The production of a dextran binding antibody by phage display library and its applications to sugar processing

A phage collection (M1114), which is a mixture of two collections, M710-3R and Silica 426 each derived from a human synthetic phage antibody library (Fab 2lox), was used further enriched for dextran binding using enzyme-linked immunosorbent assays (ELISA) screening. The effects of dextran concentration on phage binding affinity were tested using indirect sandwich ELISA on phage collections, M1114-m74 and AE-M1114-m74-2R. Most of the phage bound to dextran (T2000) coated on a sandwich ELISA. The combination of ELISA screening and a sephadex columns enriched dextran binding 7 fold over enrichment by a single ELISA screen. Phage collection (M1114-m74) produced by combination screening showed the greatest binding on ELISA. The color intensity produced by phage collection (AE-M1114-m74-2R) obtained after the 2nd round of selection was 3.5 fold higher than that of phage collections, AE-M1114-m74-1R after the 1st round. Dextran binding by phage collection (AE-M1114-m74-2R) was illustrated using image analysis of transmission electron micrographs. Sephadex bead agarose electrophoresis (SBAE) screening produced phage collections (AE-M1114-m74-1R and 2R) which were used in a paper-dip assay. A dip stick assay using a protein blocked paper with adsorbed high molecular size dextran (T10,000, 107) produced the most color (59 ±5) using anti-dextran phage enzyme linked assays. Low molecular size dextran (T40, 4x104) produced significantly lower color (15 ±1). Phage collection (AE-M1114-m74-2R) was tested for specificity against dextran (T2000), corn starch, sucrose, dextrose, and chitin. Dextran produced up to 18 fold the normalized intensity of the other carbohydrates. The presence of Fab inserts in the phage collections was confirmed using PCR, and the presence of the same insert in the host E.coli was checked using a â-galactosidase linked assay. DNA sequencing of phage collection (AE-M1114-m74-2R) confirmed that human origin antibody was present. The PCR products of ë, ê light chains and heavy chain from phage collection (AE-M1114-m74-2R) were approximately 420 bp, 550 bp and 600 bp. This research used various selection methods to isolate anti-dextran phages from a library. These were used to develop a paper-dip stick method for dextran detection used for routine screening of sugar juices

Long lived transients in gene regulation

Gene expression is regulated by the set of transcription factors (TFs) that bind to the promoter. The ensuing regulating function is often represented as a combinational logic circuit, where output (gene expression) is determined by current input values (promoter bound TFs) only. However, the simultaneous arrival of TFs is a strong assumption, since transcription and translation of genes introduce intrinsic time delays and there is no global synchronisation among the arrival times of different molecular species at their targets. We present an experimentally implementable genetic circuit with two inputs and one output, which in the presence of small delays in input arrival, exhibits qualitatively distinct population-level phenotypes, over timescales that are longer than typical cell doubling times. From a dynamical systems point of view, these phenotypes represent long-lived transients: although they converge to the same value eventually, they do so after a very long time span. The key feature of this toy model genetic circuit is that, despite having only two inputs and one output, it is regulated by twenty-three distinct DNA-TF configurations, two of which are more stable than others (DNA looped states), one promoting and another blocking the expression of the output gene. Small delays in input arrival time result in a majority of cells in the population quickly reaching the stable state associated with the first input, while exiting of this stable state occurs at a slow timescale. In order to mechanistically model the behaviour of this genetic circuit, we used a rule-based modelling language, and implemented a grid-search to find parameter combinations giving rise to long-lived transients. Our analysis shows that in the absence of feedback, there exist path-dependent gene regulatory mechanisms based on the long timescale of transients. The behaviour of this toy model circuit suggests that gene regulatory networks can exploit event timing to create phenotypes, and it opens the possibility that they could use event timing to memorise events, without regulatory feedback. The model reveals the importance of (i) mechanistically modelling the transitions between the different DNA-TF states, and (ii) employing transient analysis thereof

kk-Schur functions and affine Schubert calculus

This book is an exposition of the current state of research of affine Schubert calculus and $k$-Schur functions. This text is based on a series of lectures given at a workshop titled "Affine Schubert Calculus" that took place in July 2010 at the Fields Institute in Toronto, Ontario. The story of this research is told in three parts: 1. Primer on $k$-Schur Functions 2. Stanley symmetric functions and Peterson algebras 3. Affine Schubert calculusComment: 213 pages; conference website: http://www.fields.utoronto.ca/programs/scientific/10-11/schubert/, updates and corrections since v1. This material is based upon work supported by the National Science Foundation under Grant No. DMS-065264