Session 4 Abstracts
Session 4 Abstracts – Saturday, June 23 – 2:00 PM
BioMEMS / Instrumentation
Miniature Biosensors for Local Analysis of Cell Function
Alex Revzin, Associate Professor, Department of Biomedical Engineering, UC Davis
Cell function analysis – the type of molecules that cells release – is important for assessing cellular phenotype. Traditionally, cells are analyzed by collecting media for off-dish functional assays such as ELISA. Our lab has been interested in developing assays that may be miniaturized and placed at the site of small groups of cells for on-dish detection of cell function. In developing these assays or biosensors we strive to achieve local and continuous detection of molecules appearing in extracellular space. This presentation will highlight three categories of biosensors for cell secreted molecules under development in our lab: aptamer based sensors for protein detection, peptide-based sensors for protease activity monitoring and enzyme-based biosensors for small metabolite analysis. Applications of these technologies to diagnosis of infectious diseases and monitoring cellular responses to injury will be discussed.
A Carbohydrate-Responsive Polymer for Charge-Based Sensing
Alexander Wollenberg,1,2 Boaz Vilozny,1 Daniel Hwang,1 Bakthan Singaram,2 Nader Pourmand*1
1Department of Biomolecular Engineering, Baskin School of Engineering at University of California Santa Cruz
2Department of Chemistry and Biochemistry, University of California Santa Cruz
A boronic acid-appended polycation was synthesized, and shows distinct changes in solubility at the pKa of the boronic acid. Using spectrophotometry, the affinity for the polymer with several dyes was measured. This made it possible to quantify two different variables that affected the affinity of the PVPBA to a substrate: reversible covalent bonds between boronic acid and diols, and electrostatic interactions. The anionic dye HPTS forms a ground-state complex with the cationic polymer through electrostatic interactions, as shown in Figure 1. This also results in quenching of the fluorescence of the dye. Because the charge of the polymer can be modulated with carbohydrates, a fluorescent assay can be carried out by weakening the dye/polymer affinity. A fluorescent displacement assay was performed in solution to determine the affinity of PVPBA for glucose and fructose (Figure 1). This assay was dependent upon sugar concentration, and could detect differences in these sugars down to 1 mM. By tailoring the polymer composition, selectivity for different saccharides may be achieved. Unlike most solution-phase probes, the polymer is designed to be incorporated into a solid-state device. Taking advantage of the electrostatic properties of this polymer enable attaching it to the inside of a nanopipette, a quartz conical channel with an opening of 20-40 nm. The same electrostatic modulation exploited in the fluorescence assay can be used in modulating the nanopipette electrode. Future work will compare performance of the solution-phase probe to a conical nanochannel electrode modified with the polymer.
Genetic Detection of H1N1 Influenza Virus via Microfluidic Electrochemical Quantitative Reverse- Transcription Loop-Mediated Isothermal Amplification
Kuangwen Hsieh, B. Scott Ferguson, Ting-Ting Wu, Ren Sun, H. Tom Soh
UC Santa Barbara
Genetic detection of pathogens at the point of care (POC) has become increasingly important in applications ranging from clinical diagnostics to homeland security. For example, detection of viral RNA is critical for the diagnosis and the surveillance of emerging, pandemic-causing influenza. In such applications, the ideal detection method must be portable, rapid, and sensitive, while supporting real-time, quantitative analysis across a wide dynamic range in order to offer important information such as the viral load. To date, however, existing genetic detection systems have yet to meet all of these requirements. Motivated by this unmet need, we have developed the microfluidic electrochemical quantitative reverse- transcription loop-mediated isothermal amplification (MEQ-RT-LAMP) system – an integrated microfluidic platform for the rapid, sensitive, and quantitative detection of viral RNA directly from viral particles. Our system leverages reverse-transcription loop-mediated isothermal amplification (RT-LAMP), a powerful alternative to reverse-transcription PCR that offers advantages in terms of sensitivity, reaction speed, and amplicon yield, while operating under single-step and isothermal reaction conditions. As a further benefit, this amplification technique employs four to six different primers and thus confers exquisite specificity. Importantly, we have achieved real-time, quantitative electrochemical detection of RT-LAMP amplification by monitoring the intercalation of DNA-binding methylene blue (MB) redox reporter molecules into newly formed amplicons with a set of integrated electrodes. Our system therefore obviates bulky optical instrumentation that has plagued microfluidic devices from wide-spread adoption at the point of care. Using our platform, we demonstrate direct and quantitative detection of influenza H1N1 virus down to 1 TCID50 – approximately 5 orders of magnitude below the clinical titer for this virus — in a single step and in less than an hour. The novel functionality and the performance of our MEQ-RT-LAMP platform thus present a significant advancement in real time genetic detection toward POC applications.
