The Joint BioEnergy Institute, Emeryville, CA, USA; Biological Systems and Engineering Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA.
The Joint BioEnergy Institute, Emeryville, CA, USA; Biological Systems and Engineering Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA.
Plasmid. 2020 Jul;110:102514. doi: 10.1016/j.plasmid.2020.102514. Epub 2020 Jun 3.
Pseudomonas putida is a widely used host for metabolic engineering and synthetic biology. However, the use of P. putida has been hampered by the availability of a limited set of expression vectors for producing heterologous proteins. To widen the scope of expression vectors for gene co-expression studies, a previously established dual-inducible expression vector pRG_Duet2 developed for Corynebacterium glutamicum has been modified for use in P. putida. This expression vector, named pRGPDuo2, harbors two origins of replication, colE1 for replication in E. coli and pRO1600 for replication in P. putida. Two multiple cloning sites (MCS1 and MCS2) in pRGPDuo2 are individually controlled by inducible promoters P or P. Functional validation of pRGPDuo2 was confirmed by the co-expression of genes for the fluorescent proteins namely, superfolder green fluorescent protein (sfGFP), and red fluorescent protein (RFP). Moreover, the strength of the fluorescence signal was dependent on the inducer concentrations present in the culture medium. The expression vector pRGPDuo2 is an attractive addition to the existing repertoire of expression plasmids for expression profiling and adds to the tools available for P. putida metabolic engineering.
铜绿假单胞菌是代谢工程和合成生物学中广泛应用的宿主。然而，由于可用的产生异源蛋白的表达载体数量有限，限制了铜绿假单胞菌的应用。为了拓宽基因共表达研究的表达载体范围，对先前为谷氨酸棒状杆菌开发的双诱导表达载体 pRG_Duet2 进行了修饰，使其可用于铜绿假单胞菌。该表达载体命名为 pRGPDuo2，含有两个复制起点，大肠杆菌中的 colE1 用于复制，铜绿假单胞菌中的 pRO1600 用于复制。pRGPDuo2 中的两个多克隆位点（MCS1 和 MCS2）分别由诱导型启动子 P 或 P 控制。通过共表达荧光蛋白基因，即超折叠绿色荧光蛋白（sfGFP）和红色荧光蛋白（RFP），证实了 pRGPDuo2 的功能验证。此外，荧光信号的强度取决于培养基中诱导剂的浓度。表达载体 pRGPDuo2 是表达谱分析现有表达质粒库的一个有吸引力的补充，并为铜绿假单胞菌代谢工程提供了更多的工具。
