This study is focused on comparing the wild type and mutated versions of the Vitreoscilla hemoglobin gene (vgb). The purpose of this focus is... Show moreThis study is focused on comparing the wild type and mutated versions of the Vitreoscilla hemoglobin gene (vgb). The purpose of this focus is to find out whether any of the vgb mutations provides an advantage regarding cell growth rate, as well as on the expression level of Vitreoscilla hemoglobin protein (VHb). A negative control Escherichia coli DH5α (E. coli DH5α) bearing no pUC plasmid, as well as seven E. coli DH5α strains bearing different pUC-based plasmids were tested in the experiments. Among these were one vector-only negative control (pUC18), one wild type positive control (pUC8:16, which carries wild type vgb) and five different types of pUC-bearing vgb mutants (pUC-vgb M1, M2, M3, M4 and pUC18-vgb M3). In order to compare cell growth rate among these strains, the growth rate assay was carried out under three different conditions: (1) Luria-Bertani (LB) medium, aerobic conditions; (2) Terrific Broth (TB) medium, low oxygen conditions; and (3) TB medium, microaerobic conditions. In addition, the carbon monoxide (CO) difference spectra assay was conducted to measure functioning VHb protein expression levels for the strains grown under aerobic conditions. In contrast to the results obtained by our Australian collaborators, our growth rate assay and CO difference spectra assay showed no growth advantage or higher expression level of functioning VHb protein due to any of the vgb mutations. For the further study of the vgb mutants, four different recombinant plasmids were constructed by cloning three types of mutated vgb (vgb M1, M3 and M4) as well as wild type vgb into the prokaryotic expression vector pUC8 with ampicillin (Amp) resistance. After being transformed into competent E. coli DH5α cell, these resulting xii strains, as well as the plasmid-free negative control (E. coli DH5α) and vector-only negative control (E. coli DH5α bearing plasmid pUC8) were tested by the CO difference spectra assay. Except strain E. coli DH5α [pUC8-vgb M3], which showed a slight increase in the VHb expression level, the strain bearing other mutated vgbs did not demonstrate any elevation in VHb protein expression level, compared to the positive control containing wild type vgb. M.S. in Biology, May 2015 Show less
