Document Type

Honors Project

First Advisor

Dr. Stephen F. Baron

Degree Award Date

2022

Keywords

Cloning, Overexpression, Bacteriophage, Endolysin, Gene, Escherichia coli, Transformation, DNAM5_40, DH5α, BL21(DE3)RIL

Disciplines

Biology | Microbiology

Abstract

Endolysins are enzymes used by bacteriophages (bacterial viruses) at the end of their replication cycle to degrade the peptidoglycan layer of their host bacterium from within the cell. This action weakens the integrity of the bacterial cell wall, resulting in lysis and release of the newly synthesized bacteriophages. Research suggests that endolysins could be used in place of antibiotics to fight bacterial infections in humans by targeting disease-causing bacteria without harming desirable bacteria or human cells, circumventing some of the problems with antibiotic therapy. A bacteriophage specific to Streptomyces sp. SFB5A, Brock, was previously isolated and its genome sequenced. Its genome contained two potential endolysin genes. Our goal was to clone and overexpress one of these genes, DNAM5_40, in Escherichia coli, purify the endolysin, and evaluate its activity against bacterial cell walls. We successfully cloned the E. coli DH5α transformants into E. coli BL21(DE3)RIL but were unable to get the protein to express despite conducting a temperature study, inducing with different IPTG concentrations, and trying several different media.

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