Overview

Bovine mastitis is the most costly agricultural disease, plaguing dairy cattle worldwide. It costs the United States around $2 billion annually, half of which is due to subclinical mastitis [1]. Traditional treatments usually involve the use of antibiotics. However, the use of antibiotics has led to increasing concerns of resistance as well as negative implications on human health. Furthermore, the development and synthesis of new antibiotics is slow and may not necessarily have the desired effects.

Bacteriocins are natural antimicrobial peptides secreted by bacteria to gain competitive advantage in their natural ecosystems. Functionally, bacteriocins kill target cells via different mechanisms, including pore formation, inhibition of translation, inhibition of transcription, and inhibition of cell wall synthesis. The use of bacteriocins as antimicrobial drugs has three significant advantages. First, bacteriocins are known to not be harmful to humans. One such group of bacteriocins, the Nisin family, is approved by the Food and Drug Administration as a food preservative [2]. Second, bacteriocins have a remarkably fast evolution rate [3]. Finally, bacteriocins are known to be very specific to certain species within the same genus, although some are known to have broad spectrum activity as well. This diversity among bacteriocins, along with their safety for human consumption, makes them a promising alternative to traditional antibiotics.

The pathogens of bovine mastitis can be divided broadly into Gram positive and Gram negative species, as well as contagious or environmental species. Our choice of bacteriocins is such that we can target the pathogens from all categories. Some potent bacteriocins have incredibly complex post-translational modifications, but we have also chosen ones that don’t require any modifications. The genes will be cloned into the standard pSB1C3 vector, and the peptides heterologously expressed by inducing the expression of T7 RNA Polymerase in Escherichia coli BL21(DE3). The efficacy of these recombinant proteins will be tested via zone of inhibition assays and growth curve assays.

BioBricks

Chassis

BL21(DE3)

BL21(DE3) contains the bacteriophage T7 RNA polymerase gene under the lacUV5 promoter and is deficient in common proteases. Therefore, this strain optimizes the expression of proteins placed under a T7 promoter after IPTG induction [5].

XL1B

XL1-Blue supercompetent cells allow for blue-white screening of recombinant plasmids. In addition, XL1B cells are endonuclease and recombination deficient, which greatly improves miniprep DNA quality and yield [6].