Instrument Development

Analytical Chemistry Tools:

 LC-Tandem MS/MS: The Dismukes lab is equipped with commercial QQQ and Q-TOF mass spectrometers coupled to LC chromatographic separation systems for nano-flow LC and 2D nano-cube LC. These are among the most sensitive mass spectrometers available (lod = Femtogram). They feature robust operation and amenability towards the detection of small, polar metabolites. The use of isotopic-labeled inputs also allows for the direct time-resolved monitoring of pathway fluxes. Chemical extraction parameters have been optimized for Synechococcus sp. PCC7002. Cellular metabolites are extracted by ice cold extraction buffer. The extracts are subsequently separated using Reversed-phase Ion-pairing (RIP) chromatography, the effluent of which is analyzed using a Agilent 6400 Series Triple Quadrupole (QQQ) mass spectrometer. Targeted metabolites are detected via predetermined mass breakdown channels, and concentrations are estimated based on compound-specific calibration curves.

Clarus 680 Gas Chromatograph (GC): Small molecule metabolites like, hydrogen, oxygen and carbon dioxide, the bi-products of fermentation and/or photosynthesis are quantified using Clarus 680 GC with Argon as a carrier gas.

 

Perkin Elmer HPLC : The extracellular excretion of organic acids like lactate, acetate and succinate, by cyanobacteria are quantified using HPLC. Rezex ROA column with 0.005N H2SO4 as mobile phase are used for chromatographic separation and detected using UV detection. HPLC is also used to quantify fatty acids after methyl esterification and detected using IR and UV detection.

 

Proteins are the effectors of most cellular reactions and the main constituent of biomass. In addition, they are the main points of regulation, through transcriptional, posttranslational, and allosteric mechanisms. This regulation enables them to control the contribution of each metabolic pathway to the overall metabolism and cell growth. The abundance and identity of proteins in cells exposed to different environments is being analyzed by sensitive methods like mass spectrometry to identify the targets of regulation and relate these to kinetic choke points in metabolic fluxes.

 

Using the LC-tandem MS approach, we aim to understand the modifications in the proteome that happen under lipid accumulation conditions in diatoms.

 

Genetic Tools:We are using genetic tools that have been developed by our collaborators from Penn State University, the Bryant lab {Bryant Lab links and paper, Frigaard, Sakuragi & Bryant, 2004; Xu et al., 2011} that allow the use of Synechococcus sp 7002 as a robust platform or H2 engineering. This cyanobacteria strain is naturally transformable. Linear DNA construct for a knockout mutation is achieved by inserting an antibiotic cassette with DNA fragments that flank the two terminal regions of the target gene. Transformation is accomplished by adding linear DNA construct into young culture and selecting for transformants on the A+ agar medium supplemented with antibiotics. Additionally, a gene expression system taking advantage of this strain's multiple, endogenous plasmids is developed. The gene of interest can be firstly inserted into a pAQ1_Ex plasmid (Figure below). Then the pAQ1Ex plasmid that carries the gene of interest will be digested to yield a linear DNA fragment for transformation purpose. This linear DNA construct contains a promoter, an antibiotic cassette, the gene of interest, and two flanking regions that are used to transform pAQ1 plasmid in cells through homologous recombination. In a cell growing at exponential phase, the copy number of the pAQ1 plasmid is six times more than the copy number of the genomic DNA. Therefore the gene of interest will potentially be over-expressed through this expression system.

pAQ1Ex plasmid described in Xu et al., 2011. Bryant's Lab.

Instrumentation Development for Renewable Energy Science (NSF-IDBR, NASA-NAI, AFOSR)
Important discoveries are often made by those with the best tools. We have designed and built several powerful instruments that offer major advantages for detection of dissolved O2 and H2 gases, intracellular fluorescence detection of pigments and pyridine nucleotides, and magnetic resonance. These tools are being applied to projects in photobiology, photochemistry and geomicrobiology.

 

Detection of Dissolved Hydrogen
In another approach we are developing ultrasensitive tools for screening for microbial hydrogen production activity from diverse natural habitats. Strains isolated from these screens have been shown to possess better metabolic properties more suited for large scale H2 production. This strategy has identified novel strains from volcanic soda lakes, thermophillic sources and hypersaline aquifers. Data from one of the three powerful tools that we have built is illustrated in the figure, showing detection of dissolved H2 (at 10-8 M sensitivity) produced by induction of hydrogenase activity in the green microalga, Chlamydomonas reinhardtii. The figure illustrates how trains of light pulses of variable pulse duration give rise to different kinetics of H2 production. At higher light duration, more O2 is produced which poisons the hydrogenase enzyme thereby suppressing H2 production. This behavior differs among individual strains and between different species.

Electrochemical Detection 

Detection of Intracellular NADH+NADPH by Fluorescence.
These cofactors are believed to serve as the sole reductants for the hox-class of bidirectional NiFe-hydrogenases. This claim has been inferred almost entirely from in vitro assays. However, there is no direct evidence identifying the cognate reductants for hydrogenase in vivo. Moreover, there is ambiguity in the literature on whether NADH or NADPH or both can serve as reductant for the same hydrogenase in some species. To examine these issues with greater precision, we have constructed an instrument for detection of intracellular fluorescence emission produced by reduced pyridine nucleotide cofactors (total NADH+NADPH) (see Fig.). This instrument includes a second channel for simultaneous electrochemical detection of dissolved H2 using our homebuilt design (2 nanomolar sensitivity). Completed in May 2007, this unique instrument
is providing a wealth of new information about intracellular redox regulation via pyridine nucleotides.

Charge Separation By Fluorescence

Detection of Charge Separation by Fluorescence.
Once of these instruments is a fast-repetition rate laser-based fluorometer that has the capability of measuring the speed and error frequency of photochemical turnover of the PSII O2-evolving complex (OEC) (Ananyev and Dismukes, 2005). The instrument has increased by 100 fold the range of flash excitation rates previously examined using oximetry and allows measurements on intact cells and leaves, samples previously inaccessible without perturbation. With this tool we have been able to characterize the efficiency of PSII-OEC turnover over its full range of turnover frequencies using intact cells and leaves without the need to isolate the PSII enzymes. Fitting of the data to kinetic models for PSII turnover have revealed new control features of the OEC.