Marker-Free Chromosomal Integration of a Multigene Cassette in Escherichia coli for Heavy-Metal Bioremediation Development

Heavy metal contamination remains a significant environmental challenge due to the persistence, toxicity, and bioaccumulation of metal pollutants. Genetically engineered bacteria offer a potential approach to heavy metal remediation; however, plasmid-based expression systems can present limitations associated with plasmid maintenance and antibiotic selection. This study describes a protocol for constructing a marker-free, multigene recombinant E. coli strain designed for the chromosomal integration of genes associated with heavy-metal resistance and sequestration. A 4,741 bp multigene cassette comprising cadD, cadA, and metallothionein (mt), together with a lipoprotein outer membrane protein A (Lpp-OmpA) surface display system and a green fluorescent protein (gfp) reporter gene under the control of the constitutive pyruvate dehydrogenase (Ppdh) promoter, was designed and synthesized. The construct was cloned into the pKIKOArsBKm integration vector and introduced into E. coli MG1655 using lambda (λ) Red-mediated recombination. The kanamycin resistance cassette was subsequently excised using the flippase-flippase recognition target (FLP-FRT) recombination system. Construct assembly and chromosomal integration were evaluated by restriction enzyme analysis, plasmid isolation, and polymerase chain reaction (PCR) amplification, while loss of antibiotic resistance was assessed by antibiotic sensitivity testing. Amplification of the expected cadD, cadA, and mt fragments from genomic DNA, together with the absence of detectable plasmid DNA, provided evidence consistent with chromosomal integration of the multigene cassette. Although this protocol establishes a framework for constructing and molecularly evaluating the recombinant strain, definitive confirmation of the integration locus, functional gene expression, long-term genetic stability, and heavy metal biosorption performance requires further experimental validation.