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敲除莽草酸转运蛋白编码基因shiA对莽草酸积累影响

作　者: 库玛娜（NDIKUMANA Yvonne）
导　师: 张惠展
学　校: 华东理工大学
专　业: 生物化学与分子生物学
关键词: Shikimic acid ShiA gene knockout Shikimic acid accumulation E. coli over-expression
分类号: Q943.2
类　型: 硕士论文
年　份: 2010年
下　载: 67次
引　用: 0次
阅　读: 论文下载

内容摘要

Shikimic acid is one of the most important chiral compounds synthesized in the aromatic amino acid pathway of plants and microorganisms. It is mostly used to produce Tamiflu to treat avian flu infections. In E. coli, shiA gene is responsible for shikimic acid uptake from the culture media. In this work, shA gene has been knocked out in E. coli JM83 and JDL02 to develop new strains of E. coli JMV5 and JMC7 which can allow much more accumulation of shikimic acid in their culture supernatants. We also amplified the expression of aroG and tktA gene so as to improve the accumulation level. The assay of shikimic acid by spectrophotometric method indicated that knocked out mutants accumulated two times lower shikimic acid than wild types with an intact shA gene. The expression of aroG and tktA showed that optimization of shikimic acid accumulation can be achieved by combining gene knockout and over-expression approaches. Among the knocked out strains; namely JMV5 and JMC7, JMC7 accumulated two times more shikimic acid than the JMV5. It is believed that the high level of accumulation in JMC7 strain results from the deletion of aroL gene into the strain. The supplement of shikimic acid into culture medium elucidated that shiA gene was completely knocked out. The entire shikimic acid did not cross the membrane to enter the cell. Moreover, it was a significant reduction of shikimic acid flow from inside the cell to the culture medium into shiA gene knocked out mutant. These results suppose that shiA gene may act in both directions because its disruption prevented the flow of shikimic acid to culture medium, meaning that shiA gene did not improve the accumulation into culture medium. In this study, it was not possible to detect the in vivo shikimic acid due to too low accumulation.

全文目录

Abstract  7-12
Chapter 1:Introduction  12-21
  1.1 Literature review  13-21
    1.1.1 Shikimic acid pathway  13-14
    1.1.2 Shikimic acid  14-15
    1.1.4 Thermosensitive plasmid methods  15-17
    1.1.5 PCR technique  17-18
    1.1.6. Description of shiA gene  18-19
    1.1.7 Meaning of the study  19-20
    1.1.8 Objectives of this study  20-21
Chapter 2:Materials and Methods  21-35
  2.1 Materials  21-27
    2.1.1 Chemical and Biological reagents  21-25
    2.1.2 Instruments and equipments  25-27
  2.2 Methods  27-35
    2.2.1 Bacteriological techniques  27-28
      2.2.1.1 Bacterial growth conditions  27-28
      2.2.1.2 Presevration of Culture  28
    2.2.2 Genetics Methods  28-30
    2.2.3 Molecular biology techniques  30-33
    2.2.4 Spectrophotometric methods  33
    2.2.5 Software used  33-35
Chapter 3:Results  35-52
  3.1 Construction of shiA gene knocked out mutants of E. coli JM83 and JDL02  35-47
    3.1.1 PCR amplification of shiA (f) and shiA (b)  36-38
    3.1.2 Cloning of PCR products  38-39
    3.1.3 Construction of pMAK-shiA (f)-shiA (b) plasmid  39-40
    3.1.4 Cointegration of Plasmid into chromosome  40-43
    3.1.5 Plasmid excision from chromosome  43-44
    3.1.6 Elimination of thermosensitive plasmid  44-46
    3.1.7 Sequencing  46-47
  3.2 Analysis of Shikimic acid accumulation  47-52
    3.2.1 Standard curve of Shikimic acid  47-49
    3.2.2 Validation of spectrophotometric method  49
    3.2.3 Shikimic acid assay  49-50
    3.2.4 Optimization of shikimie acid accumulation  50
    3.2.5 Shikimic acid transport  50-52
Chapter4:Discussion  52-55
  4.1 Gene targeting  52
  4.2 Deletion of shiA gene on E. coli chromosome  52-53
  4.3 Use of expression plasmid  53-54
  4.4 Transport of shikimic acid in new strains  54-55
Conclusion  55-56
References  56-60
ACKNOWLEDGMENTS  60-61

敲除莽草酸转运蛋白编码基因shiA对莽草酸积累影响

内容摘要

全文目录

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