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Research projects from Summer 2006

Research projects for Summer 2007

1. Activation of Immune Cells by Polyanhydride Nanospheres (Narasimhan and Lapizco)

The Narasimhan lab has developed new biodegradable polyanhydride nano-adjuvants to understand the cellular and molecular mechanisms that establish immunologic memory. They have designed nanospheres based on sebacic acid (SA), 1,6-bis(p-carboxyphenoxy)hexane (CPH), and α,ω-bis(p-carboxyphenoxy)tri ethylene glycol (CPTEG), and demonstrated the stability of antigens released from these nanospheres. They are evaluating the uptake of antigen-loaded nanospheres by antigen-presenting cells (APCs, e.g., macrophages and dendritic cells) and using in vitro and in vivo approaches to discern the influence of polymer chemistry on APC activation. Lapizco has developed dielectrophoresis (DEP)-based methods for the separation and concentration of cells, proteins, and nanoparticles. They have designed a new class of micro-devices called insulator-based DEPs (iDEP). In this device, non-uniform electric fields are created with an array of insulators instead of electrodes. Separation of the particles takes place inside micro-channels within the insulating structures. Insulating materials such as polymers have excellent malleability and because they can be mass-replicated, provide for high-throughput and large-volume devices. Below, we describe a collaborative project that will provide a stimulating research experience for an REU student.

Fig. 1. Layout of micro-device geometries for iDEP experiments for cell screening. Different layouts will be employed, and each layout will be used with two of eight microchannels with varying post diameters (40-80 μm) and center-to-center distance (300-500 μm).

Example REU Project: Students will prepare 50-500 nm polyanhydride nanospheres loaded with FITC and based on copolymers of SA, CPH, and CPTEG. These nanospheres will be incubated with the J774.1A macrophage cell line to ascertain adjuvanticity (i.e., activated macrophages) and tested for uptake and cellular activation using cytokine profiles. Students will also use lithography at the ISU Keck Laboratory (consisting of class 10/100 clean rooms) to fabricate micro-devices of glass, polyimide, or poly(methyl methacrylate). These devices will contain micro-channels and an array of cylindrical posts (Fig. 1) inside the micro-channels, whose surface and geometry will affect the dielectrophoretic response obtained from the cells. This response will be used to distinguish the cells that have taken up the nanospheres. These micro-devices and the cells will be analyzed by iDEP at ITESM using synchronized video microscopy. By working on this project, the student will learn: (a) nanosphere fabrication; (b) fabrication of patterned substrates; and (c) cell screening.

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2. Micropatterned Polymer Substrates For Growth of Neural Stem Cells (Mallapragada)

We have designed micropatterned polymer substrates (Fig. 2) to control neuronal outgrowth and promote peripheral nerve regeneration. Studies are underway to extend these approaches to central nervous system regeneration, specifically in the case of the optic nerve, using astrocytes and adult rat neural stem cells and promoting their directed outgrowth and differentiation. Current knowledge suggests that the pattern formation and differentiation of stem cells into various cell types that occur during brain development is guided by a variety of signals, including preformed distributions of extracellular matrix proteins and growth factors. A thorough understanding of this differentiation is important not only to answer fundamental questions in biology related to the elaborate organization of the mammalian brain, but also to control the differentiation of neural stem cells in a tissue-engineered scaffold to promote regeneration.

Fig. 2. Neurites on polymer substrate with smooth top half and micropatterned bottom half.

Example REU Project: We have developed substrates made of polystyrene and poly(lactide-co-glycolide) with micropatterns of various proteins using photolithography. The REU student will investigate the growth and differentiation of adult hippocampal progenitor cells (AHPCs from rats that we use regularly) on these micropatterned substrates. These studies will test the hypothesis that unique combinations of signals from preformed distributions of extracellular matrix proteins will affect the fate of neural stem cells. The cell culture system will involve AHPCs growing on a polymer substrate micropatterned with proteins such as laminin and basic fibroblast growth factor (FGF). The pattern sizes and order of exposure of the cells to laminin and FGF will be interchanged, and the migration, proliferation, and differentiation of the cells will be monitored using microscopy and morphological characterization. The markers – microtubule-associated protein and glial fibrillary acidic protein - will be used to investigate whether the cells adopt neuronal or glial fates in response to the cues. This is a collaborative effort between Mallapragada’s group and researchers in biochemistry and genetics. Through this experience, the student will learn: a) mammalian cell culture on polymer substrates; b) immunocytochemistry; and c) integration of engineering and biological approaches.

