Technologies Developed at the ATCG3D
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Synthetic Gene Design and Whole Gene Synthesis |
Nanovolume Microfluidic Crystallization in Confined Geometries |
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Synthetic Gene Design and Whole Gene Synthesis:
Name: Gene Composer™ Software for Synthetic Gene Design and Construct Engineering
Description: Gene Composer™ Express and Gene Composer™ Lite software for computer aided construct engineering and expression optimized synthetic gene design are available for download from www.genecomposer.net/download. Gene Composer™ Express is a complete synthetic gene design suite for Microsoft Windows® which runs on top of a single user Access® database with an Alignment Viewer, Construct Design Module, and Gene Design Module. The Express software has recently been upgraded with features that allow virtual cloning of designed constructs into expression vectors by way of user defined adaptor assemblies that recapitulate most wet lab cloning procedures.
Gene Composer™ Lite is a Java™-based Protein-to-DNA algorithm that allows users to design codon optimized DNA sequences based on the original cDNA or amino acid sequence data. Both the Express and Lite versions of the software allow the design of synthetic genes with all “ACA” sequences silently removed from open reading frames, so that they may be used in conjunction with the MazF system for single protein production.
Once every ~2 months deCODE biostructures hosts a 2 hour on-line ATCG3D workshop on the use of Gene Composer™ software for expression optimized synthetic gene design and protocols for PCR-based gene synthesis from designed oligonucleotides and improved error reduction through the use of mis-match specific endonucleases. Workshops are typically been held on the first Friday of every other month, (June 1, August 3 etc.). Registration and video recordings of workshops are located at www.genecomposer.net/workshop.
Contact: Lance Stewart, lstewart@decode.com
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Nanovolume Microfluidic Crystallization in Confined Geometries:
Name: Nanovolume Plug-Based Microfluidic Crystallization Labcards and System
Description: Originally developed by the Ismagilov lab at the University of Chicago, nanovolume plug-based microfluidic protein crystallization is an emerging technology for efficient protein use for crystallization trials in conjunction with in situ X-ray imaging. deCODE and its microfulidic device collaborators have produced several different plastic labcards that contain of the required microfluidic circuitry to prepare several hundred nanovolume (~20 nl) protein crystallization experiments as aqueous plugs that are carried in a microfluidic channel by an inert fluoropolymeric oil (fluorinert). Three aqueous microfluidic channels for protein, buffer and precipitant converge in a mixer circuit together with the fluorinert carrier fluid (a “3+1 Mixer”). Microsyringe pumps controlled by Microplugger™ software drive the formation of nanovolume aqueous plugs into the fluorinert carrier fluid which enters a long microfluidic holding circuit where final nanovolume crystallization experiments are stored.
Functional 3+1 Mixer + channel holder labcards made out of cyclic olefin copolymer (COC) and polycarbonate (PC) material have been produced with chemical treatments that lower the surface energy of the microfluidic circuitry, making it functional for nanovolume aqueous plug formation. The labcards have proved to be of reasonable reliability in running gradient method nanovolume plug-based protein crystallizations using test proteins (lysozyme and thaumatin) as well as other experimental proteins. In situ X-ray diffraction on in-house sources have been demonstrated for both COC and PC types of labcards with reasonable success. deCODE recently presented the technology together with the Ismagilov lab at the Brookhaven Crystallization Course.
deCODE’s sister company, Emerald BioSystems is responsible for commercialization of the Ismagilov technology for nanovolume plug-based microfluidic crystallization (labcards and microfluidic pumping systems). Emerald has secured industrial and academic beta test site customers for the technology, and hopes to disseminate the technology broadly once the beta testing is complete.
Contact: Lance Stewart, lstewart@decode.com
Name: Microfluidic Hybrid Method for Protein Crystallization
Description: We developed a microfluidic "hybrid" method that combines sparse-matrix and optimization-gradient screening into one simple experiment and uses nanoliter-sized plugs to minimize sample consumption (PNAS 2006 103: 19243-19248). Many distinct reagents were sequentially introduced as “large” plugs (~ 140 nL) into a microfluidic device. Large plugs were combined with a protein sample and a diluting buffer so that multiple ~10 nL plugs of each reagent were formed over a range of concentrations. The concentrations of each reagent were well-controlled by a computer subroutine and were indexed with plug size. We validated the hybrid method by demonstrating its applicability to two challenging problems: i) crystallization of model membrane proteins and ii) handling of detergent solublized membrane protein and viscous precipitants. We overcome these challenges with two technical developments: i) the use of perfluoroamines as carrier fluids, and ii) the use of Teflon capillaries for the formation, transport, and storage of plugs. We obtained high-quality crystals of membrane proteins and solved a crystal structure. This robust method requires inexpensive equipment and supplies, is accessible to most basic individual laboratories, and could find applications in a number of areas that require chemical, biochemical or biological screening and optimization.
Contact: Rustem Ismagilov, rustem@uchicago.edu
Name: Plug-Based Microfluidic Seeding Method for Protein Crystallization
Description: We have developed a microfluidic method of seeding to separate and independently control the stages of nucleation and growth in protein crystallization (Angew. Chem. Int. Ed. 2006 45: 8156-8160). This seeding method enables the generation of high quality protein crystals in nanoliter volumes with minimal sample consumption, and it is especially suitable for crystallizing proteins that display a supersaturation gap on their phase diagram. In a typical experiment, seeds are first generated in plugs under high supersaturation conditions in the nucleation stage. Next, these seeds are merged with aqueous plugs at lower supersaturation to initiate the growth stage and allow formation of ordered crystals. This method was validated by growing crystals of Oligoendopeptidase F. The crystal structure of Oligoendopeptidease F was solved at 3.3 Ǻ.
Contact: Rustem Ismagilov, rustem@uchicago.edu
Name: DETECT-X Crystal Imaging Technology
Description: We have developed a DETECT-X (Difference Extinction for the Detection of Crystals) microscope system and associated image processing algorithms for improved crystal imaging. The DETECT-X microscope is a fully automated polarization and UV fluorescence microscope for inspecting crystallization trials. Through the analysis of four polarization images (at 0°, 45°, 90°, 135°) the system calculates quantitative values for the transmission, the birefringence, and the extinction angle at each pixel. Each of these values can then be displayed as a false-color image and together these images provide better contrast for identifying crystalline material. In its birefringence microscopy mode the DETECT-X microscope produces quantitative images of crystals depicting the orientation of the slow optical axis of protein crystals and their orientation-independent, quantitative birefringence. Such images allow the detection of crystals that are masked by precipitate. Spherulites and microcrystalline material can be readily distinguished from amorphous precipitate. As a result, there are fewer false negatives and more potential leads are identified for crystal optimization. Twinned crystals can also be readily identified, so that the best crystals for diffraction experiments can be selected. The use of in situ UV fluorescence microscopy also helps to distinguish between salt crystals and protein crystals. It is anticipated that DETECT-X microcopy improved efficiencies in optical screening of protein crystallization trials.
Contact: Peter Nollert, pnollert@emeraldbiosystems.com
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