Heterologous expression is a key technique for understanding mechanisms of bacterial pathogenesis. Many cloning and expression vectors are available for Gram-negative bacteria but due to constant advances in methods (e.g. Golden-Gate cloning) and fundamental discoveries (e.g. new regulation systems), new and better plasmid tools are desirable.
Here we report a new plasmid vector (pUS250, 4.7 kb) that combines advantageous features of prior vectors with some novel elements to yield a highly versatile and efficient tool for gene cloning and expression. The plasmid is based on the broad host range KmR plasmid pBBR1MCS-2, which replicates in most Gram negative bacteria. The mobility functions of the original vector have been removed and replaced with a more space-efficient oriT sequence, and all 'junk' regions and annoying restriction sites in the plasmid backbone have been removed. The new plasmid features an extensive polylinker (26 unique sites) which is compatible with Golden Gate (BsaI and Esp3I) and BioBrick cloning. Cloning of a foreign gene into pUS250 results in excision of the amilCP chromoprotein gene, which enables blue/white selection of recombinants in any kind of bacterial host. Inducible expression of cloned genes occurs via a novel variant of the cumate control system from Pseudomonas putida, consisting of the cymR regulator and two CymR-binding operators flanking the strong Pc promoter from a class 1 integron.
We have shown that pUS250 can be mobilised between E.coli strains, and from E.coli to Pseudomonas and Rhizobium, confirming the functionality of the oriT. The cumate regulatory system is functional in all three hosts, and gives both tight control and also very high levels of derepressed expression; experiments with superfolder GFP as a test protein indicate similar performance to pET/BL21, but with the advantages that expression is not confined to E.coli, and is inducible by a very cheap and non-toxic substrate.