Analysis and application of hyperthermophilic genes using the host-vector system of Thermus thermophilus
嗜热栖热菌宿主载体系统的超嗜热基因分析及应用
基本信息
- 批准号:12460038
- 负责人:
- 金额:$ 8.51万
- 依托单位:
- 依托单位国家:日本
- 项目类别:Grant-in-Aid for Scientific Research (B)
- 财政年份:2000
- 资助国家:日本
- 起止时间:2000 至 2002
- 项目状态:已结题
- 来源:
- 关键词:
项目摘要
I have succeeded in expressing several P. horikoshii genes in T. thermophilus HB27 efficiently by replacing promoter sequences of the expression vector. The use of Pslp promoter was most effective. By using Pslp promoter, three OT3 genes were expressed at the higher levels than those expressed in E. coli. In particular, the mannnosidase gene, which was not expressed at all in E. coli, was clearly expressed in T. thermophilus.In order to improve the expression levels of foreign genes, I tried to develop a high copy expression vector. The entire nucleotide sequences of the expression vector and its high-copy derivative were determined, and the sequences were compared. It was elucidated that integration of the fragment derived from host genome into the upstream region of a putative replication gene of the vector increased the copy number. I have constructed a new high copy expression vector, pET-Pslpm, as a shuttle vector available in both T. thermophilus and E. coli.I have already shown that integration of foreign DNA fragments into T. thermophilus genome could be carried out efficiently. But it was necessary to methylate the donor DNA prior to introduce into T. thermophilus. I have cloned the methylase gene (MTthHB27) from T. thermophilus HB27. The cloned MTthHB27 gene was introduced into E. coli expression system. Heat-treated cell-free extract prepared from E. coli recombinant showed methylation activity.Genes for restriction enzyme and modification enzyme are known to exist in tandem in various microorganisms. However, there was no possible restriction enzyme gene in the region nearby the MTthHB27. MTthHB8 and RTthHB8 genes were reported to be existing in tandem on HB8 genome. But their codon usage was far from Thermus-type. Thus, it was speculated that the restriction-modification system of HB8 was horizontally transferred from other organisms, and that HB27 has original Thermus-type one.
我已经成功地表达了几个P. horikoshii基因在T。通过替换表达载体的启动子序列,高效地表达嗜热菌HB 27。Pslp启动子的使用是最有效的。通过使用Pslp启动子,三个OT 3基因的表达水平高于在E.杆菌特别是甘露糖苷酶基因,在大肠杆菌中完全不表达。coli中表达,在T.为了提高外源基因的表达水平,我尝试开发高拷贝表达载体。测定表达载体及其高拷贝衍生物的全部核苷酸序列,并比较序列。据阐明,将来自宿主基因组的片段整合到载体的推定复制基因的上游区域中增加了拷贝数。我构建了一个新的高拷贝表达载体pET-Pslpm,作为穿梭载体,在T. thermophilus和E.我已经证明了外源DNA片段整合到T.嗜热菌基因组的研究。但在导入T.嗜热菌我从T.嗜热菌HB 27。将克隆的MTthHB 27基因导入E. coli表达系统。热处理E.已知限制性内切酶基因和修饰酶基因串联存在于多种微生物中。然而,在MTthHB 27附近的区域中不存在可能的限制性内切酶基因。MTthHB 8和RTthHB 8基因串联存在于HB 8基因组中。但它们的密码子使用与栖热菌型相差甚远。因此,推测HB 8的限制-修饰系统是从其他生物水平转移的,而HB 27具有原始的Thermus型限制-修饰系统。
项目成果
期刊论文数量(18)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
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HOSHINO Takayuki其他文献
HOSHINO Takayuki的其他文献
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{{ truncateString('HOSHINO Takayuki', 18)}}的其他基金
Electrochemical metabolic control of bacteria for nitrogen removal
细菌脱氮的电化学代谢控制
- 批准号:
15K14683 - 财政年份:2015
- 资助金额:
$ 8.51万 - 项目类别:
Grant-in-Aid for Challenging Exploratory Research
Electron-beam induced single molecular manipulation and nano processing of living cell in aqueous solution
电子束诱导水溶液中活细胞的单分子操纵和纳米加工
- 批准号:
23680052 - 财政年份:2011
- 资助金额:
$ 8.51万 - 项目类别:
Grant-in-Aid for Young Scientists (A)
On-demand wiring of electro conductive nano wire toward cell interface
导电纳米线按需布线至细胞界面
- 批准号:
23656179 - 财政年份:2011
- 资助金额:
$ 8.51万 - 项目类别:
Grant-in-Aid for Challenging Exploratory Research
Cell migration driven self-assembly of microstructure and its application for intracellular nano interface
细胞迁移驱动的微结构自组装及其在细胞内纳米界面中的应用
- 批准号:
20860031 - 财政年份:2008
- 资助金额:
$ 8.51万 - 项目类别:
Grant-in-Aid for Young Scientists (Start-up)
相似国自然基金
超嗜热古菌Pyrococcus horikoshii几丁质降解酶研究
- 批准号:30570012
- 批准年份:2005
- 资助金额:30.0 万元
- 项目类别:面上项目