Acinetobacter sp ADP1:: an ideal model organism for genetic analysis and genome engineering
Acinetobacter sp ADP1:: an ideal model organism for genetic analysis and genome engineering
复制标题
DOI：
10.1093/nar/gkh881
复制
发表时间：
2004-01-01
影响因子：
14.9
通讯作者：
de Crécy-Lagard, V
中科院分区：
文献类型：
--
作者：
Metzgar, D;Bacher, JM;de Crécy-Lagard, V
不动杆菌属。 ADP1 菌株是一种天然可转化的革兰氏阴性细菌，具有简单的培养要求、原养代谢和最近已测序的 3.7 Mb 紧凑基因组。通过将线性 DNA 构建体直接添加到对数期培养物中，可以对野生型 ADP1 进行遗传操作。这使其成为复杂菌株构建自动化的理想有机体。在这里，我们通过构建多种突变体展示了 ADP1 作为遗传模型的灵活性和多功能性。这些包括标记和未标记的插入和缺失、互补替换、染色体表达标签及其复杂组合。在这些构建过程中，我们证明ADP1可以有效表达多种外源基因，包括抗生素抗性盒、必需代谢基因、负选择分解代谢基因，甚至来自高度分化细菌的完整操纵子。所有描述的突变都是通过相同的剪接 PCR、直接转化生长培养物和在选择性培养基上铺板的过程实现的。这些工具的简单性使得具有最少微生物遗传学专业知识和很少设备的实验室可以使用不动杆菌 ADP1 进行遗传分析和工程。它们还与基因分析和工程方案的完全自动化兼容。
Acinetobacter sp. strain ADP1 is a naturally transformable gram-negative bacterium with simple culture requirements, a prototrophic metabolism and a compact genome of 3.7 Mb which has recently been sequenced. Wild-type ADP1 can be genetically manipulated by the direct addition of linear DNA constructs to log-phase cultures. This makes it an ideal organism for the automation of complex strain construction. Here, we demonstrate the flexibility and versatility of ADP1 as a genetic model through the construction of a broad variety of mutants. These include marked and unmarked insertions and deletions, complementary replacements, chromosomal expression tags and complex combinations thereof. In the process of these constructions, we demonstrate that ADP1 can effectively express a wide variety of foreign genes including antibiotic resistance cassettes, essential metabolic genes, negatively selectable catabolic genes and even intact operons from highly divergent bacteria. All of the described mutations were achieved by the same process of splicing PCR, direct transformation of growing cultures and plating on selective media. The simplicity of these tools make genetic analysis and engineering with Acinetobacter ADP1 accessible to laboratories with minimal microbial genetics expertise and very little equipment. They are also compatible with complete automation of genetic analysis and engineering protocols.