Ophthalmologist-on-a-Chip: Fully Integrated Microfluidic Tear Osmolality and Protein Biomarker Quantification for Dry Eye Stratification
Kelly Karns, Nancy McNamara & Amy E. Herr
We report on a multiplexed assay for validated autoimmune dysfunction biomarkers in human tear film fluids from healthy and Sjögren’s syndrome (SS) patients. To our knowledge, this work is the first specific and rapid (< 3 min) assay for biomarkers of dry eye disease in sparingly available ocular fluid. Our microfluidic approach combines on-the-fly quantitation of both tear fluid osmolality and lactoferrin protein concentration in microliter quantities of human tear fluid from a large patient sample repository (SICCA, UC San Francisco). Although tear protein levels change in dry eye pathogeneses, protein and osmolality measures are not used in ocular diagnostic medicine owing to a lack of appropriate clinical diagnostic technologies.
To surmount this diagnostic gap, our microfluidic assay employs a two step process: 1) tear osmolality, a putative biomarker for keratoconjuctivitis sicca, is quantified through electrical impedance measurements and 2) lactoferrin (Lf), a putative biomarker for SS, is quantified using an electrophoretic homogeneous immunoassay. Using this approach, tear osmolality and lactoferrin in tear matrix are measured in < 3 min. On- chip Lf quantitation correlates well with ELISA (R2 = 0.9941) and the lower limit of detection is clinically relevant (32 nM) with a linear dynamic range spanning two orders of magnitude (3 to 400 nM).
In a pilot retrospective clinical study of osmolality and Lf levels in healthy and SS patient samples, the assay measures significantly down-regulated Lf levels in SS tears compared to healthy controls (6.6x decreased, p<0.02 by t-test) and increased osmolarity in SS tears compared to controls (2.9x higher), as expected from literature. For the first time, we demonstrate an integrated tool for tear biomarker quantitation. Importantly, the novel measurements enabled by this tool have implications in advancing our understanding of dry eye disease pathology and may enable improved dry eye diagnostics and disease outcomes.
Bioinspired Interfaces for Detection and Identification of Bacteria in Blood
Vicente Nuñez, Jillian Larsen, Srigokul Upadhyayula, Valentine Vullev
Infectious diseases are responsible for a quarter of the deaths worldwide. Even for advanced cases of bacteremia, the count of leukocytes and erythrocytes exceeds with many orders of magnitude the count of prokaryotic cells in the infected blood. A robust and rapid way for capturing, detecting and identifying contaminants in biological and environmental fluids is of urgent need. Using surface chemistry procedures developed in our lab we designed biofunctional surfaces activated with carbohydrates that target the natural ability of bacteria to adhere, colonize and infect. These bioinspired interfaces allowed us to capture a wide range of Gram-positive and Gram-negative contaminants from blood samples. Amyloid stains allowed us to readily visualize and detect the captured bacterial cells. By imaging the dynamic staining of captured bacterial cells we were able to track the kinetics of their staining which revealed unprecedented capabilities for parallel identification of each of the bacterium in a sample containing a mixture of bacterial species. The breaking of century-old traditions of Boolean bacterial bioanalyses reflects the innovative nature of the assays based on the dynamics of staining.
Control of interparticle spacing using structured microfluidic channels
Dianne Pulido, Aram Chung, Mahdokht Masaeli, Hamed Amini, and Dino Di Carlo
UC Los Angeles
Microfluidic techniques in cell analysis such as particle separation, flow cytometry, and particle encapsulation in droplets all require manipulation of cells or particles in fluid. Implementation of a platform capable of controlling particle position and spacing would increase throughput and accuracy in such systems by allowing for operation at higher concentrations and reliable prediction of particle position. Here we present a system for the control of interparticle spacing using structured microfluidic channels with inertial effects. Using established inertial microfluidic techniques to first focus particles into desired equilibrium positions, we have evaluated how downstream localized changes to channel width affect particle ordering behavior.
