Deposition of Thin Films of Biocompatible Calcium Carbonate via Template-Driven Mineralization

Natural bone is a composite of collagen based hydrogel and inorganic dahilite crystals. The unusual combination of a hard inorganic material and an underlying elastic hydrogel network endows native bone with unique mechanical properties, such as low stiffness, resistance to tensile and compressive forces and high fracture toughness. Throughout the cavities of the bone, there are bone cells and myriads of soluble and extracellular matrix components that are constantly involved in the bone formation and remodeling process. Among the extra cellular component the acidic matrix proteins that are attached to the collagen scaffold play important templating and inhibitory roles during the mineralization process. It would be interesting to generate such functional scaffolds that mimic a template driven mineralization and which can assist cell adhesion, proliferation, migration and differentiation. Towards this direction, we have chosen one synthetic (Nylon 66 membrane) and one natural (eggshell membrane) scaffold and carried out a template driven mineralization of CaCO₃ as model systems. The surface modifications were carried out by the pre-adsorption of acidic polymers before the deposition of the CaCO₃. The deposition of the crystalline calcium carbonate on these modified templates were archived from a supersaturated solution of Ca(HCO₃)₂.Singapore-MIT Alliance (SMA

Eggshell Matrix Protein Mimetics: Elucidation of Molecular Mechanism of Goose Eggshell Calcification using Designed Peptides

Model peptides were designed, synthesized and conducted a detailed structure-property study to unravel the molecular mechanism of goose eggshell calcification. The peptides were designed based on the primary structural features of the eggshell matrix proteins ansocalcin and OC-17. In vitro CaCO₃ crystal growth experiments in presence of these peptides showed calcite crystal aggregation as observed in the case of the parent protein ansocalcin. The structure of these peptides in solution was established using intrinsic tryptophan fluorescence studies and quasi-elastic light scattering experiments. The structural features are correlated with observed results of the in vitro crystallization studies.Singapore-MIT Alliance (SMA

Designer peptides to understand the mineralization of calcium salts

Recently, we reported the extraction, purification and amino acid sequence of ansocalcin, the major goose eggshell matrix protein. In vitro studies showed that ansocalcin induces spherical calcite crystal aggregates. We designed two peptides using the unique features of the sequence of ansocalcin and the role of these peptides in CaCO₃ crystallization was investigated. The peptides showed similar activities as compared to ansocalcin, but at a higher concentration. The full characterization of the peptides and a rational for the observed morphology for the calcite crystals are discussed in detail.Singapore-MIT Alliance (SMA

SCAPA (Spacially Addressable Protein Array) – a Novel Protein Array for the Differential Profiling of Ligand-receptor Induced Signalling

While protein microarray technology has been successful in demonstrating its usefulness for large scale high-throughput proteome profiling, performance of antibody/antigen microarrays has been only moderately productive. Immobilization of either the capture antibodies or the protein samples on solid supports has severe drawbacks. Denaturation of the immobilized proteins as well as inconsistent orientation of antibodies/ligands on the arrays can lead to erroneous results. This has prompted a number of studies to address these challenges by immobilizing proteins on biocompatible surfaces, which has met with limited success. Our strategy relates to a multiplexed, sensitive and high-throughput method for the screening quantification of intracellular signalling proteins from a complex mixture of proteins. Each signalling protein to be monitored has its capture moiety linked to a specific oligo âtag’. The array involves the oligonucleotide hybridization-directed localization and identification of different signalling proteins simultaneously, in a rapid and easy manner. Antibodies have been used as the capture moieties for specific identification of each signaling protein. The method involves covalently partnering each antibody/protein molecule with a unique DNA or DNA derivatives oligonucleotide tag that directs the antibody to a unique site on the microarray due to specific hybridization with a complementary tag-probe on the array. Particular surface modifications and optimal conditions allowed high signal to noise ratio which is essential to the success of this approach.Singapore-MIT Alliance (SMA

Protein Microarray: "Theory" to "Real Practice"

