Institute of Microbiology and Molecular Biology, Justus-Liebig University Giessen, 35392 Giessen, Germany.
J Biotechnol. 2012 Nov 30;162(1):75-80. doi: 10.1016/j.jbiotec.2012.06.016. Epub 2012 Jun 26.
Synechocystis sp. PCC 6803 is a model organism for the study of photosynthetic processes. Methods to genetically manipulate this bacterium are essential to investigate these processes and to evaluate potential biotechnological applications. We developed a vector for controllable expression of proteins using a platform for stable integration of the expression cassette into the genome. The respective gene is translationally fused to the promoter of the petJ gene encoding cytochrome c(553) that is repressed by copper. Maximal expression from this promoter is achieved under copper depletion, whereas normal copper concentrations in standard medium lead to low expression rates. We show here the application of this system for construction of a conditional knockout mutant for the ferrochelatase, which is an essential enzyme in heme biosynthesis. Using different amounts of copper in the medium we were able to control the amount of ferrochelatase in the cell resulting in a varying expression of the phenotype.
集胞藻 PCC 6803 是研究光合作用过程的模式生物。对该细菌进行基因操作的方法对于研究这些过程和评估潜在的生物技术应用至关重要。我们开发了一种载体，用于使用可稳定整合表达盒到基因组中的平台来可控表达蛋白质。相应的基因与编码细胞色素 c(553)的 petJ 基因的启动子翻译融合，该基因受铜抑制。在铜耗尽的情况下，该启动子可实现最大表达，而在标准培养基中的正常铜浓度则导致低表达率。我们在这里展示了该系统在构建亚铁螯合酶条件性敲除突变体中的应用，该酶是血红素生物合成中的必需酶。通过在培养基中使用不同量的铜，我们能够控制细胞中铁氧还蛋白的含量，从而导致表型的表达变化。
