Molecular cloning of a construct for targeted RecA gene disruption via homologous recombination in Agrobacterium tumefaciens

Law, Victoria (2025) Molecular cloning of a construct for targeted RecA gene disruption via homologous recombination in Agrobacterium tumefaciens. Final Year Project, UTAR.

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Abstract

Agrobacterium tumefaciens is a cornerstone of plant genetic engineering and serves as the primary vector for delivering transgenes into a wide variety of plant species. However, the efficiency of this process is compromised by the bacterium's native homologous recombination system, which is mediated by the RecA protein. Functional RecA promotes unintended rearrangements and deletions of the transferred DNA (T-DNA) within the bacterial cell, leading to integrated transgenes that are fragmented, rearranged, or incomplete. This reduces the precision and effectiveness of Agrobacterium-mediated transformation (AMT) and necessitates screening of excessive numbers of transformants to recover a few with the correct construct, making the process inefficient and costly. This study aimed to address this limitation by constructing a targeted gene disruption vector to create a stable recA-deficient strain of A. tumefaciens. The strategy involved engineering a plasmid that upon introduction into Agrobacterium would insertionally disrupt the function chromosomal recA gene with a (cat)-repA cassette via a double-crossover homologous recombination event. To achieve this, the N-terminal and C-terminal regions of recA were amplified by polymerase chain reaction (PCR) using A. tumefaciens genomic DNA as a template. These fragments were then sequentially cloned into the pASK-KO suicide vector flanking the chloramphenicol resistance (cat)-repA cassette. The resulting construct, pASK-NrecA-cat-repA-CrecA, was assembled and propagated in Escherichia coli TOP10 cells. Putative recombinant clones were selected on kanamycin-containing media and validated using a combination of colony PCR, restriction enzyme digestion, and Sanger sequencing, which confirmed 100% identity with the expected plasmid sequence. The successful construction of this vector is a critical first step. Its subsequent introduction into A. tumefaciens is expected to generate a mutant strain with a disrupted recA locus. This engineered strain should exhibit significantly enhanced T-DNA plasmid stability, thereby minimizing rearrangements and ultimately increasing AMT fidelity and efficiency. The development of such specialized strains has significant potential to streamline plant biotechnology workflows, reducing the time and resources required to generate transgenic plants with intact functional genes.

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