VITREOSCILLA HEMOGLOBIN: STRUCTURE-FUNCTION AND GENETIC ENGINEERING STUDIES

Paenibacillus strains 32O-W and 32O-Y were attempted to be transformed by electroporation with constructed plasmid pNW33N-vgb, modified to contain the vgb gene which can be expressed as Vitreoscilla hemoglobin. Only attempts with 32O-Y were successful. Transformed 32O-Y/pNW33N-vgb was grown in CDM medium with dibenzothiophene (DBT) as the sole source of sulfur at different temperatures. Dramatic variability was observed in culture at different temperatures, so only the data at 45 °C was analyzed. The growth assay showed that the 32O-Y/pNW33N-vgb strain grew slower than untransformed 32O-Y, although Gibbs assay showed improvements in utilizing ability of DBT of 32O-Y/pNW33N-vgb compared to untransformed 32O-Y. This finding indicated that genetic engineering of introducing vgb into 32O-Y may cause deterioration in cell growth rate but improvement in desulfurization activities. Plasmid pUC-vgb-M2 was transformed into E. coli DH5α. The transformed DH5α/M2-vgb was cultured along with DH5α/pUC8:16, bearing plasmid pUC8:16 that was previously constructed in our lab and can be expressed to produce wild type VHb, and untransformed DH5α. CO-difference spectra were performed with the lysed cultures for the detection of VHb expression. As a result, DH5α/M2-vgb was confirmed to lack the ability to express functional VHb.M.S. in Biology, May 201