Results across various geometries indicate both expansion and contraction of interparticle spacing can be achieved for particles in the same stream (SS), while particles in cross streams (CS) only exhibit a weak contraction in certain designs (Figure 1A-B). Further analysis of SS spacing has demonstrated the system’s ability to meet a minimum spacing requirement, which has applications in increasing throughput in particle analysis techniques by reducing coincident detection events in highly concentrated conditions (Figure 1C). Additionally, our structured channels are observed to shift specific particle focusing positions towards the channel walls and improve the accuracy of focusing, two characteristics that are beneficial to cell and particle separation systems (Figure 1D). Fluid flow simulations demonstrate an induction of secondary flows and a net change to fluid streamlines passing through the structured region, which explain the experimental observations of particle behavior (Figure 1E).
Our structured microfluidic channels currently exhibit manipulation of particle effects that can be useful in a wide range of applications. Further analysis will be conducted towards exploring the range of control of this system, specifically how interactions scale with particle size and mixed populations of particles.
Electrophysiology-Activated Cell Sorting: A label-free, functional cell purification strategy for regenerative medicine.
Frank B. Myers, Oscar J. Abilez, Chris K. Zarins, Luke P. Lee
The application of stem cells to regenerative medicine poses a number of exciting engineering challenges for the robust production of differentiated cells. Paramount among these challenges is the need to isolate cells of a specific therapeutically-relevant phenotype and exclude all other cells which can arise during differentiation, particularly any remaining undifferentiated cells. Conventional cell purification techniques require exogenous labeling or genetic modification, neither of which is ideal for clinical applications. As many of the cell phenotypes relevant for therapy are electrically-excitable (e.g. cardiomyocytes, neurons, smooth muscle, pancreatic beta cells), we propose a new cell sorting methodology based on electrophysiological response to stimulus. Electrophysiology offers a highly-specific, non-invasive, label-free approach to cell purification. We have developed microfluidic cell sorters which electrically stimulate cells in suspension, observe their response via extracellular microelectrodes or intracellular metal indicator dyes, and sort them accordingly (see figure). We have applied these devices for the label-free purification of stem cell-derived cardiomyocytes from non- electrically excitable cells such as undifferentiated hiPSCs and fibroblasts. We believe electrophysiology- activated cell sorting (EPACS) can substantially reduce the risk of teratoma formation because undifferentiated cells do not express the necessary voltage-gated ion channels to produce electrophysiological signals. Furthermore, we hypothesize that electrophysiological homogeneity of implanted cardiomyocytes will lead to improved graft viability, improved electromechanical coupling within the host myocardium, and reduced incidence of arrhythmias when compared to other label-free sorting techniques.
Molecular, Cell and Tissue Engineering
Emergent behaviors in 3D epithelial tissues prepared by programmed assembly
Zev Gartner, Assistant Professor, Department of Pharmaceutical Chemistry, UC San Francisco
Social interactions among epithelial cells give rise to tissues with emergent behaviors that promote proper development and oppose aberrant phenotypes associated with disease. These properties of epithelial tissues can be studied in three-dimensional (3D) culture, which recapitulates many of the microenvironmental conditions encountered in vivo. However, studying the response of epithelial cells to differences in their immediate neighbors remains challenging, as we lack methods to precisely vary the composition of multicellular aggregates under 3D culture conditions. We used DNA-programmed assembly to construct MCF10A or MDCK aggregates with defined cell number, composition, and initial cell-cell connectivity. The approach allowed us to observe alterations in epithelial morphogenesis when a biochemically or genetically heterogeneous cell is incorporated into an otherwise homogeneous cellular aggregate. We found that in 3D culture, homogeneous aggregates of MCF10A cells or derivatives expressing low levels of activated H-Ras rapidly condensed into polarized microtissues and eventually formed acini with an apoptotic and hollow lumen. However, incorporation of single cells with elevated H-Ras activity into otherwise wild-type (WT) microtissues elicited two emergent behaviors during morphogenesis: H-Ras-activated cells were either basally extruded from the tissue or led the formation of multicellular protrusions and migration of the entire microtissue. Both of these emergent behaviors exhibited specific dependencies upon signalling pathways downstream of Ras. Remarkably, these emergent behaviors were not observed in homogeneous microtissues comprising only activated H-Ras cells. These data demonstrate that heterogeneity in pathway activation within local populations of epithelial cells triggers unique intercellular programs that drive emergent behaviors at the tissue level.
