed, such as phage display, compartmentalized self replication and primer extension assay. Here we report, to the best of our knowledge, the first high-throughput screening method for DNA polymerase based on cell surface display (CSD). CSD separates the polymerase from cytosolic materials which might interfere with the selection of the polymerase for desired activities. A DNA polymerase is displayed on Escherichia coli (E.coli) cell surface using an inactivated esterase autotransporter. The activity of the displayed polymerase is then detected by a dye that intercalates into elongated DNA and emits more fluorescence. Using this method, we evolved Klenow fragment of E. coli DNA polymerase I to incorporate NTP and C2'-OMe modified oligonucleotides. The CSD-based screening method provides a novel alternative for directed evolution of DNA polymerase.