Generation of Large Libraries of Random Mutants in Bacillus subtilis by PCR-Based Plasmid Multimerization

Abstract

We describe a PCR-based method for the generation of plasmid multimers that can be directly transformed into Bacillus subtilis with very high efficiency. This technique is particularly useful for the generation of large libraries of randomly mutagenized genes, which are required for the optimization of enzymes by directed evolution. We subjected the gene coding for the protease subtilisin to six consecutive rounds of PCR at three different levels of mutagenicity. The resulting 18 populations were cloned using our PCR multimerization protocol, and the mutation frequencies were determined by DNA sequencing. The resulting data demonstrate that the mutation frequency during PCR can be controlled by adding varying concentrations of manganese chloride to the reaction mixture. We observed a bias in the type of base pair changes with A and T being mutated much more frequently than C and G. We determined the fraction of active clones in all populations and found that its natural logarithm is proportional to the average mutation frequency of the populations. These data reveal that a fraction of about 0.27 of all possible mutations leads to the inactivation of the subtilisin gene, which provides a measure for its structural plasticity.

Strategies for the in vitro evolution of protein function: enzyme evolution by random recombination of improved sequences 1 1Edited by J. Wells

“…Four of the 12 new point mutations identi®ed in the ®fth and sixth generation variants, for example, are G 3 C (and C 3 G) and G 3 T (and C 3 A) transversions, which were not found at all during the ®rst four generations of pNB esterase evolution involving PCR mutagenesis (Moore & Arnold, 1996). These mutations were also generated very rarely during the error-prone PCR mutagenesis of subtilisin (Sha®khani et al, 1997). DNA shuf¯ing and errorprone PCR together may provide access to a wider range of amino acid substitutions.…”

The optimal burst of mutation to create a phenotype

“…Survival curves are thus a function of base changes, and a lethal fraction is estimated as a proportion of total base changes. Protein lethal fractions are heterogeneous, commonly near 0.3 (subtilisin, DNA glycosylase, lac repressor; Guo et al, 2004;Shafikhani et al, 1997), but as high as 0.6 (for an antibody, Daugherty et al, 2000;Drummond et al, 2005) and with lows of 0.12 and 0.03 (for T4 lysozyme and an RNAse; calculated by Guo et al, 2004, from other studies). (These lethal fractions have been converted into per nucleotide change, whereas the lethal fractions for proteins are more commonly given per amino acid change.)…”

“…A fraction L of all mutations are lethal, and a genome dies if it receives one or more lethal mutations. Thus, (1b) In this manifestation of survival, the surviving fraction of the population is the Poisson fraction that escapes lethal mutations (Shafikhani et al, 1997;Bershtein et al, 2006;Bloom et al, 2007). Log-linear survival across changes in U ̅ requires that L be constant.…”

“…There has been far less attention to the evolutionary consequences of mutation pulses, perhaps because they seem unnatural. Nonetheless, mutation pulses are used in industrial attempts to create novel molecules (Shafikhani et al, 1997;Drummond et al, 2005;Rowe et al, 2003), and they may also operate in nature, as when a cell induces an error prone DNA polymerase for repair (Tompkins et al, 2003) or during biofilm progression (Boles et al, 2004).…”

“…Viral survival is measured as the ability to produce a plaque or some other visible disturbance that stems from a single infecting virus. More recent studies have assayed retention of function in proteins as a function of numbers of mutations; strict exponential decay with increasing numbers of mutations is not typically observed Daugherty et al, 2000;Shafikhani et al, 1997).…”