A BROAD HOST RANGE MOBILIZATION SYSTEM FOR INVIVO GENETIC-ENGINEERING - TRANSPOSON MUTAGENESIS IN GRAM-NEGATIVE BACTERIA
A BROAD HOST RANGE MOBILIZATION SYSTEM FOR INVIVO GENETIC-ENGINEERING - TRANSPOSON MUTAGENESIS IN GRAM-NEGATIVE BACTERIA
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DOI：
10.1038/nbt1183-784
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发表时间：
1983-01-01
期刊：
影响因子：
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通讯作者：
PUHLER, A
中科院分区：
文献类型：
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作者：
SIMON, R;PRIEFER, U;PUHLER, A
我们开发了一种新的载体策略，用于将外源基因插入到革兰氏阴性菌基因组中，该系统由两部分组成：特异性E. colidonor菌株和E.大肠杆菌质粒供体菌株（称为动员菌株）携带整合在其染色体中的广泛宿主范围IncP型质粒RP 4的转移基因。它们可以利用任何革兰氏阴性细菌作为接合DNA转移的受体。载体质粒含有用于动员的P型特异性识别位点（Mob位点），并且可以以高频率从供体菌株动员。可移动载体来源于常用的E。大肠杆菌载体pACYC 184、pACYC 177和pBR 325，并且不能在肠道细菌群以外的菌株中复制。因此，它们可广泛地用作转座子载体复制子，用于在它们可移动进入但不能稳定维持的任何菌株中进行随机转座子插入诱变。所述载体尤其可用于在多种革兰氏阴性生物中定点转座子诱变和位点特异性基因转移。
We have developed a new vector strategy for the insertion of foreign genes into the genomes of gram negative bacteria not closely related toEscherichia coli.The system consists of two components: specialE. colidonor strains and derivatives ofE. colivector plasmids. The donor strains (called mobilizing strains) carry the transfer genes of the broad host range IncP–type plasmid RP4 integrated in their chromosomes. They can utilize any gram negative bacterium as a recipient for conjugative DNA transfer. The vector plasmids contain the P–type specific recognition site for mobilization (Mob site) and can be mobilized with high frequency from the donor strains. The mobilizable vectors are derived from the commonly usedE. colivectors pACYC184, pACYC177, and pBR325, and are unable to replicate in strains outside the enteric bacterial group. Therefore, they are widely applicable as transposon carrier replicons for random transposon insertion mutagenesis in any strain into which they can be mobilized but not stably maintained. The vectors are especially useful for site–directed transposon mutagenesis and for site–specific gene transfer in a wide variety of gram negative organisms.