Hi,

I encountered problem with my protein of interest. It's 20kDa protein which is expressed on very low lvls in M. smegmatis. During stress its lvl is elevated but not much. I wanted to visualize it by fusing it with mNeonGreen on C-term. I constructed strain with it in native locus and second strain with one copy of fusion protein in attB site under inducible promotor. In first strain I can not see anything (under the microscope ofc). I think there is more signal compare to WT and its autofluorescence but it's very empirical. On the other hand in overexpression of protein (second strain) I can see strong signal without any pattern through the cell. I know that protein is documented to bind to DNA via N-term and interact with proteins via C-term domains, e.g DnaA. I checked that on WB and it's produced (anti-mNeonGreen). I checked also semi native gel electrophoresis in order to check if it's folded properly.

All of that is not really making me sure that my fusion protein is folded properly so I was thinking if i can use BTH to check if C-term domain is working (N- term domain can be checked by purifying protein and doing EMSA with DNA). So i wanted to put my fusion protein (protein of interest fused with mNeonGreen on C term) and clone it into vector where C25 or C18 would be on N terminal region of protein. Then check if it's interacting with DnaA which would be cloned in vector with C25/C18 on C or N term region. Technically I think it's possible to do and might work but I'm just first year PhD student for now :).

I would be thankful for the answer.