Stencil Patterning Improves Robustness in Pluripotent Stem Cell Expansion and Cardiomyocyte Differentiation
Jason S. Silver, Frank B. Myers, Oscar J. Abilez, Chris K. Zarins, and Luke P. Lee
Geometric factors including the size, shape, and density of pluripotent stem cell colonies, as well as the spacing between neighboring colonies, play a significant role in the maintenance of pluripotency and in cell fate determination. These factors are impossible to control using standard tissue culture methods. As such, there can be substantial batch-to-batch variability in cell line maintenance and differentiation yield. Not only does this variability pose a challenge in the laboratory setting, but it is a significant barrier to developing large scale cell production methods for clinical applications. We have developed a simple, robust technique for patterning Matrigel using a thin silicone stencil. We have observed that patterned arrays of Matrigel spots lead to human induced pluripotent stem cell (hiPSC) colonies which are highly uniform in growth rate, size, cell density, and gene expression, using single cell suspensions without ROCK inhibitor. These colonies exhibit more homogenous intracolony pluripotency due to the uniform cell density throughout the colony. Patterned colonies exhibit greater repeatability as compared with conventional scrape-passaged colonies when undergoing directed differentiation into spontaneously beating cardiomyocytes. Therefore, this simple tool can be used to efficiently produce uniform colonies for large-scale production of stem cell derived cells for therapeutic, drug screening, or research applications.
Biomimetic Scaffolds for Enhanced Dural Regeneration
John Eng, Vaibhavi Umesh, Lissette Wilensky, Falei Yuan, Song Li, Shyam Patel
Every year in the US, there are over 250,000 neurosurgeries, all of which require the surgeon to make incisions through the dura mater in order to access the brain. As a result, post-surgical repair requires the grafting of a medical device as a “dural substitute” to patch dura mater defects. Current solutions are plagued by poor cell infiltration and tissue in-growth, causing device failure in up to 15% of surgical cases. This leads to cerebrospinal fluid leakage that result in severe infections, paralysis, and costly revision surgeries. We have addressed this need by utilizing electrospinning technology to create an aligned, nanofibrous matrix that promotes dural regeneration. Successful dural regeneration and better patient recovery outcome is dependent on cell infiltration and tissue ingrowth within the device. To fulfill these criteria, we have explored multiple design formulations to modulate scaffold porosity, testing the hypothesis that increased porosity will lead to enhanced tissue integration. In order to assess tissue ingrowth, scaffolds were subcutaneously implanted in Sprague-Dawley rats and explanted at multiple time points ranging from 1 week to 1 month. At each time point, the tissue was cryosectioned and assessed by histological staining for cell infiltration and tissue integration. The results demonstrated that cell infiltration and collagen matrix deposition within the thickness of the scaffold was improved by tailoring its chemical and physical properties. We plan to build on these results by assessing the performance of the nanofibrous dural substitute in a large animal duraplasty model and translating the technology for clinical use.
Interaction Affinity Determination of SUMO E1 Heterodimer Aos1/Uba2 and intermediates of activation cascade by Quantitative FRET Analysis
Ling Jiang, Sophie Qu, Amanda N.Saaredra, Nidhanjali Bansal, Jiayu Liao
Ubiquitin-like modifiers, such as SUMO, play important roles in regulating protein activities, half-life and interactions with other proteins in vivo, and are involved in many physiological and pathological processes, such as cancers, immune response, neurodegerative diseases and metabolic diseases. The conjugation of SUMO peptide to substrates is carries out by a multi-enzyme catalyzed cascade, involving E1, E2 and E3 ligases in vivo. In the SUMO conjugation cascade, multiple covalent and non-covalent protein-protein interactions play critical roles for a successive activation and transferring of SUMO peptides to substrates. The kinetics and protein interaction affinities in the cascade were not determined. The SUMO E1 ligase consisting of a heterodimer, Aos1 and Uba2, plays critical roles in the initial SUMO activation and transferring the activated SUMO to E2 ligase. Here, we report the first determinations of Aos1 and Uba2 interaction dissociation constant (Kd) with our recently developed novel quantitative FRET assay. The Kd values from four different assays with various concentrations of Aos1 were confirmed by a surface resonance plasma (SPR) assay. This Kd value of Aos-Uba2 interaction provides an unique perspective for the first step to dissect the complex UBL conjugation cascade quantitatively. The good agreement of Kd values between the Quantitative FRET assay and SPR demonstrates reliability and accuracy of the Quantitative FRET assay that can be expand to other protein interactions in general. We also determined the effects of SUMO and ATP on E1 heterodimer interactions using our novel quantitative FRET assay. The results show that the formation of thioester bond of SUMO and Uba2 led to increase of FRET signal which indicates E1 heterodimer more stable in the presence of SUMO and ATP. These results suggest that our novel quantitative FRET is not only useful to determine protein interaction affinity but also track protein conformational and dynamics changes.
