Constructing a Library of Stable Mutations for Enhanced Bioethanol Production by Saccharomyces Cerevisiae Using Various Molecular Biology Methods
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- Saccharomyces Cerevisiae, SPT15, Error Prone PCR, Manganese Chloride, Random Mutants, Gtme
- El-Rotail, Ashraf; Shia, Gui Yang; Rashed, Marwan M. A.; Al-Farga, Ammar; Mousa, Ahmed; Bakry, Amr M.; Korma, Sameh A.; Linghuana, Zhu; Zhia, Gao
- The aim of this study was to investigate theeffects of MnCl2 concentration instead of MgCl2 to construct libraries of mutant from Saccharomyces cerevisiae with error-Prone PCR protocol to prepare a novel random mutagenesis characterized by a high mutation rate and stable better than wild-type strain. In our work, the genetic strategies were utilized to construct libraries from SPT15 and TAF23 mutant genes. Those genes are basically existed in the haploid (MAT-a [CICC 1374]) and (MAT-α [CICC 31144]) of S. cerevisiae. The diploid mutants were generated after the mating process. Through our novel technique and integration gTME and Error Prone PCR protocol, differentconcentrations of MnCl2 instead of MgCl2 were used to adjust the technique.The results showed an average 12-fold increasing for mutation rate when addition different concentrations of MnCl2 instead of MgCl2 with using Error prone PCR reaction and traditional enzymes such as rTaq. In addition to, Mutants recorded the highest stability level by using 3 % MnCl2. In conclusions, there have been many previous studies presented methods to get mutations by using PCR Random Mutagenesis Kit for commercial companies, while in this study we adopted the construction of a new protocol using a combination of items to get random mutagenesis-Super; which were characterized by a high mutation rate, and simultaneously be stable better than wildtype strain.
Full text: IJRAS_547_FINAL.pdf