A comparison of mutant versus wild type versions of the Vitreoscilla hemoglobin gene (vgb) were the focus of this study. E. coli DH5a cells... Show moreA comparison of mutant versus wild type versions of the Vitreoscilla hemoglobin gene (vgb) were the focus of this study. E. coli DH5a cells were transformed with recombinant pUC plasmids containing either the wild type or one of a series of mutated versions of the vgb gene. The study was conducted in two pmis. In the first part, eight strains in total were tested (one plasmid-free negative control, one vector-only (pUC18) negative control, one wild type positive control (pUC8: 16), and five mutants (pUC vgb M1-4 and pUC18 vgb M3)) and comparisons were made to reveal growth advantage or possible increased Vitreoscilla hemoglobin protein (VHb) expression due to vgb gene mutation. Three different growth assays were carried out to compare growth rates among the eight strains under different oxygen concentration conditions: (1) Luria-Bertani (LB) medium, aerobic growth assay, (2) Terrific Broth (TB) medium, low-oxygen growth assay, and (3) TB medium, microaerobic growth assay. Additionally, a series of carbon monoxide (CO) difference spectra were run to quantify functioning VHb protein concentrations in the different strains grown under each of the aforementioned oxygen concentration conditions. In the second part of the study, the two vgb gene mutants that exhibited the highest growth rates and the wild type version were subcloned into the vector pUC8, transformed into E. coli DH5a cells, and then compared using the CO difference spectrum assay. Five E. coli DH5a strains were tested (one plasmid-free negative control, one vector-only negative control (pUC8), one vgb wild type positive control (pUC8 vgb WT), and two vgb mutants (pUC8 vgb M1 and M3)). Transfmmation of plasmids containing the mutated or unmutated version of the vgb gene was verified through E.Z.N.A. plasmid miniprep with gel electrophoresis and additionally through growth on selective medium containing ampicillin (Amp) [50 micrograms (ug)/ milliliter (ml)]. Contrary to the results obtained by our Australian collaborators, our growth assays and CO difference spectra revealed no growth advantage or increased expression of functioning VHb protein due to any of the vgb mutations. M.S. in Biology, May 2015 Show less
