DNA operon structure optimization for the expression of fluorescent proteins in mycobacteria

Introduction. The use of bacteriophage derivatives, whose growth indicator is a fluorescent signal (fluorophages), seems to be very promising in microbiological analyses. The first mandatory stage of their design (in particular, of fluoromycobacteriophages) is the optimization of the structure of the fluorescent protein operon (FPO) with control of its ability to provide fluorescence of mycobacteria The aim of the study is to construct different variants of FPO in plasmids and compare their expression in mycobacteria by the magnitude of the fluorescent signal. Materials and methods. As part of the pMind plasmid, which is capable of replicating in both Escherichia coli and mycobacteria, FPOs of different structures with the hsp60 promoter producing the TurboGFP protein have been constructed using conventional genetic engineering methods. Plasmids with FPOs were introduced into Mycobacterium smegmatis cells by elecroporanion. Results. The greatest fluorescent signal was provided by FPO, in which the hsp60 ribosome binding site in the 5’-untranslated mRNA region was replaced by that for the gp9 gene of the TM4 phage. The necessity of the presence of a transcription terminator in FPO is shown, as well as the importance of preserving the mRNA structure before the point of translation initiation. Conclusion. The optimal structure of FPO has been selected for its subsequent incorporation into the phage genome. Elements of the operon structure important for the expression of a foreign protein in mycobacteria have been identified.