Construction and application of an expression vector from the new plasmid pLAtc1 of Acidithiobacillus caldus
Construction and application of an expression vector from the new plasmid pLAtc1 of Acidithiobacillus caldus
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Acidithiobacillus caldus新质粒pLAtc1表达载体的构建及应用
DOI：
10.1007/s00253-014-5507-z
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发表时间：
2014-01
影响因子：
5
通讯作者：
Liu Shunag-Jiang
中科院分区：
文献类型：
--
作者：
zhang Ming-Jiang;Jiang Cheng-Ying;You Xiao-Yan;Liu Shunag-Jiang
在这项研究中，一个最近测序的9.8 kb的质粒，pLAtc 1，从嗜热硫杆菌菌株SM-1的特点和发展成为一个表达载体。pLAtc 1骨架携带一个oriV、三个rep基因、五个mob基因、一个Nic位点和一个成瘾系统。多位点序列分析表明，pLAtc 1与IncQ类广宿主范围质粒的亲缘关系比与其他IncQ质粒的亲缘关系更近。pLAtc 1能够复制和驻留在革兰氏阴性大肠杆菌，睾丸酮丛毛单胞菌，但不能在革兰氏阳性谷氨酸棒杆菌。pLAtc 1通过接合进入E. coli BL 21和A. caldus SM-1; coli S17-1。定量PCR显示A. caldus和E. coli细胞。表达载体pLAtcE由pLAtc 1通过引入可调控启动子（PtetH）、转录终止子、多克隆位点、卡那霉素抗性基因和链霉素抗性基因而构建。通过在大肠杆菌中表达编码增强型绿色荧光蛋白的基因证明了pLAtcE的功能性。coli和A.卡尔杜斯。利用pLAtcE在A.卡尔杜斯。将sucAB和sdhA整合到质粒pLAtcE-sucA-sucB-sdhA上的新工程菌在连四硫酸盐和葡萄糖培养基中的生长优于亲本菌株SM-1/pLAtcE，并产生更高的酸度，导致更强的氧化环境。本研究为嗜热嗜酸自养A.卡尔杜斯。
In this study, a recently sequenced 9.8-kb plasmid, pLAtc1, from Acidithiobacillus caldus strain SM-1 was characterized and developed into an expression vector. The pLAtc1 backbone carried an oriV, three rep genes, five mob genes, a Nic site, and an addiction system. Multilocus sequence analysis indicated that pLAtc1 was phylogenetically more related to the IncQ-like broad host range plasmids than to other IncQ plasmids. pLAtc1 was able to replicate and reside in Gram-negative Escherichia coli, Comamonas testosteroni, but not in Gram-positive Corynebacterium glutamicum. pLAtc1 was mobilized via conjugation into E. coli BL21 and A. caldus SM-1 from E. coli S17-1. Quantitative PCR revealed seven and four copies of plasmid in A. caldus and E. coli cells, respectively. The expression vector pLAtcE was constructed from pLAtc1 by introducing a regulatable promoter (P tetH ), a transcriptional terminator, a multiple cloning site, a kanamycin resistance gene, and a streptomycin resistance gene. The functionality of pLAtcE was demonstrated by expressing a gene encoding enhanced green fluorescence protein in E. coli and in A. caldus. pLAtcE was used to express α-ketoglutarate dehydrogenase (sucAB) and succinate dehydrogenase (sdhA) genes in A. caldus. The newly engineered strain that harbored sucAB and sdhA on a plasmid pLAtcE-sucA-sucB-sdhA grew better than the parent strain SM-1/pLAtcE in tetrathionate and glucose-supplemented medium and produced more acidity and resulted in a more oxidative environment. This study created a useful molecular tool for genetic manipulation of the thermoacidophilic and autotrophic A. caldus.
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影响因子：
11.4
作者：
W. Zeng;G. Qiu;Hong-bo Zhou;J. Peng;Miao Chen;S. Tan;W. Chao;Xue-duan Liu;Yansheng Zhang
通讯作者：
W. Zeng;G. Qiu;Hong-bo Zhou;J. Peng;Miao Chen;S. Tan;W. Chao;Xue-duan Liu;Yansheng Zhang
影响因子：
10.3
作者：
C. Sansom
通讯作者：
C. Sansom
DOI：
10.1038/nbt1183-784
发表时间：
1983-01-01
期刊：
BIO-TECHNOLOGY
影响因子：
--
作者：
SIMON, R;PRIEFER, U;PUHLER, A
通讯作者：
PUHLER, A
DOI：
--
发表时间：
2006
期刊：
--
影响因子：
--
作者：
David W. Russell;J. Sambrook
通讯作者：
David W. Russell;J. Sambrook
影响因子：
2.8
作者：
Jian-feng Wu;Cui-wei Sun;C. Jiang;Zhipei Liu;Shuangjiang Liu
通讯作者：
Jian-feng Wu;Cui-wei Sun;C. Jiang;Zhipei Liu;Shuangjiang Liu