pTn5cat:: A Tn5-derived genetic element to facilitate insertion mutagenesis, promoter probing, physical mapping, cloning, and marker exchange in phytopathogenic and other gram-negative bacteria
pTn5cat:: A Tn5-derived genetic element to facilitate insertion mutagenesis, promoter probing, physical mapping, cloning, and marker exchange in phytopathogenic and other gram-negative bacteria
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DOI：
10.1006/plas.1998.1340
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发表时间：
1998-01-01
期刊：
影响因子：
2.6
通讯作者：
Alvarez-Morales, A
中科院分区：
文献类型：
--
作者：
Marsch-Moreno, R;Hernández-Guzmán, G;Alvarez-Morales, A
构建了一个Tn 5衍生的移动的元件，用于鉴定假单胞菌致病性和毒力相关的基因和启动子。菜豆为了提高突变率，构建了携带突变转座酶的Tn 5衍生物，该突变转座酶顺式放置在转座因子上，但正好在反向重复序列之外，因此消除了二级转座并增加了插入的稳定性。新元件还包含一个无启动子的cat（氯霉素乙酰转移酶）基因作为报告基因，以允许在特定条件下表达的启动子的阳性选择。为了便于在大肠杆菌中的克隆和操作，ColE 1复制起点已被包括在转座因子以及来自广宿主范围质粒RP 4的Mob区域内，这使得该元件能够通过三亲交配或通过使用大肠杆菌来有效地移动。大肠杆菌菌株如S17-1，以提供tra功能。罕见切割酶PacI和PmeI的位点也被包括在内，以便于在PacI和/或PmeI物理图上定位插入。这种结构结合了可移动质粒和转座子的性质，因此被称为pTn 5cat。其大小与野生型Tn 5（5877 bp）几乎相同，并且已成功地在P. s.菜豆黄单胞菌（Xanthomonascampestris pv.）野油菜(C)北京：科学出版社.
A Tn5-derived mobile element has been constructed to identify genes and promoters related to pathogenesis and virulence in Pseudomonas syringae pv. phaseolicolo. To enhance the rate of mutation this Tn5 derivative was constructed carrying a mutant transposase which was placed in cis to the transposable element, but just outside the inverted repeats, therefore eliminating secondary transposition and increasing the stability of the insertion. The new element also contains a promoterless cat (chloramphenicol acetyltransferase) gene as reporter to allow for positive selection of promoters bring expressed under specific conditions. To facilitate cloning and manipulations in Escherichia coli, a ColE1 origin of replication has been included within the transposable element as well as the Mob region from the broad-host-range plasmid RP4, which allows this element to be efficiently mobilized by a triparental mating or by using an E. coli strain such as S17-1 to provide the tra functions. Sites for the rare cutters PacI and PmeI have also been included to facilitate locating the insertions on a PacI and/or PmeI physical map. This construction combines the properties of both a mobilizable plasmid and a transposon and therefore has been termed pTn5cat. It is almost the same size as the wild-type Tn5, 5877 bp, and has successfully been tested in P.s. phaseolicola and Xanthomonas campestris pv. campestris. (C) 1998 Academic Press.