Ligase-Independent Cloning in Synthesis of Recombinant Bacteriocins

Probiotics are live bacteria organisms used to maintain the balance of gut microflora and they inhibit the growth of pathogenic microorganisms. This in turn improves the performance of the host directly or indirectly by preventing or treating diseases, especially those of bacterial origin. Probiotics show their inhibitory effect to pathogenic microorganisms through various mechanism of which production of bacteriocins is among those. Bacteriocins are protein substances produced by different bacterial species against other bacteria. The most widely used bacteriocin in food and pharmaceutical industry is Nisin which is produced by bacillus species and it has activity against gram positive bacteria. The L-1077 is a bacteriocin produced by Bacillus Salivarius whose spectrum of activity is tilted more towards gram negative bacteria such as Campylobacter jejuni which is widely associated with food borne illnesses. Bacteriocins have narrow spectrum of activity, they are usually effective against either more on gram positive or gram negative bacteria, but not both. The objectives of this research were to produce a recombinant bacteriocin that would be effective on both gram negative and gram positive bacteria. To achieve this goal the genetic sequences of Nisin and L-1077 were identified, these sequences were synthesized, producing the recombinant DNA fragment which was then inserted in plasmid with ligase independent cloning (LIC). This recombinant plasmid will be transfected into E. coli to express the recombinant protein. The recombinant bacteriocin DNA fragment was achieved using fusion PCR. Specific primers were designed to extend complementary overhangs on the C-terminal of Nisin and N-terminal of L-1077. Fusion of the PCR products was successful and the resultant recombinant sequence bearing the C-terminus of Nisin and N-terminus of L-1077 was cloned into linearized pUC57 plasmid vector