论文总字数：24954字

摘 要

脂多糖(lipopolysaccharide, LPS)，是分布于所有革兰氏阴性细菌细胞壁中的一种成分，一般由三部分组成，由内向外依次是疏水性的类脂A(lipid A)、核心寡糖和由重复性糖单元构成的O抗原(或称O多糖)。脂多糖抑制营养的进入，阻止产物的释放。降低这种障碍将可以提高整个细胞的代谢反应和工业微生物的产量。

野生型大肠杆菌渗透性较小，而经过敲除不同基因或表达某些基因可以使得脂多糖上的磷酸基团、脂肪酸链和多糖链结构发生改变，影响到大肠杆菌的渗透性。本实验即是通过基于λ噬菌体Red重组系统的同源重组技术使Escherichia coli BW25113的一些脂多糖相关基因发生失活，研究是否能够增强其细胞膜的通透性。

利用含有质粒pKD46 的菌株BW25113 ，在阿拉伯糖诱导后，表达λ噬菌体的3 个重组蛋白，宿主菌就具有了同源重组的能力。设计的引物5’端有~55 bp的拟敲除基因的同源臂，3’端为扩增引物，以pIJ773为模板，扩增两侧含FRT 位点的安普拉霉素(Apr)抗性基因，将此线性片段电转入具重组功能的感受态细胞，利用安普拉霉素平板就可以筛选到阳性转化体.

关键词：脂多糖 膜渗透性 Red重组

Changes on E.coli’s construction by Red Recombination

Abstract

Lipopolysaccharide (LPS), as the major component of outer membrane in most gram-negative bacteria, is significant to the permeability of outer membranes. LPS consists of three moieties: the hydrophobic lipid A, the core region and the O-antigen repeats. It retards the entry of substrate into the cell and prevents the product from being released. Reducing those barrier would accelerate the efficiency of whole-cell catalyzed reactions and the production yield of industrial microorganism.

According to the research named The effect of the structure of lipopolysaccharide on the permeability of Escherichia coli cell membranes did by State Key Laboratory of Food Science and Technology ,we can get that wild type E. coli showed the least permeability, while mutants in which LPS structures were changed by the deletion or expression of related genes showed higher permeability. The number of phosphate groups and the acyl chains, and the length of the polysaccharide of lipopolysaccharide all affect the permeability of E. coli. So based on this, our research is going to achieve changes in permeability of outer membranes through deletion of some genes in chromosome of E. Coli BW25113(PKD46) by Red Recombination.

BW25113 with pKD46 has the function of recombination when induced by L2arabinose. PCR products were obtained by using primers with 55bp extension which were homologous to the target genes and by using template plasmid named pij113 carrying chloramphenicol resistance gene flanked by FRT sites. The PCR products were introduced into BW25113 by electroporation. Using this system ,some specific genes in chromosome of Escherichia coli was deleted.

Key Words: Lipid A; Permeability of cell membranes; Red Recombination

目 录

摘要 I

ABSTRACT II

第一章 文献综述 1

1.1革兰氏阴性菌LPS 1

1.1.1 LPS的结构 1

1.1.2 LPS的生物功能 1

1.1.3 LPS的生物合成与结构修饰 2

1.2革兰氏阴性菌的外膜渗透性 3

1.2.1革兰氏阴性菌细胞外膜结构 3

1.2.2 改变细胞外膜渗透性的方法 4

1.2.3细胞外膜渗透性在工业生物技术中的意义 4

1.3 Red重组系统在基因敲除上的应用 5

1.3.1基因敲除 6

1.3.2 Red重组系统 6

1.4研究的内容和意义 7

1.4.1研究内容 7

1.4.2研究意义 8

第二章 利用Red重组系统改造大肠杆菌的脂多糖结构 9

2.1 实验材料 9

2.1.1 菌株与质粒 9

2.1.2 主要试剂 9

2.1.3 主要设备及仪器 10

2.1.4 培养基 11

2.2 实验方法 11

2.2.1 引物设计 11

2.2.2 载体质粒（pIJ773）DNA的提取 12

2.2.3 以质粒pIJ773为模板进行PCR 12

2.2.4 离心法胶回收 12

2.2.5 DpnI酶切去除模板 13

2.2.6 离心法胶回收 13

2.2.7 大肠杆菌E.coli BW25113感受态细胞的制备 13

2.2.8 pKD46热休克法转入E.coli BW25113 14

2.2.9 大肠杆菌E.coli BW25113/pKD46电转化感受态细胞的制备 14

2.2.10 胶回收PCR产物电转化入BW25113/pKD46电转感受态 14

2.2.11 筛选、测序 15

2.3 结果与讨论 17

2.3.1 结果 17

2.3.2 讨论 21

第三章 结论与展望 25

3.1 结论 25

3.2 展望 25

参考文献 24

致 谢 27

第一章 文献综述

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