优化易错PCR条件以提高毕赤酵母GAP启动子文库突变效率

生物技术通报 ›› 2014, Vol. 0 ›› Issue (6): 211-217.

优化易错PCR条件以提高毕赤酵母GAP启动子文库突变效率

秦秀林¹, 钱江潮², 储炬²

（1.广西大学生命科学与技术学院，南宁 530004；2.华东理工大学生物反应器工程国家重点实验室，上海 200237）

收稿日期:2014-02-22 出版日期:2014-06-25 发布日期:2014-06-25
作者简介:秦秀林，女，讲师，博士，研究方向：生物化学与分子生物学；E-mail：xiulinqin@gxu.edu.cn
基金资助:
广西自然科学基金项目（2013GXNSFBA019096）

High-error-rate Random Mutagenesis of GAP Promoter in Pichia pastoris Using an Optimited Error Prone PCR

Qin Xiulin¹,Qian Jiangchao²,Chu Ju²

（1. College of Life Science and Technology，Guangxi University，Nanning 530004；2. State Key Laboratory of Bioreactor Engineering，East China University of Science & Technology，Shanghai 200237）

Received:2014-02-22 Published:2014-06-25 Online:2014-06-25

摘要/Abstract

摘要： 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-prone PCR，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1 ng/μL、25和10 mmol/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。

关键词: 易错PCR, 突变效率, 随机突变, 毕赤酵母, GAP启动子

Abstract: The first important step toward a successful preparation of large and diverse promoter library with desired complexity, is to select a suitable mutagenesis strategy. To generate a promoter library of GAP promoter（pGAP）variants, mutations were introduced using error-prone PCR. After optimization of the conditions for EP-PCR random mutagenes, high mutation（error rate 1.1%）frequence was obtained using 1 ng/μL template and 10 mmol/L Mg²⁺, in combination with 25 thermal cycles. To increase mutational diversity and reach an appropriate error rate, three consecutive rounds of EP-PCR were carried out under the same conditions. After random sequencing of 10 clones from each round, an overall range of mutation rates from 1.1% to 4.0% was observed. Then, 250 clones containing pGAP variants were screened using the highthroughput screening approach in 48-deep-well plates. Among them, 5 mutants exhibited higher fluorescent intensity compared to the wild-type promoter.

Key words: Error-prone, PCR, Mutation frequency, Random mutagenesis, Pichia pastoris, GAP promoter

秦秀林, 钱江潮, 储炬. 优化易错PCR条件以提高毕赤酵母GAP启动子文库突变效率[J]. 生物技术通报, 2014, 0(6): 211-217.

Qin Xiulin,Qian Jiangchao,Chu Ju. High-error-rate Random Mutagenesis of GAP Promoter in Pichia pastoris Using an Optimited Error Prone PCR[J]. Biotechnology Bulletin, 2014, 0(6): 211-217.

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