Mutant library

The BFD mutant library generated by epPCR was expressed in microtiter plates and 8000 clones were subjected to the carboligation assay with benzaldehyde 4 and dimethoxyacetaldehyde 8 as the substrates. The reaction was incubated for 24 h at 30 °C as suggested from experiments using the positive control strain... 

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. 

Generation of Mutant Libraries Using Random Mutagenesis... 

Lewenza, S., Falsafi, R.K., Winsor, G et al. (2005) Construction of a mini-Tn5-luxCDABE mutant library in Pseudomonas aeruginosa PAOl a tool for identifying differentially regulated genes. Genome Res. 15, 583-589. 

Fig. 4.3.1. Schemes for genetic selection of DNA polymerase function based on complementation, (a) Host cells of E. coli recA718po/A12 that encodes a temperature-sensitive variant of DNA polymerase I (Pollts) are transformed with a polymerase mutant library. Active polymerase mutants substitute for DNA polymerase I at the non-permissive temperature (37 °C). (b) The host strain E. coli...

Functional tolerance is a significant factor for the success of directed evoluiton. A protein that is functionally tolerant allows many mutations without disrupting the fitness, making it more likely that there is a connected path in sequence space of single mutations that leads to regions of higher fitness. Tolerance also affects the quality of the mutant library. If the protein is functionally intolerant, the mutant library will consist of mostly inactive proteins. 

The parameters to be optimized for experimental recombination include the crossover frequency, the number of parents, and the sequence similarity between parents. Additionally, it is important to understand the conditions under which recombination is useful. For all these questions, the optimal parameters represent a balance between the exploration and exploitation capabilities of the search algorithm. Any process that creates more diversity, such as using many parents, very disparate parents, or small fragment sizes, will improve exploration at the cost of exploitation. The balance between these effects will shift according to the landscape ruggedness and the sampling ability of the mutant library. 

Macken and Perelson studied antibody affinity maturation as a random walk on the random energy landscape (Macken and Perelson, 1989 Macken et al., 1991 Macken and Perelson, 1991). The total number of mutants tried before a positive mutation is discovered T(F) is a measure of the change in the necessary size of the mutant library. The expected value of T, given that F is not a local optimum, is derived as... 

For the inner region 0 < 1, Eq. (23) reduces to E[T(F)] 1/(1 — G(F)). The total number of mutants tried increases exponentially as the sequence approaches the global optimum (Fig. 12). Outside of the boundary, the mean number of trials approaches the asymptote of 0.78D, indicating that the required mutant library size is on the order of the dimensionality of the landscape (Macken et al., 1991). 

The optimal size of the mutant library is a parameter that has immediate physical meaning. A more challenging parameter to define is the quality of the library. For example, quality can be broadly interpreted as the structural robustness or the fraction of positive mutants. The short-term goal is to produce a library that maximizes the probability of finding fitter mutants and the long-term goal is to maximize the total fitness improvement after multiple generations. Research in statistical mechanics and information theory has introduced methods that can be used to quantify the quality of a mutant library. 

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