Generation of Marked and Markerless Mutants in Model Cyanobacterial Species
Generation of Marked and Markerless Mutants in Model Cyanobacterial Species
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DOI：
10.3791/54001
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发表时间：
2016-05-01
影响因子：
1.2
通讯作者：
Howe, Christopher J.
中科院分区：
文献类型：
--
作者：
Lea-Smith, David J.;Vasudevan, Ravendran;Howe, Christopher J.
蓝藻是重要的生态生物，也是生产生物燃料和有用工业产品的潜在平台。蓝藻的遗传操作是基础研究和应用研究的重要手段，尤其是模式生物聚胞藻6803和聚球藻7002。产生未标记的突变体，即通过插入抗生素抗性盒（含有一个或多个基因的DNA的可操作片段）将染色体改变引入菌株，然后使用阴性选择标记去除该盒，是一种特别强大的技术。未标记的突变体可以重复进行遗传操作，从而可以根据需要将尽可能多的改变引入到菌株中。此外，突变菌株中不存在编码抗生素抗性蛋白的基因是期望的，因为它避免了抗生素抗性生物体“逃逸”到环境中的可能性。然而，在科学文献中没有很好地描述用于蓝藻的重复轮遗传操作的详细方法。在这里，我们提供了一个全面的描述，这种技术，我们已经成功地用于产生多个缺失，单点突变的目的基因和插入新的基因盒内的突变体。
Cyanobacteria are ecologically important organisms and potential platforms for production of biofuels and useful industrial products. Genetic manipulation of cyanobacteria, especially model organisms such as Synechocystis sp. PCC6803 and Synechococcus sp. PCC7002, is a key tool for both basic and applied research. Generation of unmarked mutants, whereby chromosomal alterations are introduced into a strain via insertion of an antibiotic resistance cassette (a manipulatable fragment of DNA containing one or more genes), followed by subsequent removal of this cassette using a negative selectable marker, is a particularly powerful technique. Unmarked mutants can be repeatedly genetically manipulated, allowing as many alterations to be introduced into a strain as desired. In addition, the absence of genes encoding antibiotic resistance proteins in the mutated strain is desirable, as it avoids the possibility of 'escape' of antibiotic resistant organisms into the environment. However, detailed methods for repeated rounds of genetic manipulation of cyanobacteria are not well described in the scientific literature. Here we provide a comprehensive description of this technique, which we have successfully used to generate mutants with multiple deletions, single point mutations within a gene of interest and insertion of novel gene cassettes.