Abstract
A method is described for using 96-well plates to prepare libraries of Escherichia coli cultures for screening a library of gene variants. This approach bypasses colony-picking to allow standard molecular biology laboratories to carry out directed evolution efficiently with a 96-well plate-reader and multichannel pipettes. Initial screens are applied to cultures that are rapidly prepared by diluting transformed cells so that an average of four cells starts each culture. Subsequent screens are used to isolate individual enzyme-expressing clones that exhibit activity higher than the parental clone. The outlined method also includes guidelines for preparing a library of gene variants and for optimizing a screening method.
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References
Stevenson BJ, Liu JW, Ollis DL (2008) Directed evolution of yeast pyruvate decarboxylase 1 for attenuated regulation and increased stability. Biochemistry 47:3013–3025
Yip SH-C, Foo J-L, Schenk G, Gahan LR, Carr PD, Ollis DL (2011) Directed evolution combined with rational design increases activity of GpdQ toward a non-physiological substrate and alters the oligomeric structure of the enzyme. Protein Eng Des Sel 24:861–872
Studier FW, Rosenberg AH, Dunn JJ, Dubendorff JW (1990) Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol 185:60–89
Leung DW, Chen E, Goeddel DV (1989) A method for random mutagenesis of a defined DNA fragment using a modified polymerase chain reaction. Technique 1:11–15
Zhao H, Giver L, Shao Z, Affholter JA, Arnold FH (1998) Molecular evolution by staggered extension process (StEP) in vitro recombination. Nat Biotechnol 16:258–261
Salazar O, Sun L (2003) Evaluating a screen and analysis of mutant libraries. In: Arnold FH, Georgiou G (eds) Directed enzyme evolution: screening and selection methods. Humana Press, Totowa, NJ, p 383
Sambrook J, Russell DW (2001) Molecular cloning. A laboratory manual, 3rd edn. Cold Spring Harbor Laboratory Press, New York, p 13.19
Neylon C, Brown SE, Kralicek AV, Miles CS, Love CA, Dixon NE (2000) Interaction of the Escherichia coli replication terminator protein (Tus) with DNA: a model derived from DNA-binding studies of mutant proteins by surface plasmon resonance. Biochemistry 39:11989–Â11999
Studier FW (2005) Protein production by auto-induction in high-density shaking cultures. Protein Expr Purif 41:207–234
Khlebnikov A, Keasling JD (2002) Effect of lacY expression on homogeneity of induction from the Ptac and Ptrc promoter by natural and synthetic inducers. Biotechnol Prog 18:672–674
Stevenson BJ, Liu JW, Kuchel PW, Ollis DL (2012) Fermentative glycolysis with purified Escherichia coli enzymes for in vitro ATP production and evaluating an engineered enzyme. J Biotechnol 157:113–123
Moore JC, Arnold FH (1996) Directed evolution of a para-nitrobenzyl esterase for aqueous-organic solvents. Nat Biotechnol 14:458–467
Acknowledgements
This work was supported by the Australian Research Council. We thank Nicholas Dixon for providing pETMCSIII and the E. coli strains.
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Stevenson, B.J., Yip, S.HC., Ollis, D.L. (2013). In Vitro Directed Evolution of Enzymes Expressed by E. coli in Microtiter Plates. In: Samuelson, J. (eds) Enzyme Engineering. Methods in Molecular Biology, vol 978. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-293-3_18
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DOI: https://doi.org/10.1007/978-1-62703-293-3_18
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Publisher Name: Humana Press, Totowa, NJ
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