You can also take advantage of the lacUV5 promoter driving the restriction endonuclease. Excise the RE with PmeI/PstI and replace it with your gene; just remember to include a stop codon before the PstI site and the sequence between the PmeI site and the ATG (containing the RBS) in your PCR primers.

In general, using cloning vectors for expression is not recommended. The copy number is too high, repressing basal expression is hard, etc. Christian's last remark resonates with my own experience.

The vector works in principle, but there are a few caveats:

First, you might want to include an RBS in your coding sequence, as there is none at the location where your insert goes.

A T7 terminator at the 3'-end might be necessary, as the T7 polymerase is usually quite strong. Thus, upon proper induction in a strain carrying the T7 polymerase, the run-through transcription might negatively affect plasmid stability as you will get interference with rep and bla transcription. When we tried this approach a few years back, we got pretty strange effects that I would attribute to that.

Otherwise, this should work.

Thanks for the input Christian, adding a T7 terminator makes a lot of sense and is something i will do. As I say its really a to gain a rough idea of activity but you certainly have helped clarify the technique for me! Thank you.

You can also take advantage of the lacUV5 promoter driving the restriction endonuclease. Excise the RE with PmeI/PstI and replace it with your gene; just remember to include a stop codon before the PstI site and the sequence between the PmeI site and the ATG (containing the RBS) in your PCR primers.

In general, using cloning vectors for expression is not recommended. The copy number is too high, repressing basal expression is hard, etc. Christian's last remark resonates with my own experience.

Hi Alejandro-thanks for your advice, yes it seems we will have to go down the route of using expression vectors for our future work!