If you want to express multiple proteins, consider cloning multiple genes into one vector. You can add terminators between genes if you don't want them to influence each other.

Hi,

As a suggestion if nobody else replies, you could clone your genes in tandem within your plasmid, with their promoters and terminators. To do this, methods like Golden Gate are quite interesting, and you'll also find kits for Gibson Assembly (NEB), Infusion (Clonetech), PCR-based cloning (http://dx.plos.org/10.1371/journal.pone.0003827.g007), overlap extension PCR cloning (e.g. http://www.ncbi.nlm.nih.gov/pmc/articles/pmc3121328/. http://link.springer.com/protocol/10.1007/978-1-62703-625-2_4), exponential megapriming PCR (http://dx.plos.org/10.1371/journal.pone.0053360.g006), etc.

The other alternative is that you replace the selectable cassette yourself, either by restriction digestion or, for instance, by overlap extension PCR cloning.

Cheers,

R.

You could try using the Mini-Tn7 system to create a stable integration of your protein of interest. There are numerous papers using this system to introduce genes into a conserved and generally accepted neutral site on the bacterial chromosome. You could include in your construct an inducible promoter.

There are also some broad host range vectors out there, such as pBBR that are able to replicate in many different gram negative bacteria. I don't know if Pseudomonas is on that list. You could try other plasmids to, incompatibility groups P (incP) and Q (incQ) plasmids are generally broad range and work in many bacterial species.