KAIROS Scientific Research and Development

Selected Grant Abstracts

NIH RO1 "Optimization of Combinatorial Mutagenesis"

The principal goal of this work is to obtain antibody Fab fragments to targets of medical importance, including small molecules which are acute toxins or other substances which are abused. Specific Fabs would become lead molecules for the development of therapeutic antibodies. Most of this work now focuses on optimizing mutagenesis procedures.

DOE "Macromolecular Scaffolds for Energy Studies"

Combinatorial chemistry techniques will be applied to the problem of isolating macromolecular scaffolds (either protein or DNA) that bind ligand pairs which interact to achieve photochemical reactions. Ultimately, these studies could lead to a photocatalytic scaffold that mediates turnover of photochemical reactants. These 'photobodies' are analogous to catalytic antibodies and ribozymes, given that light rather than thermal energy is used. Fluorescence Resonance Energy Transfer experiments on GFP are supported by this grant.

NASA Phase 2 SBIR, "High Resolution Imaging Microscope"

The feasibility of obtaining high resolution spectra of individual marine and freshwater microorganisms was demonstrated in Phase 1. To perform this task, a digital imaging spectrophotometer (DIS), previously configured for macroscopic samples, has been redesigned and constructed. The prototype instrument, HIRIM (High Resolution Imaging Microscope) has been used to simultaneously acquire spatial and spectral information of hundreds of algae and cyanobacteria immobilized on slides. Peaks due to chlorophyll, phycobilins, carotenoids and other pigments can be readily determined from individual cell spectra. In Phase 2, we will expand this spectral database of known microbial samples as well as continue HIRIM hardware and software developments. The success of the Phase 1 feasibility trials has motivated us to broaden our Phase 2 goals and include the spectral analyses of other targets ranging from mineralogy to plant sciences to pathology. All of these fields have the common denominator of using HIRIM to determine the spectra of complex groupings of microscopic features in a massively parallel manner.

NIH Phase 2 SBIR, "Multispectral Fluorescent Proteins"

High through-put mutagenesis and screening methods were developed during Phase 1 for isolating spectral and kinetic variants of the Green Fluorescent Protein (GFP). Mutants were found that show large spectral shifts and accelerated fluorophore formation relative to wild-type. Optical screening of approximately 500,000 combinatorial mutants near the chromophore site yielded two main classes of proteins with blue-shifted emission (BFP) or red-shifted excitation (RSGFP). By shifting the excitation maximum from 390 to 490 nm, the RSGFP spectrum is well separated from cellular autofluorescence, making it vastly superior to wild-type GFP for epifluorescence microscopy and fluorescence activated cell sorting (FACS). In Phase 2, we plan to search for red-shifted emission derivatives of RSGFP using a method capable of negotiating very rough fitness landscapes. RSGFP and a red-shifted emission derivative would constitute an extremely valuable pair of molecular reagents, making two channel FACS, dual color epifluorescence microscopy, and fluorescence resonance energy transfer measurements readily available to cell biologists. In conjunction with imaging spectroscopy, the construction of a series of spectroscopically distinct red-shifted emission derivatives (e.g., green, yellow, orange, and red) would enable simultaneous analysis of several tagged promoters, fusion proteins, and/or cell types.

NIH Phase 2 SBIR, "Fluorescence Imaging MicroSpectrophotometer: FIMS"

The prototype Fluorescence Imaging MicroSpectrophotometer (FIMS) developed in Phase 1 will be reduced to a turn-key system for applications in a variety of cell biology experiments which require quantitative analysis of complex spatial and spectral patterns. Spatially coregistered fluorescence excitation, fluorescence emission, and absorption image stacks will be acquired on a microscope using fully tunable wavelength selection devices for both incident and emitted light. Special emphasis will be placed on multispectral fluorescence experiments using the Green Fluorescent Protein (GFP) as a tag for promoters and proteins in eukaryotic cells. Because of the spectral overlap in excitation and/or emission of GFP derivatives, quantitative methods will be implemented to determine the relative concentration of each fluorophore for every pixel in the scene. Hardware and software will be developed to combine fluorescence image stacks with brightfield image stacks to show the distribution of fluorophores in various tissues or cell types against a background stained by conventional dyes (e.g., H&E). Quantitative imaging methods will also be used to determine whether two proteins are interacting, as indicated by fluorescence resonance energy transfer (FRET) between tagged proteins. In all applications, hyperspectral pseudocoloring techniques will be further developed so as to provide a concise and quantitative representation of the critical information within the image stack.

NIH Phase 2 SBIR, "Solid Phase Enzyme Screening"

During Phase 1, a prototype MicroColonyImager (MCI) and colorimetric solid phase assays were developed to screen bacterial libraries expressing mutagenized enzymes for enhanced activity. This high-throughput assay can detect less than a 3-fold difference in enzyme rates within microcolonies grown at a nearly confluent density of 500 colonies per cm² on an assay disk. Each microcolony is analyzed simultaneously at single-pixel resolution (1.5 megapixels; 75 micron/pixel), requiring less than 100 nanoliters of substrate per measurement, a 1000-fold reduction over current liquid phase assays. By simultaneously assaying two different substrates tagged with spectrally distinct chromogenic reporters, we have successfully used this technique to change the substrate specificity of a beta-glucosidase from glucoside to its epimer, galactoside. In Phase 2, we will refine MCI hardware and software to enable high-throughput screening of enzyme libraries by time course analyses of single-pixels, using either absorption, fluorescence or FRET. Membrane disk size and colony density will be increased to image ~50,000 microcolonies simultaneously, while decreasing substrate volumes to ~10 nanoliters per measurement. Concurrent with instrument development, we will demonstrate the commercial value of this technology by screening libraries undergoing directed evolution, including four model enzymes. This technology should lead to the isolation of new enzyme activities that are useful in the synthesis of various substances, including pharmaceuticals.

NIH Phase 2 SBIR,  "Multispectral Bacterial Identification"

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