Bacteria depend on tightly regulated genetic systems to survive and respond to changing environments, and understanding how bacterial genes can be manipulated has become essential for studying both basic cellular processes and applied microbiology. The ability to modify specific genes directly in the chromosome provides a powerful way to investigate: gene function, alterations of defined regions of the bacterial genome and interactions between genes and their pathways. As gene‑editing tools have continued to advance, evaluating how these methods perform across different loci has become an important part of modern bacterial genetics.

Among bacterial species, Escherichia coli (E. coli) remains one of the most widely used models for gene editing. Its well‑characterized genome, rapid growth, and compatibility with recombineering‑based approaches make it a valuable platform for testing genetic tools that are later adapted for other organisms. In this project, E. coli was used to assess the practical performance of the DIRex λ‑Red recombineering system when applied across multiple chromosomal targets. Five genes bipA, hfq, deaD, rimJ, and ybeY were selected and they all contribute to RNA‑ and ribosome‑related processes. Using several genes with related cellular roles made it possible to examine how chromosomal deletions behave across different regions of the genome.

The project used a recombineering‑based deletion workflow that removes specific DNA sequences without leaving a selectable marker. Each gene was followed through the main steps of the process: constructing the required DNA fragments, introducing them into the chromosome, removing the temporary cassette, and verifying the final allele. Monitoring multiple targets in parallel made it possible to see where difficulties arose, such as challenges in fragment amplification, differences in recombination efficiency, or failure to recover the intended deletion during screening.

After generating a confirmed deletion of bipA, the project also tested how this mutation could be moved into other genetic backgrounds. The intermediate form of the edited locus allowed the ΔbipA allele to be transferred into several rRNA‑modification‑deficient strains using P1 bacteriophage transduction. This step showed that a successfully created deletion can be introduced into new ribosomal contexts without repeating the entire editing procedure.

Overall, the work uses E. coli to explore the practical steps involved in generating multiple chromosomal deletions in genes linked to RNA and ribosome function. By comparing how each target progressed through the workflow and by transferring ΔbipA into additional strains, the project provides a clear and accessible look at the challenges and possibilities of multi‑locus gene editing in bacteria.