4. Mission Impossible The genetic shotgun cloning and chemical extraction from a novel Zooshikella species

Mission Impossible: The genetic shotgun cloning and chemical extraction from a novel antibiotic-producing Zooshikella species

Aubrey Carey, Magnolia Valdez, Brittany Gasper, and Eric Warrick
State College of Florida

Antibiotic resistance has become a prevalent problem in recent decades, as fatalities from previously preventable infections continue to rise. If no significant action is taken by 2050, multidrug resistant bacteria will result in more deaths than cancer and diabetes combined (Small World Initiative). While it is not a matter of “if” bacteria will become resistant to new antibiotics, but “when”, we hope to prepare ourselves for the next lethal strain of resistance. Following protocols outlined by the Tiny Earth Initiative, student Danielle Pearman isolated a bacterium, MI3, from North Beach Florida in Fall 2015, which we believe to be a novel species of Zooshikella based on previous sequencing results. When initially isolated, the bacteria displayed antimicrobial properties against both Gram-positive and Gram-negative bacteria. The research conducted and shown in this presentation focuses on MI3’s antimicrobial activity against Staphylococcus epidermidis and further genetic analysis of the genome. An initial chemical extraction in a mid-polar solvent, Ethyl Acetate, was performed using a rotary evaporator. Further extractions were performed using an affinity purification column and an SPE manifold. The column was run using prepared solvent blends varying in polarity from least polar, hexane, to most polar, ethanol. Fractionations were tested for antibacterial activity against S. epidermidis. All fractionations showed zones of inhibition except for one solvent blend, 50% Ethyl Acetate and 50% Ethanol. Fractionations were then sent off to our collaborator at Florida State University for LCMS. Returned data provides us with known weights of molecules that could be the antibiotic(s). Further Analysis of MI3’s genome was conducted by shotgun analysis, also discussed in this presentation.