Fueled by ever-growing genomic information and rapid developments of proteomics–the large scale analysis of proteins and mapping its functional role has become one of the most important disciplines for characterizing complex cell function. For building functional linkages between the biomolecules, and for providing insight into the mechanisms of biological processes, last decade witnessed the exploration of combinatorial and chip technology for the detection of bimolecules in a high throughput and spatially addressable fashion. Among the various techniques developed, the protein chip technology has been rapid. Recently we demonstrated a new platform called “Spacially addressable protein array” (SAPA) to profile the ligand receptor interactions. To optimize the platform, the present study investigated various parameters such as the surface chemistry and role of additives for achieving high density and high-throughput detection with minimal nonspecific protein adsorption. In summary the present poster will address some of the critical challenges in protein micro array technology and the process of fine tuning to achieve the optimum system for solving real biological problems.Singapore-MIT Alliance (SMA

The future of metabolic engineering and synthetic biology: Towards a systematic practice

Industrial biotechnology promises to revolutionize conventional chemical manufacturing in the years ahead, largely owing to the excellent progress in our ability to re-engineer cellular metabolism. However, most successes of metabolic engineering have been confined to over-producing natively synthesized metabolites in E. coli and S. cerevisiae. A major reason for this development has been the descent of metabolic engineering, particularly secondary metabolic engineering, to a collection of demonstrations rather than a systematic practice with generalizable tools. Synthetic biology, a more recent development, faces similar criticisms. Herein, we attempt to lay down a framework around which bioreaction engineering can systematize itself just like chemical reaction engineering. Central to this undertaking is a new approach to engineering secondary metabolism known as ‘multivariate modular metabolic engineering’ (MMME), whose novelty lies in its assessment and elimination of regulatory and pathway bottlenecks by re-defining the metabolic network as a collection of distinct modules. After introducing the core principles of MMME, we shall then present a number of recent developments in secondary metabolic engineering that could potentially serve as its facilitators. It is hoped that the ever-declining costs of de novo gene synthesis; the improved use of bioinformatic tools to mine, sort and analyze biological data; and the increasing sensitivity and sophistication of investigational tools will make the maturation of microbial metabolic engineering an autocatalytic process. Encouraged by these advances, research groups across the world would take up the challenge of secondary metabolite production in simple hosts with renewed vigor, thereby adding to the range of products synthesized using metabolic engineering.National Institutes of Health (U.S.) (1-R01-GM085323-01A1)Special Research Funds BOF (BOF08/PDO/014)Research Foundation Flanders (FWO-Vlaandern V.4.174.10.N.01

Design, Fabrication and Functional Analysis of a New Protein Array Based on ssDNA-based Assembly

In the post genomic era, proteomics has enormous potential in biology and medicine. Among the various bioanalytical tools developed, protein microarray is one of the recent advancements which offer high throughput profiling of cellular proteins to provide insights into the mechanisms of biological processes. Fundamentally, the protein microarray involves the immobilization of interacting elements, proteins, on a few square microns of a solid support and in principle, it is capable of detecting analytes with a higher sensitivity than conventional macroscopic immunoassays. Here in the present report we delineates the design, fabrication and functional analysis of protein microarray using semi-synthetic ssDNA tagged-proteins as capturing moiety as well as address on a solid support. Optimization of the platform has been carried out by investigating various parameters such as surface chemistry, signal amplification, and conditions for homogenous liguid phase protein-protein interaction.Singapore-MIT Alliance (SMA

DNA Directed Assembly Probe for Detecting DNA-Protein Interaction in Microarray Format

Quantifying DNA-protein interaction using DNA microarrays are gaining increasing attention due to their ability to profile specificity of interactions in a high-throughput manner. This paper describes a new approach that used the ability of ssDNA-dsDNA probe to complex with DNA binding proteins in the solution phase and then spatially immobilized onto microarray through specific DNA hybridization. In one case, the Spatially Addressable DNA Array (SADA) approach demonstrated that enzymatic cleavage in solution is more efficient than if conducted heterogeneously. In addition, binding of RNA polymerase with promoter DNA could be detected with this strategy.Singapore-MIT Alliance (SMA

Microbial metabolic engineering: Methods and protocols

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