The primary goal of the Advanced Technology Platform Centre is to accelerate innovation in biology and biotechnology and thus contribute towards improving the Indian economy. The Centre will plug a huge gap in the innovation pipeline that has previously attenuated the ability of Indian researchers to realize their true potential. Towards this end, the ATPC will house cutting edge technologies to enable researchers within all the constituent partner institutes to conduct experiments that will provide deep insight in biological processes and provide the best opportunity to translate these discoveries for commercialization. At present, all the six facilities are operational in ATPC and these include Protein interaction and Purification Facility, Flow Cytometry facility Mass Spectrometry Facility, Genomics Facility, Optical Imaging facility and Electron Microscopy Facility. Access to these cutting edge facilities will help Indian researchers find new solutions to problems faced by the nation in public health, agriculture and skill development.
Flow Cytometry Facility
The BD Influx™ fluidics system features a unique acoustical coupling in the nozzle assembly to reliably create droplets for sorting, while ensuring low shear stress to optimize cell viability, even at high pressures. To support aseptic sorting, the fluidics path is easily removed and replaced with the optional disposable fluidics kit. Our facility is having Influx Systems with 5 lasers that support 16 simultaneous parameters and 16 x 16 compensation.
The BD Influx™ system features two-, four-, and six-way sorting, as well as plate sorting. The BD Influx electronics support high-speed sorting, and BD FACS™ Accudrop technology helps simplify sort optimization.
(https://www.bdbiosciences.com/us/instruments/research/cell-sorters/bd-influx/m/744777/overview)
A dedicated FACS facility exists at ATPC. The facility has 1) a 2-lasers and 4-colours FACS analyzer (Becton Dickinson), 2) a 3-lasers and 8-colours FACS analyzer (Becton Dickinson), 3) a 3-lasers and 10-colours FACS analyzer (Beckman Coulter), and 4) a high-end, 6-ways cell sorter (Becton Dickinson). The facility is designed for cell counting, sorting, biomarker detection and protein engineering, and multiparametric analysis of the physical and/or chemical characteristics of cells routinely used for the diagnosis of health disorders, and several applications in basic research, clinical practice and clinical trials.
(https://www.beckman.com/coulter-flow-cytometers/gallios)
Mass Spectrometry Facility
Mass Spectrometry enables determination of the relative molecular masses of small molecules as well as of large biomolecules such as proteins and peptides. The ABSCIEX Triple TOF plus 5600 system housed at ATPC is highly sensitive, high-resolution LC-MS/MS mass spectrometer with high mass accuracy and equipped with smart speed acquisition. The Triple TOF 5600 has both ESI and APPI ionization sources and is hyphenated with a Shimadzu UFLC for LC-MS analysis of small molecules and also with an Eksigent Nano LC-Ultra system for use in demanding proteomic applications.
The Advanced Technology Platform Centre (ATPC) of Bioscience cluster is armed with AB SCIEX TOF/TOF 5800 System with ekspert nanoLC 400 and EKSpot MALDI Spotter for comprehensive proteomics applications both qualitative and quantitative. This system facilitates the identification and analysis of the total complement of proteins and other biomolecules. The system also capable of performing to the identification of intact protein, protein/peptide identification and characterization, protein biomarker research, PTM identification and analysis, De novo sequencing, MALDI imaging, gel based and non-gel based applications, quantitative proteomics including labelled (iTRAQ, SILAC etc.), lipid and polymer analysis.
We have set up for sample preparation for protein/peptide identification and cation exchange /high pH fractionation of complex proteome using our HPLC system which is coupled with automated fraction collector for wider coverage of proteome.
Protein Purification and Molecular Interactions Facility
The instruments available in this facility include Bioreactors, FPLC system, Cell homogenizer and High Speed Centrifuge. The facility will aid users in recombinant expression and purification of proteins at preparative and analytical grades.
Monolith NT.115 uses MicroScale Thermophoresis (MST) technology to quantify binding events. One partner is typically labeled with a fluorescent dye and becomes an extremely sensitive reporter for all binding interactions. Monolith is all about accurately determining binding affinity in minutes. The strength of the interactions between a fluorescently labeled or intrinsically fluorescent sample and a binding partner (or ligand) are measured while a temperature gradient is applied over time. These events, weak or strong, cause a change in MST signal that is easily detected by plotting the normalized fluorescence against concentration of ligand. The results are automatically translated into precise and quantitative Kd values.