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3. Rational Design of Heterogeneous Catalysts for Biorenewable Conversions (B. Shanks)

We use advanced material synthesis to rationally design heterogeneous catalysts to convert biorenewable feedstocks. The high degree of functionality in biological molecules and the liquid phase reaction environment needed for processing them creates new challenges in the design of solid catalysts. Fig. 3 shows a nanostructured organic-inorganic hybrid catalyst designed for reversible esterification of fatty acids with methanol to produce methyl esters. The organosulfonic acid moieties were selected to provide sufficient acidity to achieve the esterification reaction and the hydrophobic organic groups create a reaction environment that excludes water from the active acidic sites. The resulting catalyst was more active in the esterification reaction than conventional solid acid catalysts. We are extending this approach of molecular control of the reaction domain via multi-functionalization of the pores in nanostructured metal oxides to other biorenewable systems.

Fig. 3. Nanostructured organic-inorganic hybrid catalyst

Example REU Project: The student will synthesize organic-inorganic hybrid catalysts, using techniques available in the Shanks group for application in the dehydration of carbohydrates and their derivatives. She/he will attach catalytic organic moieties within the pores of nanostructured silicas through co-condensation or grafting and will characterize the resulting materials using adsorption-desorption isotherms for surface area, pore volume and size distribution, x-ray diffraction for metal oxide structure, scanning electron microscopy of catalyst particle morphology, and reactor studies for kinetics. The project will allow the student to learn: (a) synthesis and characterization of organic-inorganic hybrid catalysts; and (b) about the issues involved in solid catalysts for biorenewables conversion.

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4. Molecular Simulation of Protein Crystallization (Lamm)

The Lamm group uses molecular and mesoscale simulations to discover and interpret relationships between molecular structure and the thermodynamic and transport properties of biological and bio-inspired materials. Building on our previous work in solid-fluid equilibria calculations, we are developing simulation strategies to predict crystal growth rate and structure in pharmaceutical compounds and proteins. The goal of this work is to develop a set of guidelines for the rational design of crystallization processes. In collaboration with the Glatz group, we are studying the crystallization behavior of mutants of the protein subtilisin.

Example REU Project: Experiments by the Glatz group have demonstrated that subtilisin crystal growth rate can be altered by changing amino acid groups on the surface of the protein. Using surface contact patches determined from X-ray diffraction data, the student will construct a coarse-grained, anisotropic patchy particle model for each subtilisin mutant. The student will then perform Brownian dynamics simulations on the model subtilisin mutants to observe crystal growth rates. Using a low-resolution protein model rather than an atomistically detailed protein structure will allow the student to simulate many proteins and quantify the competition between binding of properly oriented and misoriented molecules. The student will gain experience with molecular simulation tools and high performance computing as well as a familiarity with experimentally gathered protein crystallization data.

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5. Catalytic Conversion and Enhanced Metabolic Utilization of Bio-Derived Nutrients (Schrader)

The Schrader group is developing new bioprocessing pathways based on innovative extraction technology and catalyst development. These efforts are directed towards improving the recovery and biological utilization of carotenoids, which are receiving new attention for human nutritional needs. A new catalytic pathway promises to greatly increase the availability of lutein and lycopene, which are antioxidants with proven efficacy in preventing macular degeneration of the eye and in inhibiting the development of some forms of cancer.

Example REU Project: This project will identify, quantify, and convert key bio-based nutrients, especially carotenoids, in diverse plant systems such as maize, tomatoes, and even marigolds. Lutein, lycopene, and other anti-oxidants are important human nutritional supplements effective in improving heath and preventing disease. The student will use laser Raman spectroscopy and HPLC to quantify these carotenoids for two feedstocks based on biorenewable resources. A fixed bed micro-reactor will be used to explore converting these extracted mixtures by selective oxidation. These studies will quantify the yield of partially oxidized products that could be useful nutritional supplements in themselves or that could be converted to lutein and other compounds. The student would gain an appreciation of the challenges in converting bio-based feedstocks, which typically are more complex and which usually involve aqueous processing routes.

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6. Recovery of high-value biological products (Glatz and Rito)

The Glatz and Rito laboratories both work to develop optimal conditions for the recovery of macromolecular products (e.g. proteins, aroma compounds, colorants, inclusion bodies, virus like particles) of biological expression hosts and bioprocessing streams using aqueous two-phase (ATP) partitioning in PEG-salt and PEG-dextran systems. Intensive study of the behavior of specific proteins, previously well characterized in conventional analyses and purifications, have helped to establish ground rules to predict molecular partition in novel systems based upon quantifying physical and biochemical parameters of system components. Each is interested in further steps for product recovery from these systems and reuse of phase components, which will be the focus of their joint project.

