Transfer of megaplasmid pKB1 from the rubber-degrading bacterium Gordonia westfalica strain Kb1 to related bacteria and its modification
Transfer of megaplasmid pKB1 from the rubber-degrading bacterium Gordonia westfalica strain Kb1 to related bacteria and its modification
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橡胶降解菌 Gordonia westfalica Kb1 菌株巨质粒 pKB1 向相关菌的转移及其修饰
DOI：
10.1007/s00253-007-1262-8
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发表时间：
2008
影响因子：
5
通讯作者：
A. Steinbüchel
中科院分区：
文献类型：
--
作者：
Bröker;Arenskötter;A. Steinbüchel
由于缺乏有效的转移系统，威斯特戈登氏菌Kb 1的101.016bp大质粒pKB 1的工程改造失败，pKB 1通过接合转移从G. WestfalicaKb 1转化为不透明红球菌PD 630的卡那霉素抗性突变体，频率约为每个受体细胞6.2 × 10− 8个事件。将pKB 1转移到G.以5.5 × 103、1.9 × 103和8.3 × 102个转化子/μ g质粒DNA的频率电穿孔多聚异戊二烯噬菌体VH 2和Y2K和耻垢分枝杆菌。在这些实验中使用pKB 1编码的镉抗性基因cadA进行选择。重组质粒pKB 1-containingG.聚异戊二烯类VH_2和M. smegmatiswere then used to engineer工程pKB 1.将卡那霉素抗性盒插入pKB 1编码的cadA基因中，连接到自杀质粒pBBR 1 MCS-5上，并将所得质粒电穿孔到携带质粒的菌株中。在自杀质粒cadAon与pKB 1中相应序列之间的同源重组导致其整合到pKB 1中。因此，两个选择标记被容纳在pKB 1中，以监测质粒转移到戈登氏菌和相关分类群中，用于分析橡胶降解和其他必需基因。本研究成功地应用了两种大质粒的转移方法和pKB 1的工程改造策略，从而扩展了戈登氏菌的工具箱。
Because engineering of the 101.016-bp megaplasmid pKB1 ofGordonia westfalicaKb1 failed due to the absence of an effective transfer system, pKB1 was transferred by conjugation fromG. westfalicaKb1 to a kanamycin-resistant mutant ofRhodococcus opacusPD630 at a frequency of about 6.2 × 10−8events per recipient cell. Furthermore, pKB1 was transferred toG. polyisoprenivoransstrains VH2 and Y2K and toMycobacterium smegmatisby electroporation at frequencies of 5.5 × 103, 1.9 × 103, and 8.3 × 102transformants per microgram plasmid DNA. The pKB1-encoded cadmium resistance genecadAwas used for selection in these experiments. Recombinant pKB1-containingG. polyisoprenivoransVH2 andM. smegmatiswere then used to engineer pKB1. A kanamycin resistance cassette was inserted into the pKB1-encodedcadAgene, ligated to suicide plasmid pBBR1MCS-5, and the resulting plasmid was electroporated into plasmid-harboring strains. Homologous recombination betweencadAon suicide plasmid and the respective sequence in pKB1 led to its integration into pKB1. Thus, two selection markers were accommodated in pKB1 to monitor plasmid transfer intoGordoniaand related taxa for analysis of genes essential for rubber degradation and others. In this study, two transfer methods for large plasmids and strategies for engineering of pKB1 were successfully applied, thereby, extending the tool box forGordonia.
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影响因子：
4.4
作者：
Q. Banh;M. Arenskötter;A. Steinbüchel
通讯作者：
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影响因子：
3.2
作者：
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通讯作者：
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DOI：
10.1099/00207713-49-4-1785
发表时间：
1999-10-01
期刊：
INTERNATIONAL JOURNAL OF SYSTEMATIC BACTERIOLOGY
影响因子：
--
作者：
Linos, A;Steinbüchel, A;Kroppenstedt, RM
通讯作者：
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影响因子：
5
作者：
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通讯作者：
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DOI：
--
发表时间：
1998
期刊：
影响因子：
--
作者：
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通讯作者：
A. Steinbüchel