Rapid cloning of random mutagenesis libraries using PTO-QuickStep

Jajesniak, P., Tee, K. L. and Wong, T. S. (2022) Rapid cloning of random mutagenesis libraries using PTO-QuickStep. In: Currin, A. and Swainston, N. (eds.) Directed Evolution: Methods and Protocols. Series: Methods in molecular biology (2461). Springer: New York, pp. 123-135. ISBN 9781071621516 (doi: 10.1007/978-1-0716-2152-3_8)

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.

• Item Type: Book Sections
• Additional Information: 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.
• Status: Published
• Glasgow Author(s) Enlighten ID: Jajesniak, Dr Pawel
• Authors: Jajesniak, P., Tee, K. L., and Wong, T. S.
• College/School: College of Science and Engineering > School of Engineering > Biomedical Engineering
• Publisher: Springer
• ISSN: 1064-3745
• ISBN: 9781071621516

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