Remineralization of Decellularized Bone Collagen Matrix for Use as a Novel Structural Biomaterial
1Soicher, MA; 2Garcia, TC; 1Christiansen, BA; 2Stover, SM; +1Fyhrie, DP
+1Lawrence J Ellison Musculoskeletal Research Laboratory, Department of Orthopaedic Surgery, UC Davis School of Medicine. Biomedical Engineering Graduate Group, UC Davis; 2JD Wheat Veterinary Orthopedic Research Laboratory, School of Veterinary Medicine, UC Davis
Polymer scaffolds used for bone tissue engineering often lack desired mechanical properties. There is a trend towards using composite materials to better match bone’s biphasic composition. We have developed a novel technique for remineralization of demineralized bone matrix (DBM). This abstract describes our method for producing constructs with potential for use in allograft or xenograft applications. Cortical bone was cut from equine third metacarpal (MC3) and demineralized using formic acid (22.5%). Separate solutions of calcium chloride and sodium phosphate were prepared. DBM were sequentially exposed to each solution for 45 minutes, allowing time for ions to diffuse throughout the matrix. Alternating saturations continued for 96 hours. An automated system was designed using pinch valves and a LabView program to regulate the flow of solutions. Beams were imaged throughout the study via x-ray and microCT. Beams were mechanically tested in 3 point bending using a Bose Enduratec system. Tests were performed on non-demineralized, demineralized, and remineralized specimens, with stiffness calculated as the slope of the force-displacement curve. Serial x-rays indicated accumulation of mineral within the DBM with treatment (microCT confirmed). Bending tests determined that remineralization increased beam stiffness approximately 13 fold (p<0.05). We have incorporated calcium phosphate (CP) mineral throughout equine DBM. Qualitatively, DBM is rubbery and flexible while our remineralized construct is palpably more rigid. Quantitatively, we demonstrated an order of magnitude increase in the stiffness of DBM. We have begun to optimize the process by altering specific parameters (i.e. time, temperature, pH). One benefit we envision for this technique is the ability to shape DBM to fit a defect, remineralize to hold the new shape, and fabricate customized, mechanically stable bone defect fillers. Preliminary experiments have demonstrated cell viability on DBM. We are expanding these experiments to remineralized matrix in order to further evaluate these remineralized matrices.
Cell Line and Stage-Specific Optimization for Enhanced Endothelial Differentiation of Mouse and Human Embryonic Stem Cells
William S. Turner, Drew E. Glaser, Sarah J Parkhurst, Andrew B. Burns, Kara E. McCloskey
Both mouse and human embryonic stem cells (ESC) can be differentiated into functional endothelial cells (EC) in vitro. Currently, monolayer differentiation is performed by a step-wise process with endothelial specific signals from both extracellular matrix (fibronectin and collagen-type IV) and soluble factors (VEGF, BMP-4, and FGF). Individual ESC human lines (H7 and H9) and mouse (commercially available: E14 and R1, as well as, our own ESC lines: A3 and B2) were examined. Based on the expression of Flk-1/KDR VEGF receptor, we have optimized the differentiation protocols for each of the above ESC lines varying initial seeding density, matrix substrate, and VEGF concentration. The first stage of differentiation of human ESC was found to express maximum numbers of KDR positive cells on days 14 and 12, for H7 and H9 ESC lines respectively. The four mouse ESC lines expressed maximum numbers of Flk-1 positive cells between 2 and 4 days. Although the optimal matrix and VEGF concentration varied slightly between cell lines, the most significant variable for increasing the number of Flk-1/KDR+ cells was found to be density of the initial cell seeding. Most importantly, we found that the extracellular matrix signaling for directing EC fate is conserved between murine and human, however the time period required for development is distinct between the two species.
