I'm trying to purify a natively expressed protein from a large operon, native expression levels are very low, does anyone have any experience with changing the innate promoter with let's say an inducible one? Is it possible? I'm trying to find any literature on that, but don't see much.
I'd appreciate any help :)
(heterologous expression from a vector is not an option)
All the best!
Its is possible, however, more information is needed what organism are working with and what is the strain background?
alright then you can use lamda red to take the regulatory region of a plasmid for induction and recombine it instead of the native promoter. You can for example use pQE-5 as template (https://www.snapgene.com/resources/plasmid-files/?set=qiagen_vectors&plasmid=pQE-5) you add homology arms that are 50bp start from the atg codon from your target locus and extend 50bp and do the same with the other homology arm (I cannot advice where to stop since I do not know the locus you are targeting therefore you will have to use your own judgement for that). The pQE-5 plasmid has an IPTG regulated t5 promoter hence no need for a t7 strain and it also has an amp maker close to the promoter add your homology to this fragment allows to easily identify the recombinants that took up the promoter + amp (hence the wt promoter has been switched). Of course you can use whatever plasmid you want as template depending on the system you want to incorporate just make sure you gel purify your fragment before electroporation otherwise you will end you with the plasmid itself rather than the promoter swapped region.
Is your operon really too large too clone? By putting on a plasmid you have the benefit of having whatever promoter you like but you also have the advantage of increased gene dosage by the higher plasmid copy number. Are you wanting to express all the proteins or just one, which would be even easier.
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