pOPARA: Vectors for Golden Gate assembly of expression constructs containing the araBAD promoter

Abstract

AbstractHeterologous protein production is often required to investigate the structural, biochemical, and biophysical properties of a protein of interest. Frequently, optimisation of expression conditions is required to obtain soluble protein and maximise yield. Trialling a variety of solubility and purification tags, as well as constructs containing different regulatory elements, is desirable. Golden Gate cloning allows modular assembly of different constructs using Type IIS restriction enzymes. The pOPIN vector suite, which utilises the T7 expression system, has been adapted to be compatible with Golden Gate assembly. Here, we present the pOPARA vectors. Expression from pOPARA vectors is driven by the araBAD promoter (pBAD) and is induced by addition of arabinose to the culture medium. pOPARA allows modular assembly of expression constructs using Golden Gate cloning with the CDS of interest and an optional C-terminal tag. pOPARA1 contains a carbenicillin resistance cassette flanked by restriction sites to allow exchange of the selectable markers. In pOPARA2, the carbenicillin resistance cassette has been exchanged for a spectinomycin resistance cassette. We demonstrate that both vectors can be used to express and produce a control protein.