Fig. 4. Recombinant protein purification from plants

Example REU Project: The two labs have considerable expertise in precipitation, ultrafiltration and chromatography to serve in subsequent process steps. Each lab also has product proteins (Fig. 4) of particular interest: high-value colored proteins (c-phycocyanin and B-phycoerytrin) from microbial sources and recombinant pharmaceutical/industrial enzymes from corn. The student project will be to take the source material and partitioning step from one lab into the other lab where they will evaluate the subsequent downstream step. That step will be either precipitation (ISU), where the phase system polymers and salts might aid in reducing solubility or ultrafiltration (ITESM), where differences in size of phase components and the product will be exploited. Back extraction with ATP and expanded bed adsorption also will be evaluated. Students will obtain protein extracts, follow recovery by enzymatic or ELISA assays, and interpret results in terms of solubility, partition coefficient, or retention time, depending on the separation method.

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7. Computation of Enzyme Structure and Function (Reilly)

The Reilly group has performed extensive computational analysis of amino acid sequences of the same enzyme produced by different organisms and of 3-D enzyme structures obtained by crystallography. The crystallography experiments to determine enzyme structure and function are difficult and time-consuming, and computation, which is relatively fast and quite accurate, can greatly expand what is known about the specific enzyme. We have used automated docking with glucoamylase, β-amylase, α-1,2-mannosidase, phospholipase D (Fig. 5), several cellulases, and the non-enzymatic mannose-binding protein C.

Fig. 5. Streptomyces phospholipase D, showing a phosphate group (red and gold) in the active site between the catalytic proton donor (histidine 165) and catalytic nucleophile (histidine 438), and with the essential binding residues lysine 167 and lysine 440.

Example REU Project: The student will study hydrolases, which cleave the glycosidic bonds between sugar residues. She/he will perform two activities: a) Use existing algorithms to align amino acid sequences obtained by different research groups and found in databases. Such alignments will distinguish different subfamilies within an enzyme family, such as glucoamylase, and from this the student can infer that structures and properties of the different forms of the same enzyme produced by different organisms are closer or more distant from each other. b) Use automated docking with the algorithm AutoDock of different ligands, either already known substrates, potential substrates, or inhibitors, into the active sites of enzymes with known structures. This allows the student to understand enzyme function supplement knowledge gained by experimentation.

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8. Metabolic Engineering of Alkaloids in Catharanthus roseus (J. Shanks)

The periwinkle, Catharanthus roseus, is the source of several alkaloids with pharmaceutical activity. However, yields of the compounds in the plant are very low, resulting in high expense for commercial production (e.g., vincristine) or nonuse of otherwise promising compounds. This project is designed to exploit opportunities to improve the productivity of indole alkaloids in a well-characterized model plant tissue culture system, C. roseus hairy roots. The overall goals are to determine the metabolic effects of genetic manipulations of some key enzymes in the indole and non-mevalonate terpenoid pathways on flux through the indole alkaloid synthesis pathways, with special emphasis on the interplay between primary and secondary metabolism on the final alkaloid productivity. We have generated several transgenic C. roseus hairy roots lines that over-express one or two enzymes in the tryptophan and/or the terpenoid pathways [45,46]. Each line requires several characterization methods, including growth rates and yields, enzyme activities, and alkaloid yields. These enzymes are under the control of an inducible promoter, and thus controls of uninduced versus induced state, as well as negative controls, are important, but these additional steps greatly increase the amount of experimentation.

Example REU Project: The student will perform characterization studies on one engineered line plus the negative control. Our lab has extensive experience in all of the methods, and she/he will work closely with a graduate student. The latter will have the necessary three subculture cycles of the line done before the undergraduate arrives, so that she/he will be able to complete this plant tissue culture experiment and chemical measurements in six weeks, with two weeks left for data analysis and presentation. The REU student will make biomass measurements, extract alkaloids from the root tissues, and run and interpret the HPLC chromatograms. Other measurements (enzyme activities, mRNA levels) will be performed by the graduate student. The REU student will also participate in a multidisciplinary team, as collaborators at Rice (Bioengineering) and Minnesota (Plant Biology) exchange information every 3 weeks and have a yearly group meeting.

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9. Aptamer-Based Biomolecular Recognition Systems (Porter)

Immunological recognition is among the most used labeling approaches in bioassays, relying on the high specificity of antibody-antigen binding. Examples include detecting large biomolecules like insulin, small molecules like TNT, and pathogens like E. coli (Fig. 6). These assays, however, suffer from several limitations (batch-to-batch variability, temperature and matrix effects, and cross-reactivity), which may be overcome by a new class of molecular recognition ligands based on nucleic acid oligomers. These ligands, referred to as aptamers, are typically 20-100 base pairs long and are isolated from pools of oligomers with random sequences. Through iterative combinatorial processes known as SELEX (systematic evolution of ligands by exponential enrichment), which entail a series of binding affinity and PCR amplification steps, oligos with high affinity and specificity for a target are isolated from large libraries. Furthermore, aptamers are synthesized by automated processes, which may reduce binding diversity complications when using antibodies.

