Conception of conjugative cloning vectors.

Bacteria Conjugation is the primary means of transfer of genetic material among prokaryotes. We have designed a conjugation system to work this to our advantage. Using a tri-parental mating system, we intended to test the feasibility to clone large genes fragments in vivo solely through conjugation and homologous recombination. Unlike conventional methods of cloning genes, which usually involve the use of restriction enzymes to clone large gene fragment into the target organism, our triparental system can override this laborious process using simple genetic tools constructed synthetically. Combining DNA sequencing technology with conjugation protocols, tri-parental mating was performed using 3 different Escherichia coli strains, S17-1 strain as the donor of a pUC vector with RP4 OriT incorporated, a PHL 628 strain possessing the ~7.8kb large fragment(MalA gene) and lastly a S17-1 Rifamycin-resistant strain being the collector of the recombinant shuttle plasmid. Our results had indicated that it is possible for genetic exchange with our triparental system. We were able to obtain recombinant clones containing the phenotypes markers of all 3 strains. Yet, genetic analysis failed to reveal large fragment of the involved gene. Further experimentation is still required to verify the sustainability and success of this triparental system.Bachelor of Science in Biomedical Science