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The objective of this study is to create a spectrophotometric assay that can measure quinone reduction in the HbRC. The key techniques used in the project consisted of a PCR, a pseudo golden gate, a transformation into E. coli, a conjugation into Heliomicrobium modesticaldum, a growth study, a HbRC prep, and absorbance spectroscopy. PCR was crucial for amplifying the Cyt c553-PshX gene for the pseudo golden gate. The pseudo golden gate ligated Cyt c553-PshX into the plasmid pMTL86251 in order to transform the plasmid with the desired gene into the E. coli strain S17-1. This E. coli strain allows for conjugation into H. modesticaldum. H. modesticaldum cannot uptake DNA by itself, so the E. coli creates a pilus to transfer the desired plasmid to H. modesticaldum. The growth study was crucial for determining if H. modesitcaldum could be induced using xylose without killing the cells or inhibiting the growth in such a way that the project could not be continued. The HbRC prep was used to isolate and purify the Cyt c553-PshX protein. Absorbance spectroscopy and JTS kinetic assay was used to characterize and confirm that the protein eluted from the affinity column was Cyt c553-PshX. The results of the absorbance spectra and JTS kinetic assay confirmed that Cyt c553-PshX was not made. The study is currently being continued using a new system that utilizes SpyCatcher SpyTag covalent linkages in order to attach cytochrome to reduce P800 to the HbRC.
The estimates of metal-ligand equilibrium constants at 25°C and 1 bar were made using multiple linear free energy relationships in accordance with the metal-coordinating properties of ligands such as denticity, identity of electron donor group, inductive effects and steric hindrance. Analogous relationships were made to estimated metal-ligand complexation entropy that facilitated calculation of equilibrium constants up to 125°C using the van’t Hoff equation. These estimates were made for over 250 ligands that include carboxylic acids, phenols, inorganic acids, amino acids, peptides and proteins.
The stability constants mentioned above were used to obtain metal speciation in several microbial growth media including past bioavailability studies and compositions listed on the DSMZ website. Speciation calculations were also carried out for several metals in blood plasma and cerebrospinal fluid that include metals present at over micromolar abundance (sodium, potassium, calcium, magnesium, iron, copper and zinc) and metals of therapeutic or toxic potential (like gallium, rhodium and bismuth). Metal speciation was found to be considerably dependent on pH and chelator concentration that can help in the selection of appropriate ligands for gallium & rhodium based anticancer drugs and zinc-based antidiabetics. It was found that methanobactin can considerably alter copper speciation and is therefore a suitable agent for the treatment of Wilson Disease. Additionally, bismuth neurotoxicity was attributed to the low transferrin concentration in cerebrospinal fluid and the predominance of aqueous bismuth trihydroxide. These results demonstrate that metal speciation calculations using thermodynamic modeling can be extremely useful for understanding metal bioavailability in microbes and human bodily fluids.