Here is a link to a previous post related to your question:

https://www.researchgate.net/post/Which_plasmid_to_use_for_expression_of_two_proteins

What is the reason you can't use the T7 promoter? If you can work around that I would recommend the pET duet system:

http://www.emdmillipore.com/US/en/product/pETDuet-1-DNA-Novagen,EMD_BIO-71146

May be a bit more work, but if the restriction sites work out and you already have one or both genes on individual plasmids you can build your own plasmid:

For example, if you have one cloned into a pBAD24 at EcoRI and NcoI, you can amplify, digest, and insert the other downstream of the first at NcoI and HindIII (in this case arabinose would induce expression of both) You can also amplify the second gene with a (constitutive) promoter if you want the second gene to be expressed independent of the first. I did something similar to this, as described here (look for "Construction of a chromosomal construct for inducing 2AA formation" in the methods):

http://jb.asm.org/content/199/14/e00140-17.long

BL 21dE 3 and Rosetta are both excellent for protein expresison

Here is a link to a previous post related to your question:

https://www.researchgate.net/post/Which_plasmid_to_use_for_expression_of_two_proteins

What is the reason you can't use the T7 promoter? If you can work around that I would recommend the pET duet system:

http://www.emdmillipore.com/US/en/product/pETDuet-1-DNA-Novagen,EMD_BIO-71146

May be a bit more work, but if the restriction sites work out and you already have one or both genes on individual plasmids you can build your own plasmid:

For example, if you have one cloned into a pBAD24 at EcoRI and NcoI, you can amplify, digest, and insert the other downstream of the first at NcoI and HindIII (in this case arabinose would induce expression of both) You can also amplify the second gene with a (constitutive) promoter if you want the second gene to be expressed independent of the first. I did something similar to this, as described here (look for "Construction of a chromosomal construct for inducing 2AA formation" in the methods):

http://jb.asm.org/content/199/14/e00140-17.long

Hi Andrew. Thanks you for your advices. The T7 promoter needs a bacterial strain that expresses the T7 RNA polymerase, for instance, the E. coli BL21 (DE3) strain. However, we work with E. coli strains isolated from clinical cases. In addition, we need constitutive promoters to mantain the expression of some genes during long-term infection experiments in animals.

I see.

If you are doing long term infection experiments in animals I might recommend that if you are worried about gene maintenance you can just insert your genes of interest directly into the chromosome, using whatever constitutive promoter you need (my paper that I cited did exactly this). That way you don't need to worry about plasmid maintenance.

Good luck!

@ Andrew Borchert.

Which one has better expression results (assuming no position effect)? Gene on the chlormosome or gene on the plasmids in the cells? Usually the plsamid number is greater than chlomosome number. Do you think that the number will affact their expression level?

@Yuan-Yeu Yau

I would say that off of a plasmid is always going to express higher, unless you use a plasmid with a copy number of 1 in the cell. However, depending on the promoter used in the chromosomal construction you may not need higher expression than that. I suppose the best option really depends on what you are looking for, so you have to weigh whether it is more important to have the highest expression possible (and express off of a plasmid) or whether you want to avoid any issues with plasmid maintenance (and insert the construct into the chromosome)