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Controllable yeast mutagenic strains build its cut in the T. reesei cellulase EG III in vivo directed evolution

Author: SunJinTang
Tutor: QuYinBo；ZhuangGuoQiang
School: Shandong University
Course: Microbiology
Keywords: mutator strain in vivo directed evolution reversion mutation EndoglucanaseIII
CLC: Q933
Type: Master's thesis
Year: 2006
Downloads: 226
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Abstract


Nowadays the application of mutator strain is a in vivo method in molecular biology for directed evolution of proteins. The construction of most mutator strain is based on knocking out the key gene of the enzyme which plays an important role in DNA replication or repair, then the process of DNA replication is effected , so that its correctness and fidelity decrease and more random mutagenesis than wild type strain occur . After selective screening we can get the mutator expected. That’ s the essence principle of mutator strain as useful tool for directed evolution in vivo.The research of prokaryote mutator strain has been deepgoing and several kinds of mutator strain are produced as commodity, for example Stratagene has developed a mutator strain, called XLl-Red Competent cell, which is deficient in three of the primary DNA repair pathways. MutS, 3’-to 5’-exonuclease of DNA polymerase III and the enzyme to hydrolyze 8-oxodGTP were knocked out in the XLl-Red strain. The random mutation rate in this triple mutant strain was measured to be 5000-fold higher than that of wildtype. This strain is particularly suitable for generating random mutations within a target gene and the method does not require extensive genetic or biochemical manipulations.E. coli XL1 red, as a prokaryote mutator strain, is applicable maturely in in vivo directed evolution of proteins. But because of its prokaryote background, it is inadequate for the expression of eukaryote proteins, and it’ s difficult to control the mutation rate to a proper level according to our need . To overcome these drawbacks in former mutator strain , we construct three mew mutator strain in Saccharomyces cerevisiae RDKY3615(ura3-52, trp1△63, leu2△1, his3△200) by knocking out its sodl,cttl and srxl genes which act in preventing oxidative damage. The

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CLC: > Biological Sciences > Microbiology > Microbial Genetics
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