(https://nanotempertech.com/monolith/)
Bio-Layer Interferometry (BLI) is a label-free technology for measuring biomolecular interactions. It is an optical analytical technique that analyzes the interference pattern of white light reflected from two surfaces: a layer of immobilized protein on the biosensor tip, and an internal reference layer. Any change in the number of molecules bound to the biosensor tip causes a shift in the interference pattern that can be measured in real-time. The binding between a ligand immobilized on the biosensor tip surface and an analyte in solution produces an increase in optical thickness at the biosensor tip, which results in a wavelength shift, Δλ. Unbound molecules, changes in the refractive index of the surrounding medium, or changes in flow rate do not affect the interference pattern. This is a unique characteristic of BLI and extends its capability to perform in crude samples used in applications for protein:protein binding, quantitation, affinity, and kinetics.
Assays can now be performed over a wider temperature range of 15-40°C on the Octet RED96e system, allowing for kinetics measurement of unstable proteins at lower temperatures, or biologically relevant molecules at physiological temperatures. An evaporation cover for microplates also allows for greater walk-away run time, with minimal sample evaporation for up to 12 hours. The new Octet Data Analysis HT software version released with the Octet RED96e system brings custom reporting options and multi-experiment analysis that reduces analysis time from hours to minutes.
(https://www.fortebio.com/octet-RED96e.html)
Biosensor Selection guide can be used to find out senor for particular ligands.
(https://www.fortebio.com/biosensor-types.html)
Genomics Facility
Automated DNA sequencing facility at ATPC is equipped with The Applied Biosystems 3500 Genetic Analyzer is fluorescence based DNA analysis instrument using capillary electrophoresis technology with 8- capillaries. Big Dye terminator chemistry version 3.1 is being used for cycle sequencing . Cloned DNA, PCR products and BAC ends are analyzed using POP7 polymer and 50cm array capable of producing 900 base reads.
ddPCRTM is an advanced version of PCR that provides simultaneous clonal amplification and fluorescence-based quantification of nucleic acids. A typical reaction in ddPCRTM is partitioned into several thousands of water-in-oil droplets where individual reactions are carried out, providing absolute, precise and reliable quantification. This system has demonstrated usage in the detection of rare DNA targets, determination of copy number variations and measurement of gene expression levels.
The system includes: i) QX 200TM Droplet Generator; ii) T100TM Thermal Cycler; iii) PX1TM PCR Plate Sealer; and iv) QX 200TM Droplet Reader. The reaction utilizes nucleic acids, primer sets, fluorescent probes (TaqMan probes with FAM and HEX or VIC), and proprietary supermix for droplet generation. The QX 200TM Droplet Generator generates ~20,000 nanoliter-sized uniform droplets which are transferred to a 96 well plate for clonal amplification. After the reaction, the droplets are read in the QuantasoftTM software bundled in QX 200TM Droplet Reader that counts PCR-negative and PCR-positive droplets and provides an initial DNA concentration of target molecule by statistical analysis in terms of copies/µL."
Optical Microscopy Facility
The High content screening system is custom-designed, fully automated, epi-illumination fluorescence microscope and spinning disk confocal microscopy system with pinhole sizes of 50 µ and 60 µ. The rapid autofocus and precision sample movement features of the microscope allow large numbers of high-resolution images to be acquired in the shortest possible time. All key optical and mechanical elements are motorized, with asynchronous command execution, allowing complete real-time control of the instrument configuration through the MetaXpress® High-Content Image Acquisition and Analysis Software.
• Preferably 2, black 96 well plate clear bottom and one plate for calibration.
Super-Resolution Microscope helps to quantify and comprehend the finest details of cellular and sub-cellular components at a higher resolution beyond the diffraction limits. This microscope is configured for structured Illumination Microscopy (SR-SIM), Total Internal Reflection Fluorescence Microscopy (TIRF), Photo Activation Localization Microscopy (PALM-3D) and Stochastic Optical Reconstruction Imaging (STORM) modules. The system is equipped with 4 diode laser lines- 405, 488, 561, 640 nm and can be used with 4 different objectives- 20x Plan-Apochromat (NA 0.80), 63x C-Apochromat (NA 1.2), 63x Plan-Apochromat NA 1.4), 100x alpha Plan Apochromat (NA 1.46).
Laser Scanning Confocal Microscope is utilized for high optical resolution and contrast of microscopic samples by blocking out-of-focus light with the help of a spatial pinhole. The instrument enables optical sectioning and reconstruction of 3D images by capturing multiple 2D images. This system is equipped with three different objectives 10x, 40x, 63x and lasers for 405, 458, 488, 514, 543, 594, and 633 nm wavelengths for confocal imaging. The system is also supplemented with CO2, O2, humidifying unit and temperature control units and can be used for time lapse/live cell imaging.
CHARGES OF SERVICES AND ACCESS OFFERED BY ATPC
"For more information, please write to services_atpc@rcb.res.in."
