The Synthesis of Colicin M Using Cell-Free Protein Synthesis

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The Synthesis of Colicin M using Cell-Free Protein Synthesis Reyes, Reyvin (School: Whitney M. Young Magnet High School) Bacteriocins are proteins that are toxic to bacteria, yet are made by them. These proteins may provide an alternative to traditional antibiotics but synthesizing them using recombinant methods is problematic. A bacteria transformed with a plasmid containing a bacteriocin gene can potentially kill itself by expressing the gene; thus, making it impossible to sustain a growth of living cells and to produce the desired bacteriocin protein. The use of cell-free protein synthesis (CFPS) to create bacteriocins, such as colicin M, avoids this problem. Since the synthesis process does not require live cells, viability is not a concern. This allows for a high yield of the bacteriocins. To conduct CFPS , two major components, a plasmid containing the target gene and bacterial extract containing ribosomes and enzymes, were mixed with amino acids, energy sources, and other reagents to create an artificial cell. The plasmid was created through PCR and Gibson Assembly, and the extract was created by lysing bacteria and isolating relevant cellular components. Both the plasmid and the bacterial extract were successfully made and were mixed with the reagents to synthesize colicin M. After a day of incubation, the protein was purified and the concentration of the protein was found using a NanoDrop. The total concentration of the protein synthesized was 736.67ug/mL. This supports the idea that bacteriocins, can be created using cell-free protein synthesis. Further investigation would involve synthesizing different types of bacteriocins and testing their efficacy at killing bacteria. Awards Won: Fourth Award of $500.

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Colicin E2 Is a DNA Endonuclease
Proc. Nati. Acad. Sci. USA Vol. 73, No. 11, pp. 3989-S993'November 1976 Biochemistry Colicin E2 is a DNA endonuclease (colicin-E2-immunity protein/colicin E3/colicin-E3-immunity protein) KLAUS SCHALLER AND MASAYASU NOMURA University of Wisconsin, Institute for Enzyme Research, 1710 University Avenue, Madison, Wisc. 53706 Communicated by Henry Lardy, September 10, 1976 ABSTRACT Colicin E2 purified by conventional methods immunity protein (20). These authors demonstrated that the contains a tightly bound low-molecular-weight protein, as has immunity protein could be separated from the E3 protein by been found with purified colicin E3 [Jakes, N. & Zinder, N. D. preparative electrophoresis in sodium dodecyl sulfate/poly- (1974) Proc. Nat. Acad. Sci. USA 71, 3380-33841. Such E2 preparations do not cause DNA cleavage in vitro. After sepa- acrylamide gels and that E3 protein (called "ES*") free of ration from the low-molecular-weight protein, colicin E2 re- immunity protein was much more active than the "complexed" tained the original in vivo killing activity, and in addition ES preparation in ribosome inactivation in vitro (20). They also showed a high activity in vitro in cleaving various DNA mole- noted the presence of a small-molecular-weight protein in cules, such as a ColE1 hybrid plasmid and DNAs from Esche- highly purified E2 preparations (cited in ref. 20). From the richia coli, X phage, 4X174 phage, and simian virus 40. The low-molecular-weight protein ("E2-immunity protein") specif- analogy of the colicin E3-immunity protein complex, they ically prevented this in vitro DNA cleavage reaction, i.e., had inferred that this small-molecular-weight protein is probably an "immunity function." The results demonstrate that colicin the E2-immunity protein.
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