Lower Seeding Density Increases Functionality of Self-Assembled Meniscus-Shaped Fibrocartilage
Timothy Yeh, Pasha Hadidi, Kyriacos A. Athanasiou
As a frequently injured tissue with limited healing capacity, the knee meniscus is a prime candidate for tissue engineering . In particular, our group has developed a self-assembly process which can produce constructs with quantifiable mechanical properties and appropriate biochemical content . However, an optimal seeding density, which is important to translatability, has yet to be found . This study characterized the properties of self-assembled fibrocartilage as a function of initial cell seeding density in a meniscus shape. It was hypothesized that, within the densities explored, construct properties would display an increasing trend until a certain density, at which point they would plateau.
Methods: A 1:1 ratio of isolated bovine articular chondrocytes and meniscal cells were seeded into meniscus-shaped agarose molds with serum-free chondrogenic media at the following densities: 1.25, 2.5, 5, 10, 15, and 20 million cells per 180μL. At day 28, constructs were assayed for compressive and tensile properties, collagen, glycosaminoglycan (GAG), and DNA content. ANOVA followed by a Tukey’s HSD post hoc analysis as appropriate was performed to determine significance (p < 0.05)
Results: Constructs seeded with fewer than 5 million cells did not form contiguous tissue. Increasing numbers of cells produced successively greater amounts of extracellular matrix (ECM), until a plateau in ECM production was reached at 10 million cells per construct. Notably, collagen/wet weight and GAG/wet weight were greatest in constructs seeded with 5 million cells. ECM per wet weight correlated with mechanical properties in compression (GAG) and tension (collagen). Constructs seeded with 5 million cells had the greatest mechanical properties.
Conclusions: This work advances translational potential of the self-assembly process by demonstrating meniscus-shaped constructs may be seeded with 75% fewer cells than previously thought, while increasing biomechanical and biochemical properties. Future work will pursue the relationship between cell density, adhesion receptors, and ECM synthesis.
 Makris EA, Hadidi P, Athanasiou KA. Biomaterials 2011;32:7411-31.
 Hoben GM, Hu JC, James RA, Athanasiou KA. Tissue Eng 2007;13:939-46.
 Revell CM, Reynolds CE, Athanasiou KA. Annals of Biomed Eng 2008;36:1441-48.
Synthetic Biology / Systems Biology / Computational Biology / Genomics
Engineered Gene Circuits: From Oscillators to Synchronized Clocks and Biopixels
Jeff Hasty, Professor, Departments of Molecular Biology and Bioengineering, UC San Diego
Synthetic biology can be broadly parsed into the “top-down” synthesis of genomes and the “bottom-up” engineering of relatively small genetic circuits. In the genetic circuits arena, toggle switches and oscillators have progressed into triggers, counters and synchronized clocks. Sensors have arisen as a major focus in the context of biotechnology, while oscillators have provided insights into the basic-science functionality of cyclic regulatory processes. A common theme is the concurrent development of mathematical modeling that can be used for experimental design and characterization, as in physics and the engineering disciplines. In this talk, I will describe the development of genetic oscillators over increasingly longer length scales. I will first describe an engineered intracellular oscillator that is fast, robust, and persistent, with tunable oscillatory periods as fast as 13 minutes. Experiments show remarkable robustness and persistence of oscillations in the designed circuit; almost every cell exhibits large-amplitude fluorescence oscillations throughout each experiment. Computational modeling reveals that the key design principle for constructing a robust oscillator is a small time delay in the negative feedback loop, which can mechanistically arise from the cascade of cellular processes involved in forming a functional transcription factor. I will then describe an engineered network with intercellular coupling that is capable of generating synchronized oscillations in a growing population of cells. Microfluidic devices tailored for cellular populations at differing length scales are used to demonstrate collective synchronization properties along with spatiotemporal waves occurring on millimeter scales. While quorum sensing proves to be a promising design strategy for reducing variability through coordination across a cellular population, the length scales are limited by the diffusion time of the small molecule governing the intercellular communication. I will conclude with our recent progress in engineering the synchronization of thousands of oscillating colony “biopixels” over centimeter length scales through the use of redox signaling that is mediated by hydrogen peroxide vapor. We have used the redox communication to construct a frequency modulated biosensor by coupling the synchronized oscillators to the output of an arsenic sensitive promoter that modulates the frequency of colony-level oscillations due to quorum sensing.
