文档介绍：学号:201024430127
HEBEI UNITED UNIVERSITY
毕业论文
GRADUATE THESIS
设计题目:大肠杆菌DH5α菌株pykA基因片段的敲除
学生姓名:沈广阔
专业班级:10生物技术1班
学院:冀唐学院
指导教师:卢育新李明
2014年05月06日
摘要
目的: DH5α菌株的pykA基因敲除,从而降低菌株在作为质粒宿主菌,生产发酵时的产酸量,提高质粒生产效率。方法:(1) DH5α菌株内, DH5α菌株。(2)以质粒pkD3为模板, PCR扩增两端带有与目的基因上下游同源片段的抗性基因cat。 DH5α(含pkD46)菌株,使cat基因替换并敲除pykA基因, DH5α(含cat)菌株。(3) DH5α(含cat)菌株,在FLP重组酶的作用下,消除cat抗性基因, DH5α菌株的pykA基因敲除。结果: DH5α菌株的pykA基因成功敲除。
关键词:Red重组;基因敲除;pykA基因; DH5α菌株;FLP 重组酶;
Abstract
Objective: To construct an strain DH5α of gene pykA -knocked out by using Red bination , which as the host strain could produce less acid and raise the yield of plasmid DNA after fermentation . Methods: (1)We transformed the plasmid pkD46 into DH5α by heat shock, then the positive strains with pkD46 was gotten by screening on the AMP(ampicillin) resistant plate. (2) Using plasmid pKD3 as a template,the cat-resistant gene flanked by homologues of pykA gene was amplified by PCR products were electro - transformed into DH5α strain with pKD46. With the help of the Red bination system,pykA gene was replaced with the cat-resistant positive strains with was obtained by screening on the cat (chloram phenicol) resistant plate.(3)The plasmid pCP20 was electro - transformed into the above DH5α strain with cat-resistant gene. Then the cat-resistant gene was eliminated by FLP-promoted bination system. Results: The pykA gene of DH5α strain pletely deleted.
Key words:Red b inant system; gene knockout; pykA gene; DH5α; FLP binase;
目录
摘要 I
Abstract II
第1章前言 1
1
1
3
6
7
第2章材料与方法 8
8
8
仪器设备 8
方法 9
DH5α 9
12
DH5α(含cat) 16
第3章结果 18
质粒pkD46及酶切凝胶电泳检测结果 18
质粒pkD3凝胶检测结果 19
PCR扩增抗性基因片段,凝胶电泳检测结果 20
P