Mutant Transformation of Pathogenic Neisseria Strains

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Ajudua, Emmanuel
The University of Maryland McNair Scholars Undergraduate Research Journal, Vol. 3, 2011: 25-33.
Neisseria gonorrhoeae is a bacterium that produces ~700,000 new gonococcal diseases in the US each year. While gonorrhea is localized to the urethra in men, in women it can spread to the cervix, uterus and fallopian tubes. Lipooligosaccharide (LOS) is a surface antigen embedded in the outer membrane of all Neisseria strains that causes disease. However, with commensal Neisseria strains disease does not occur. The commensal strains, with the exception of two, all contain a third heptose attached to their core. We hypothesized that if a third heptose could be added to the LOS Beta chains of Neisseria gonorrhoeae, then the LOS would become less virulent. The proposed gene sequence that codes for the HepIII transferase (enzyme that adds heptose) was located. The DNA was extracted and run in a PCR reaction to amplify the proposed gene sequence, which was then purified. PLES2 plasmid DNA was introduced to competent DH5aMCR cells by heat shock transformation to make multiple copies. The resultant cells were plated onto LB+Ampicillin+Xgal for selectivity and restreaked for growth. As research continues the plasmid will be isolated from the E. coli and the purified gene will be inserted into the plasmid. After a few tests, the plasmid with the gene will be inserted into Neisseria gonorrhoeae to observe effects.