Abstract
PTO-QuickStep is a quick and easy molecular cloning technique that allows seamless point integration of a DNA fragment, encoding either a tag or a protein, into any position within a target plasmid. The entire process is conducted in a time-efficient and cost-effective manner, without the need of DNA gel purification and enzymatic restriction and ligation. PTO-QuickStep further innovates protein engineering by providing the possibility of integrating a random mutagenesis step (e.g., error-prone PCR) into the workflow, without compromising the time duration required. Random mutagenesis libraries can be quickly and efficiently cloned into a plasmid of interest, thereby accelerating directed evolution. On top of that, PTO-QuickStep can be utilized for rapid integration of noncoding DNA fragments to modify existing plasmids, making it an excellent tool for synthetic biologists.
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Acknowledgments
We thank the Department of Chemical and Biological Engineering, ChELSI, EPSRC (EP/E036252/1), BBSRC (BB/R020183/1), RAEng|The Leverhulme Trust Senior Research Fellowship (LTSRF1819\15\21; to TSW), the University of Sheffield GCRF Fellowship (to KLT), and the University of Sheffield PhD scholarship (to PJ) for financial support.
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Jajesniak, P., Tee, K.L., Wong, T.S. (2022). Rapid Cloning of Random Mutagenesis Libraries Using PTO-QuickStep. In: Currin, A., Swainston, N. (eds) Directed Evolution. Methods in Molecular Biology, vol 2461. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2152-3_8
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DOI: https://doi.org/10.1007/978-1-0716-2152-3_8
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