Mutagenesis Protocols in Saccharomyces cerevisiae by In Vivo Overlap Extension
A high recombination frequency and its ease of manipulation has made Saccharomyces cerevisiae a unique model eukaryotic organism to study homologous recombination. Indeed, the well-developed recombination machinery in S. cerevisiae facilitates the construction of mutant libraries for directed evolution experiments. In this context, in vivo overlap extension (IVOE) is a particularly attractive protocol that takes advantage of the eukaryotic apparatus to carry out combinatorial saturation mutagenesis, site-directed recombination or site-directed mutagenesis, avoiding ligation steps and additional PCR reactions that are common to standard in vitro protocols.
Key wordsIVOE Saccharomyces cerevisiae Combinatorial saturation mutagenesis In vivo recombination Directed evolution
This work was supported by the Spanish Ministry of Science and Innovation (projects CCG08-CSIC/PPQ-3706; PIE 200880I033) and EU project FP7-NMP4-SL-2009-229255.
- 4.Zumárraga M, Domínguez CV, Camarero S, Shleev S, Polaina J, Martínez-Arias A, Ferrer M, de Lacey AL, Fernández V, Ballesteros A, Plou FJ, Alcalde M (2008) Combinatorial saturation mutagenesis of the Myceliophthora thermophila laccase T2 mutant: the connection between the C-terminal plug and the conserved 509VSG511 tripeptide. Comb Chem High Throughput Screen 11:807–816PubMedCrossRefGoogle Scholar
- 5.Arnold FH, Georgiou G (eds) (2003) Directed evolution: library creation, methods and protocols, vol 231. Humana Press, Totowa, NJGoogle Scholar
- 13.Okkels JS (2004) In vivo gene shuffling in yeast: a fast and easy method for directed evolution of enzymes. In: Svendsen A (ed) Enzyme functionality: design, engineering, and screening. Marcel Dekker, New York, pp 413–424Google Scholar
- 14.Arnold FH, Georgiou G (eds) (2003) Directed enzyme evolution: screening and selection methods, vol 230. Humana Press, Totowa, NJGoogle Scholar