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A number of projects are available in our Lab:
Mentor:
Characterization of Photosynthetic Water Oxidation Efficiency in the Hydrogen-Producing Green Alga Chlamydomonas reinhardtii
Optimizing primary photosynthetic productivity is a key step in applying aquatic microbial oxygenic phototrophs (AMOPs) to bio-energy solutions. In this project, the student will work with a graduate student mentor to characterize the efficiency of photosynthetic water oxidation in strains of the green alga Chlamydomonas reinhardtii using laser-based fast repetition rate fluorometry (FRRF) and amperiometric oxygen detection. The student’s primary focus will be a mutant randomly generated in a –hydA2 background that does not produce photo-hydrogen in vivo, but has been shown have in vitro hydrogenase activity and can grow photoautotrophically. This observation indicates that the molecular machinery needed to directly produce hydrogen is present in the cell, but some electron carrier between Photosystem II and hydrogenase is defective. Using FRRF and inhibitors of key steps in the photosynthetic electron transport chain, we aim to understand the molecular basis of this interesting phenotype. A student working on this project can expect to learn methods related to algal cell culturing (including the use of photobioreactors and turbidostats) and sophisticated biophysical techniques (primarily those based on fluorescence and electrochemistry).
Mentor: Anagha Krishnan
Using Adenylate Kinase deletion mutants to increase ATP availability:
One of the enzymes that affect the ATP/ADP ratio, adenylate kinase (ADK), acts on ATP to convert to it to ADP.
2 ADP ⇔ ATP + AMP
During photosynthesis, ATP levels increase and therefore adenylate kinase would act to decrease the ATP levels. Thus we propose that a deletion/knockdown of this enzyme may be useful in directing the available ATP towards ATP utilizing pathways like Glycogen accumulation rather than getting converted to ADP. The proof of concept experiments have been done in Arabidopsis where, deletion mutants of Arabidopsis have shown an increase in biomass accumulation and growth when the mutant plants were grown in the presence of continuous illumination as compared to wild type plant. In potato plants carrying a knockdown mutation of this gene starch accumulation in the tubers was increased. Adenylate kinase deletion mutants would be useful in not just increasing the biomass accumulation in the form of Glycogen but also in generating higher nitrogenase based hydrogen production. Adenilate kninase knockout mutants will be generated and characterized in order to test this hipothesis.
Mentor: Tiago Guerra
Nitrogen depletion has been described as a condition capable of increasing the lipid accumulation in several algal strains including Phaeodactylum tricornutum. However the pathways and the molecular mechanism that leads to this higher lipid accumulation is still elusive.
Real-time qPCR was performed for a short list of 21 genes that can provide indication of the pathways that are activated and the genes that play a key role in increasing lipid accumulation under several nitrogen availability conditions (+NO3, +NO3+W, -NO3, +NH4 and +NH4+W). Several genes showed high levels of regulation in response to the different nitrogen conditions. Most notably many genes of the TCA cycle and glycolysis are up regulated upon nitrogen depletion (-NO3 and +NO3+W cases), nitrate reductase is highly upregulated in the same conditions. In contrast, there was a general downregulation of gene expression in the ammonia case. In all conditions tested however there was an upregulation of one of the gene models coding for the DAGAT (an essential gene for TAG synthesis) but not of Acetly-CoA Synthase (the enzyme responsible for the committed step of lipid biosynthesis).
Nitrogen is a main component of amino acids and limiting the availability of this nutrient might limit protein synthesis. Thus, one must investigate if the differential gene expression observed is translated into protein levels. For this purpose we propose to investigate the levels of select proteins by tandem mass spectrometry utilizing the quadrupole time-of-flight (Q-TOF) instrument. The student will be involved in protein sequence bioinformatic analysis, protein extraction methods, gel elecrtophoresis, mass spectrometry procedures and data analysis
Mentor: Nicholas Skizim
Cyanothece is a genus of unicellular cyanobacteria which are capable of fixing atmospheric nitrogen. This capability (and necessity under nitrogen deplete growth conditions) requires a unique metabolism because of the ATP demand and O2 sensitivity of nitrogenase (the enzyme responsible for the reduction of N2 to NH3). One can take advantage of the inducible expression of nitrogenase under dark (and often anoxic) conditions and observe photo-H2 production from the strain by supplying the organism with intense light. The light energizes the necessary reductant for nitrogenase, which produces hydrogen concomitant with N2 reduction. When N2 is limiting, a larger fraction of the light-generated reductant can be directed to proton reduction, and higher yields of hydrogen can be achieved. We have observed high rates of photo-H2 production in Cyanothece sp. Miami BG043511, and even higher rates have been reported (but not confirmed by our lab as of yet) in Cyanothece sp. ATCC 51142. We maintain both of these strains in the lab.
This project aims to study the proteome of Cyanothece under light/aerobic conditions and compare it to that of dark/anoxic conditions. We will be able to quantify the increase or decrease in protein concentration within the cell, particularly nitrogenase, uptake hydrogenase, and enzymes involved in cellular respiration and glucose oxidation. Changes in the proteins comprising the photosynthesis apparatus (PSI and PSII) will also be noted. Observed differential protein concentration will aid us in understanding how such high rates of hydrogen production can be achieved, and how the nitrogenase can protect itself from O2 poisoning. These levels will also suggest pathways which are limiting hydrogen metabolism in the strain and thus will serve as future targets for optimization.
Specific Hypothesis: The differential proteomes under (a) photoautotrophic and (b) dark/anoxic conditions will reveal the mechanism of (a) O2 protection of nitrogenase, (b) the high yield of photo-H2 observed in the strain(s), and (c) the limiting steps in reductant transfer to nitrogenase, which could serve as future targets for optimization.
Mentor: Paul Smith
The focus of this project will be on cubane syntheses and characterization.
1. Mn cubes with substituted ligands which offer potential for the attachment to TiO2, ITO, etc.
Where R = NO2, NH2, amide groups.
In this project I aim to also include phosphorylation of organic linker molecules. From this perspective, we would thus have two concepts: a) target molecules to make specifically for testing attachment, and b) target molecules which are known to attach, but do not have the PO2 substituent on the other end. Path b) can potentially lead to catalyst assembly on TiO2 surfaces, which can allow for future robust syntheses.
2. Development of an original, Ruettinger-style synthesis of Co cube derivatives, and expand to include a variety of substituted pyridine complexes for similar reasons.
Mentor: Graeme Gardner
Abstract: For the purposes of advising an undergraduate student this spring, it would be prudent to use the added help to push forward on coupling the LiCoO2 powders to electrodes using an electrophoretic deposition methodology. The student would be responsible for examining the effect of electrolyte concentration, electrolyte type, suspension concentration, and applied voltage on the deposition thickness and time. The LiCoO2 suspensions would be inherently neutral non-ionic solutions and therefore necessitate the use of either acid or electrolyte to initiate the migration through surface charging. Also, since these catalysts are in fact active for water oxidation, the deposition suspension must be a non-aqueous solvent (applied voltages far exceed the overpotential required for oxidizing water ~10 V), potentially complicating issues further but through properly designed experiments, this technique holds promise for quickly fabricating uniform, physically-adsorbed layers of LiCoO2 and the like across an appropriate surface. Ultimately, the newly formed electrodes would be tested for oxygen evolution and catalytic current stability over a reasonable number of turnovers.
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