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In situ conversion of recalcitrant petroleum sources, such as heavy oil, oil sands, and asphalts, to methane by microorganisms would have significant environmental and economic advantages over current mining and extraction methods.  For such an approach to become practical, an understanding of the natural biogeochemical cycles in such environments is required, including the roles and characteristics of the indigenous microorganisms.

Toward that end, we are investigating mechanisms of metal uptake and regulation in Methanocorpusculum labreanum Z, a methanogenic archaeon from the Tar Pits at Rancho La Brea, Los Angeles.

Using the publicly available genomic data, a putative metalloregulatory protein (MarR) was identified in Methanocorpusculum labreanum Z.  Undergraduate research student Elizabeth Edwards used PCR to amplify the gene for MarR from genomic DNA generously provided by Dr. Iain Anderson, Lawrence Berkeley National Laboratory.  The resulting PCR product was purified using a PureLink PCR Purification Kit (Invitrogen) and characterized by gel electrophoresis.  The purified PCR product was cloned into an E. coli plasmid using TOPO cloning techniques (Invitrogen).  Successful cloning was confirmed by DNA sequencing (GeneWiz, Inc.).

The MarR protein was then overexpressed in E. coli.  Preliminary experiments using lysozyme to effect cell lysis were unsuccessful, as the lysozyme interfered with subsequent protein characterization by gel electrophoresis (SDS-PAGE).  An alternate protocol using ultrasonication for cell lysis was developed and was highly successful.  Following lysis, the cell debris were removed by centrifugation.  The crude cell extract was purified by FPLC using a combination of size exclusion chromatography and ion exchange chromatography and was characterized by SDS-PAGE.  Mass spectrometric characterization is currently underway.

Preliminary results from this project have been presented at two Gordon Research Conferences in the past year: the Inorganic Reaction Mechanisms GRC in Galveston, TX, in March 2009, and the Vitamin B₁₂ and Corphins GRC in Oxford, England, in August 2009.  PRF  funds paid the stipend of one undergraduate research student for Summer, 2009.  Two other undergraduates have also been working on the PRF-supported project; their stipends were provided by an institutional grant to Barnard College from the Howard Hughes Medical Institute.  In addition, these two students have been performing undergraduate research for course credit during the academic year.

Since July 2008, seven undergraduate students (six female and one male) have worked in my research group.  Three of those students have worked on the PRF-funded project, and one has been directly supported by the PRF grant.  All seven, however, have benefited from PRF support of my group’s research.  Five of these students are continuing to work in my laboratory this year, one is now working on a senior thesis with Prof. Christian Rojas at Barnard, and one is now a graduate student in chemical engineering at MIT.

Based on preliminary results from the PRF-funded research project, we have submitted  proposals to NSF to acquire additional instrumentation to support this work.   Progress to date on this project would not have been possible without the support of PRF; with PRF support, we now have a novel purified protein in hand and are beginning detailed studies of its coordination chemistry and metal-binding thermodynamics.  Continued rapid progress is anticipated in year two of the funded project.