GoldenBraid酶切连接反应体系的优化  

Optimization of GoldenBraid Digestion-ligation Reaction System

作　　者：徐美慧 张耀杰 唐克轩[1,2] 苗志奇 XU Mei-hui;ZHANG Yao-jie;TANG Ke-xuan;MIAO Zhi-qi(College of Agriculture and Biology,Shanghai Jiao Tong University,Shanghai 200240;不详)

机构地区：[1]上海交通大学农业与生物学院,上海200240 [2]不详

出　　处：《生物技术通报》2020年第9期266-274,共9页Biotechnology Bulletin

基　　金：国家重点研发计划(2018YFA0900600)。

摘　　要：旨在获得更为高效稳定的GoldenBraid(GB)酶切连接组装反应体系。通过优化GB反应中酶切时间、缓冲体系、限制性内切酶和底物配伍来提高有效克隆的效率,确定最佳反应体系。结果显示,4 min酶切时间,1μL FastDigest Buffer(10×)缓冲体系,1 mmol/L ATP,0.5μL FastDigest Esp3I或FastDigest Eco31I,元件供体载体与受体载体的摩尔比3∶1时,有效克隆比例接近99%,是优化前(BsmBI 8%、BsaI 23%)的4倍以上,此时酶切连接效率接近100%。与原始方案相比,通过体系的优化,可以在更少内切酶用量(原方案为1μL)的情况下,显著提高有效克隆比例,表明本方法能提高GB系统的酶切连接效率和实用性。The objective of this work is to establish an efficient and stable GoldenBraid(GB)restriction-ligation assembly reaction system.In this study,enzyme digestion time,buffer system,restriction enzymes and ratio of substrates were optimized to improve the efficiency of effective cloning,and the optimal reaction system was determined.Results showed that the percentage of effective cloning was nearly 99%under 4 min enzyme digestion time,1μL FastDigest Buffer(10×)buffer system,1 mmol/L ATP,0.5μL FastDigest Esp3I or FastDigest Eco31I,and 3∶1 molar ratio of the inserts and acceptor plasmid in reaction system,which was over 4 times of that before optimization(BsmBI 8%and BsaI 23%),and digestion-ligation efficiency was nearly 100%.The percentage of effective cloning significantly increased by optimizing reaction system even with less volume of restriction enzymes,compared to the original protocol with 1μL restriction enzymes.This indicates that the efficiency and practicability of digestion-ligation in GB system may be improved via the optimized process.

关 键 词：GoldenBraid 优化 有效克隆 酶切连接效率 

分 类 号：Q789[生物学—分子生物学]