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Submitted to: Plasmid Journal
Publication Type: Peer Reviewed Journal Publication Acceptance Date: 12/12/2016 Publication Date: 1/1/2017 Citation: Nakata, P.A. 2017. Construction of pDUO: A bicistronic shuttle vector series for dual expression of recombinant proteins. Plasmid Journal. 89:16-21. Interpretive Summary: Microorganisms are used in the production of useful medicine, food, agriculture, and specialty chemicals. The recombinant DNA revolution of the mid 1970's was a primary driving force responsible for this rapid biotechnological evolvement. This technological evolution was dependent on the development of suitable vectors that allowed the engineering of these microbes. Today, wide arrays of vectors exist that allow engineering to occur in model bacteria such as E. coli. With the list of bacterium useful for biotechnological applications ever expanding, there is a continuing need for the development of vectors especially a set capable of expressing multiple genes in a broader range of hosts. Here, the construction of such a vector series named pDUO is reported. The pDUO vector series offers the ability to co-express two genes in a highly regulated fashion in a range of bacterial hosts. The utility of this vector series was demonstrated by the co-expression and functional assessment of two recombinant proteins in both E. coli and P. fluorescence. The ability to make recombinant manipulations in E. coli and then directly express the generated constructs in a range of bacterial hosts makes this versatile vector series an attractive addition to our molecular tool box. It is anticipated that this vector series, which includes multiple selectable markers, will find use in functional studies and biotechnological applications in a range of bacteria. Technical Abstract: Our ability to genetically manipulate microbial systems is often hampered by the availability of genetic tools. Thus, there is a need for the continued expansion of our molecular tool box. In support of this expansion, this study reports the design, construction, and validation of a new shuttle vector series, pDUO, for the co-expression of genes and affinity purification of gene products from a range of host bacteria. Each vector was designed and constructed to contain two araC-pBad inducible promoter systems for tight control over gene expression. Each araC-pBad promoter precedes a ribosomal binding site and a multicloning site (MCS). MCS1 contains an affinity HIS-tag N-terminus and MCS2 terminates with an affinity S-tag C-terminus for one-step purification of recombinant proteins encoded by the inserted genes. Both MCS are followed by an rrnBT1 and T2 transcriptional terminator sequence. Each vector in this series also contains a PBR322 and pRO1600-derived replicon to support replication in a variety of host bacteria along with one of four different selectable markers. The functionality of the pDUO vector series was validated through the co-expression of oxalate biosynthetic component (obc) 1 and mCherry in Escherichia coli and Pseudomonas fluorescens. It is anticipated that this new vector series will facilitate functional studies as well as the engineering of bacterial strains for biotechnological purposes. |
