Recombination in-vitro

It is possible to recombine any number of parent genes with the available methods, which raises the question of what is the optimal number. Similar to determining the optimal mutation rate for random mutagenesis, the answer will depend on the number of screened mutants and the additivity of the combined mutations. It could be advantageous to screen all the permutations of mutations from the parents. Assuming independent and additive recombination, the probability Pd that an offspring has d mutations is given by 

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Stemmer, W.P.C. DNA shuffling by random fragmentation and reassembly In vitro recombination for molecular evolution. Proc. Natl. Acad. Sci. USA 91 10747-10751, 1994. 

ZHAO H, GIVER L, SHAO z, AFFHOLTER J A and ARNOLD F H (1998) Molecular evolutiou by staggered extension process (StEP) in vitro recombination . Nature Biotech, 18, 258-61. 

Shao, Z., Zhao, H., Giver, L. and Arnold, F.H. (1998) Random-priming in vitro recombination an effective tool for directed evolution. Nucleic Acids Research, 26, 681-683. 

Key words In vitro recombination, PCR, cDNA library, High throughput, GST, MBP... 

Fig. 1. Outline of the strategy to construct GST-fused expression plasmids by the in vitro recombination-assisted method. Am, Gm, and Cm are abbreviations for ampicillin-, gentamicin-, and chloramphenicol-resistance, respectively. The figure also indicates the ccdB gene encoding a toxin targeting the co//essential DNA gyrase and the phage X recombination sites (attB, attP, attL, and attR).

PCR and in vitro recombination reactions are quite simple and straightforward for generating multiple expression plasmids in parallel, e.g., in a 96-well plate see Fig. 3a). The first preliminary expression experiment was done to evaluate the production level of each GST-fused protein. In this step, we compared the staining patterns of E. coli proteins harboring expression plasmids with the patterns of proteins harboring empty vectors on sodium dodecyle sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under the same culture conditions see Fig. 3b). In addition to... 

Transfer PCR products into expression vector using in vitro recombination reactions in a 96-weii piate... 

The sequence of chapters mirrors the steps in a standard directed-evolution experiment. In the beginning, various methods for the creation of molecular diversity are considered. S. Brakmann and B.F. Lindemann (Chapter 2) present protocols for the generation of mutant libraries by random mutagenesis. Two chapters deal with the particularly powerful approach of in-vitro recombination. H. Suenaga, M. Goto, and K. Furukawa (Chapter 3) describe the application of DNA shuffling, and M. Ninkovic (Chapter 4) presents DNA recombination by the S tEP method. 

The efficiency of S tEP recombination is similar to that of other in vitro recombination methods, but an advantage of this method lies in the fact that the reaction can be carried out in a single test tube. Template DNA can be double-stranded as well as single-stranded, and there is no significant restriction to the number of parent templates to be recombined. 

Ninkovic, M., R. Dietrich, G. Aral and A. Schwienhorst 2001. High fidelity in vitro recombination using a proofreading polymerase. BioTechniques 30 530-536. 

Lieska, N., Yang, H.-Y., and Goldman, R. D. (1985). Purification of the 300K intermediate filament-associated protein and its in vitro recombination with intermediate filaments./. Biol. Chem. 101, 802-813. 

Balaguer, P., A. Joyeux, M.S. Denison, R. Vincent, B.E. Gillesby, and T. Zacharewski (1996). Assessing the estrogenic and dioxinlike activities of chemicals and complex mixtures using in vitro recombinant reporter gene assays. Can. J. Physiol. Pharmacol., 74 216-222. 

During the evolution of S41, selective pressure was applied to both stability and activity. Stabilized mutants were accepted only if they showed no or little decrease in activity. Wintrode et al. (2000) adopted a different strategy for the evolution of SSII s activity at low temperature. Random mutant libraries of SSII were prepared either by random mutagenesis or in vitro recombination and screened for catalytic activity at 10°C. Libraries were not screened for thermostability, and mutants that showed improved activity at 10°C were selected regardless of changes, positive or negative, in stability. This experiment addressed the question of what happens to thermostability when no selective pressure is applied. 

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