Fig. 6. E. coli O157:H7 tagged with fluorescently labeled (hexachlorofluore-scein phosphoramidite) aptamers: Light microscope image (left); fluorescence microscope image (right)

Example REU Project: The student will apply SELEX and counter-SELEX to develop aptamers for the target pathogen, E. coli O157:H7, central to human health and water supply contamination. The student will perform the following tasks: (a) Design and synthesize an oligo library composed of an internal random region of 40-mers (~1015 sequences after accounting for synthesis inefficiency) flanked by PCR primers; (b) Use SELEX and counter-SELEX to identify most effective binding sequences by: (1) exposing the library to the target organism or cross-reactors (e.g., Salmonella choleragesius) that have been immobilized in a flow-through column, allowing oligos with an affinity to the target to bind; (2) separation of unbound oligos by low stringency washes; (3) elution of bound oligos, now called aptamers, from the target under conditions of varied stringency (pH and salt concentration); and (4) aptamer amplification by PCR (these four steps are repeated 4-8 times to grow sufficiently the most effective binding populations); and (c) Clone the ‘winning’ aptamers into vectors to produce material for sequencing, followed by production in quantities sufficient to further select against the sample preparation environment. In working with the Porter group and collaborators in veterinary medicine, the student will be exposed to several areas of biotechnology and to an environment that will develop life-long learning and working skills.

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10. Improving Carbon Conversion in Biomass Gasification Reactors (Brown)

The Brown group works on gasification technology for use in biorefineries. The main issues in gasifying biorenewable feedstocks include ash agglomeration, contaminant generation in the syngas, low hydrogen content, and modest carbon conversion. The latter problem accounts for cold gas efficiencies typically being only 65% and overall thermodynamic efficiencies <90% in converting solid biomass to syngas. We have improved carbon conversion by studying the interaction of fluid dynamics and reaction in fluidized bed reactors. We have designed a laboratory-scale (50 kW) bubbling fluidized bed reactor to simulate near-adiabatic operation that can measure fuel, air, and/or steam flow into the reactor and exit gas composition.

Example REU Project: During the project, the student will learn the operating principles of the fluidized bed reactor and perform system measurements. Based on our coal combustion modeling work on, she/he will formulate a reactor population balance model accounting for changes in reactor char concentration due to attrition, elutriation, and gas-solid reactions. She/he will perform semi-quantitative comparisons of the model predictions of carbon conversion with laboratory data.

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11. A Bayesian Approach for Mutation in Escherichia coli (Rollins)

The Rollins group has expertise in statistics and process modeling to reduce high dimensional data sets and linear/non-linear stochastic modeling relevant to Bayesian Independent Component Analysis. In collaboration with Gonzalez, we are developing a framework for the analysis and integration of large data sets generated by the use of functional genomics.

Example REU Project: In mutagenic polymerase chain reaction (PCR) researchers are interested in the probabilistic prediction of certain types of mutation. In this work, the assumption of independence appears to be quite common but the reality more closely follows dependence behavior (i.e., future mutations depend on what has mutated). The REU student will incorporate statistical modeling using conditional probabilities to model mutations in a specific system based on E. coli. Simultaneously, the student will compare them with real data (obtained from the literature) and make adjustments in the theoretical models. The student will learn the importance of statistical modeling in systems biology and use this to make more realistic comparisons between theory and experiment, thus leading to deeper insights.

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12. Stochastic Modeling of Carrier-Cell Interactions in Targeted Drug Delivery (Vigil)

One promising method for increasing the targeting of drugs to cells has been to attach molecules to the surface of the drug carrier (using a poly(ethylene glycol) tether) that bind to specific receptors on the target cells. Usually, when only a single receptor is targeted, off-target drug uptake is significant. However, diseased cells often over-express multiple receptor types, so attaching different ligands to carriers could potentially lead to selective targeting. We have developed stochastic models to simulate carrier-cell interactions and predict the number/type of ligands that optimize the rate and selectivity of drug uptake by target cells.

Example REU Project: Current simulations of ligand-receptor binding assume that receptors remain in fixed positions on the cell surface. However, it is known that receptors can and do move in response to binding events of other receptors. The REU student will carry out a literature search and develop and test appropriate algorithms for simulating receptor mobility. Next, the student will test the effect of receptor mobility on carrier targeting.

 

Research projects for Summer 2007