CONSTRUCTION OF A BROAD HOST RANGE COSMID CLONING VECTOR AND ITS USE IN THE GENETIC-ANALYSIS OF RHIZOBIUM MUTANTS
CONSTRUCTION OF A BROAD HOST RANGE COSMID CLONING VECTOR AND ITS USE IN THE GENETIC-ANALYSIS OF RHIZOBIUM MUTANTS
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DOI：
10.1016/0378-1119(82)90167-6
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发表时间：
1982-01-01
期刊：
影响因子：
3.5
通讯作者：
AUSUBEL, FM
中科院分区：
文献类型：
--
作者：
FRIEDMAN, AM;LONG, SR;AUSUBEL, FM
通过插入含有.lambda的1.6 kb[千碱基]BgII片段，构建了低拷贝数宽宿主范围克隆载体pRK290 （Ditta et al., 1980）的cosmid衍生物。cos进入pRK290独特的bgi位点。新载体pLAFR1长21.6 kb，具有四环素耐药性，含有独特的EcoRI位点，可被调动到许多革兰氏阴性宿主中并在其内稳定复制。利用部分EcoRI酶切法在pLAFR1中构建了一个克隆库。平均插入大小为23.1 kb。将大肠杆菌克隆库与各种异养突变体进行大规模杂交，获得四环素耐药（Tcr）转偶联体的频率为0.1-0.8，其中获得原生营养菌落的频率为0.001-0.007。pLAFR1 cosmids从R. meliloti原生菌落中被动员到大肠杆菌中，然后再被重新引入R. meliloti营养不良菌中。在大多数情况下，这些后一种Tcr转共轭体100%是原生营养的。
A cosmid derivative of the low copy number broad host-range cloning vector pRK290 (Ditta et al., 1980) was constructed by inserting a 1.6 kb [kilobase] BgII fragment containing .lambda. cos into the unique BgII site in pRK290. The new vector, pLAFR1, is 21.6 kb long, confers tetracycline resistance, contains a unique EcoRI site, and can be mobilized into and stably replicates within many Gram-negative hosts. A clone bank of R. meliloti DNA was constructed in pLAFR1 using a partial EcoRI digest. The mean insert size was 23.1 kb. When the clone bank was mated (en masse) from Escherichia coli to various R. meliloti auxotrophic mutants, tetracycline-resistant (Tcr) transconjugants were obtained at frequencies ranging from 0.1-0.8, and among these, prototrophic colonies were obtained at frequencies ranging from 0.001-0.007. pLAFR1 cosmids were mobilized from R. meliloti prototrophic colonies into E. coli and then reintroduced into R. meliloti auxotrophs. In most cases, 100% of these latter Tcr transconjugants were prototrophic.