Selenocysteine (SeC), known as the 21st amino acid, plays a pivotal role in several enzymes, particularly those involved in redox reactions. Its biosynthesis and incorporation into proteins depend on the coordinated action of several genes: selA and selD (biosynthesis), and selB and selC (incorporation). In Escherichia coli, SeC is incorporated at UGA codons through a specific mRNA structure known as the SECIS element. In contrast, Mycobacterium marinum lacks this element within its selAB operon, suggesting the presence of alternative regulatory mechanisms. This study aimed to clone and express the selA gene from M. marinum in E. coli to explore whether feedback regulation mediated by SelB levels influences selA and selB expression. The selA gene was PCR-amplified, digested, ligated into the pIGN plasmid, and transformed into E. coli. Colony PCR and sequencing confirmed the presence of the insert in selected clones. These findings underscore the challenges of cloning efficiency and sequencing accuracy and provide a foundation for investigating SECIS-independent SeC regulation pathways.
Niloofar Sadeghi (Wed,) studied this question.