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优化易错PCR条件以提高毕赤酵母GAP启动子文库突变效率

, PP. 211-217

Keywords: 易错PCR,突变效率,随机突变,毕赤酵母,GAP启动子

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Abstract:

高效的随机突变策略对于构建含有丰富突变体的启动子文库至关重要。为了建立一个突变率适中并能获得较多有益突变子的易错PCR(error-pronePCR,EP-PCR)条件,实现毕赤酵母GAP启动子的高效突变,对EP-PCR反应条件进行了优化。考察了模板浓度、反应循环数和Mg2+浓度对EP-PCR的产物得率和突变率的影响后,确定了适于GAP启动子突变的EP-PCR条件:模板浓度、反应循环数和Mg2+浓度分别为1ng/μL、25和10mmol/L。优化EP-PCR条件后,GAP启动子突变率为1.1%,连续进行3轮EP-PCR后突变率可达到4.0%。利用优化后EP-PCR对GAP启动子进行随机突变,筛选了250个突变子,获得5个启动子强度高于野生型GAP启动子的突变体,有益突变达到了2%,可用于构建GAP启动子文库。

References

[1]  Braatsch S, Helmark S, Kranz H, et al. Escherichia coli strains with promoter libraries constructed by Red/ET recombination pave the way for transcriptional fine-tuning[J] . Biotechniques, 2008, 45(3):335-337.
[2]  Siegl T, Tokovenko B, Myronovskyi M, et al. Design, construction and characterisation of a synthetic promoter library for fine-tuned gene expression in actinomycetes[J] . Metabolic Engineering, 2013, 19:98-106.
[3]  Rytter JV, Helmark S, Chen J, et al. Synthetic promoter libraries for Corynebacterium glutamicum[J] . Applied Microbiology?and?Biotechnology, 2014, Published online.
[4]  Alper H, Fischer C, Nevoigt E, et al. Tuning genetic control through promoter engineering[J] . Proceedings of the National Academy of Sciences of the United States of America, 2005, 102(36):12678-12683.
[5]  Cox RS 3rd, Surette MG, Elowitz MB. Programming gene expression with combinatorial promoters[J] . Molecular?Systems Biology, 2007, 3:145-156.
[6]  Murphy KF, Balazsi G, Collins JJ. Combinatorial promoter design for engineering noisy gene expression[J] . Proceedings of the National Academy of Sciences of the United States of America, 2007, 104(31):12726-12731.
[7]  Ellis T, Wang X, Collins JJ. Diversity-based, model-guided construction of synthetic gene networks with predicted functions[J] . Nature Biotechnology, 2009, 27(5):465-471.
[8]  Nevoigt E, Fischer C, Mucha O, et al.Engineering promoter regulation[J] . Biotechnology and Bioengineering, 2007, 96(3):550-558.
[9]  Drummond DA, Iverson BL, Georgiou G, et al. Why high-error-rate random mutagenesis libraries are enriched in functional and impro-ved proteins[J] . Journal of Molecular Biology, 2005, 350(4):806-816.
[10]  Kunichika K, Hashimoto Y, Imoto T. Robustness of hen lysozyme monitored by random mutations[J] . Protein Engineering, 2002, 15(10):805-809.
[11]  Qin X, Qian J, Xiao C, et al. Reliable high-throughput approach for screening of engineered constitutive promoters in the yeast Pichia pastoris[J] . Letters?in?Applied Microbiology, 2011, 52(6):634-641.
[12]  Klein-Marcuschamer D, Stephanopoulos G. Assessing the potential of mutational strategies to elicit new phenotypes in industrial strains[J] . Proceedings of the National Academy of Sciences of the United States of America, 2008, 105(7):2319-2324.
[13]  Cadwell RC, Joyce GF. Randomization of genes by PCR mutagenesis[J] . PCR Methods?& Applications, 1992, 2(1):28-33.
[14]  Leung DW, Chen E, Goeddel DV. A method for random mutagene-sis of a defined DNA segment using a modified polymerase chain reaction[J] . Technique, 1989, 1:11-15.
[15]  Chusacultanachai S, Yuthavong Y. Random mutagenesis strategies for construction of large and diverse clone libraries of mutated DNA fragments[J] . Methods in Molecular Biology, 2004, 270:319-334.
[16]  Wilson DS, Keefe AD. Random mutagenesis by PCR. Current Protocols?in Molecular Biology, 2001, 51:8.3:8.3.1-8.3.9.
[17]  Balci H, Ozturk MT, Pijning T, et al. Improved activity and pH stability of E. coli ATCC 11105 penicillin acylase by error-prone PCR[J] . Applied Microbiology?and?Biotechnology, 2014, 98 (10):4467-4477.
[18]  Akbari M, Hansen MD, Halgunset J, et al. Low copy number DNA template can render polymerase chain reaction error prone in a sequence-dependent manner[J] . The Journal of Molecular Diagnostics, 2005, 7(1):36-39.
[19]  Trollope KM, Nieuwoudt HH, G?rgens JF, et al. Screening a random mutagenesis library of a fungal beta-fructofuranosidase using FT-MIR ATR spectroscopy and multivariate analysis[J] . Applied Microbiology?and?Biotechnology, 2014, 98(9):4063-4073.
[20]  McCullum EO, Williams BA, Zhang J, et al. Random mutagenesis by error-prone PCR[J] . Methods Mol Biol, 2010, 634:103-109.
[21]  Shafikhani S, Siegel RA, Ferrari E, et al. Generation of large libraries of random mutants in Bacillus subtilis by PCR-based plasmid multimerization[J] . Biotechniques, 1997, 23(2):304-310.

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