Programming Cells with Synthetic RNA Regulatory Elements
Lei Qi, Adam Arkin
Prokaryotic noncoding RNAs (ncRNAs) are versatile controllers of cellular networks and key organizers of genomes. They perform a wide array of functions from environmental sensing to genetic regulation and are amenable to forward designs, which present intriguing engineering substrates for programming cells. In this work, we study the engineering rules of creating large families of orthogonal synthetic ncRNA elements that can control prokaryotic transcription or translation. We develop mathematical models to predict interaction between ncRNA and the mRNA target, and show that new elements could be designed in silico using computer algorithms. These synthetic ncRNAs can be fused to RNA aptamers as modular chimeras, which could detect cellular signals including endogenous proteins and small molecules. Multiple ncRNA elements can be further inter-connected into complex synthetic circuits to perform computations such as multi-input logic, feedbacks, and cascading. To improve the predictability of circuitry engineering, we develop a synthetic RNA processing platform from the bacterial CRISPR immune system. We show that RNA processing allows quantitative programming of gene expression and sophisticated genetic systems. Thus, our work establishes a versatile and powerful platform for programming cells based on synthetic RNA molecules, which is useful for applications from chemical production to therapeutics.
The role of Zinc and Calcium ions in MMP-14 and TIMP-2 Association
Zied Gaieb,1 Peter Nam,2 Xin Ge,2 Dimitrios Morikis1
1Department of Bioengineering and 2Department of Chemical and Environmental Engineering, University of California, Riverside
The role of proteins MMP14 and TIMP2 in tumor angiogenesis has been established, making the MMP14-TIMP2 complex crucial in development and invasion of tumors. Both proteins are highly and oppositely charged and, as a complex, regulate the activation of MMP2 (gelatinase A). MMP14 contains a catalytic zinc ion and another zinc and two calcium structural ions. We used Poisson-Boltzmann electrostatic calculations, together with computational ion removal perturbations and a computational alanine scan, to delineate the contribution of each ion and each charged amino acid in MMP14 stability and MMP14-TIMP2 interactions. Our results suggest that the ions influence nonspecific binding of TIMP2 to MMP14. In addition, the catalytic zinc ion loses its capacity for catalysis when bound to TIMP2 and becomes structurally significant. Surprisingly, despite the complementarily and excess of charge of the two proteins, we found that the binding interface is mostly hydrophobic in nature, with only one mutation of an acidic amino acid producing significant binding perturbation during the alanine scan. This study will serve as the foundation for investigating the mechanism of MMP14-TIMP2 association and to gain insight on the underlying physicochemical properties contributing to the broad specificity of TIMP2.
Web App BioInterface Simulator of Human Thyroid Hormone Feedback System Dynamics
Simon X. Han, Marisa Eisenberg and Joseph J. DiStefano III
UC Los Angeles
We have implemented an easy-to-use and easily accessible web-based application for the simulation of human thyroid hormone (TH) feedback regulation dynamics. Teachers, students, researchers, and clinicians can simulate common thyroid diseases by adjusting TH secretion or absorption rates. Easy-to- program and adjustable oral input regimens are implemented to simulate treatment options; and various intravenous inputs can also be added for research purposes. Simulated blood plasma hormone levels in response to these input treatments are displayed on the same page in separate graphs. The program was developed using common and open source components, including the Linux OS, Apache web server, and Perl programming language. Modern web technology AJAX is used for asynchronous communication with the server, JSON is used for data transfer, and JQuery helps generate dynamic content. The simulator is designed to be used especially as a tool for understanding normal and abnormal thyroid hormone dynamics and is compatible with most browsers. Currently, the interface is complete (see the figure below) and the simulator equation engine is being translated from MATLAB to an open source alternative called Octave. We expect to be online by Symposium time.
COMBOS: A Web App for Computing Global Identifiability Properties of Nonlinear Dynamic Systems Biology Models
Christine Kuo*, Nikki Meshkat and Joe DiStefano III
UC Los Angeles, * Computational & Systems Biology Undergraduate Interdepartmental Program, UCLA
Parameter identifiability problems can plague biomodelers when they reach the quantification stage of development, even for relatively simple models. Novice modelers often don’t even recognize that identifiability properties of the model are the problem. This highly technical subject is also difficult to teach. COMBOS is a user-friendly web app that addresses and solves key aspects of the structural identifiability (SI) problem for a practical class of nonlinear (and linear) ordinary differential equation (ODE) systems biology and other models. Given the ODE and measurement models (user input), it uniquely provides not only a list of SI model parameters, but also the combinations of parameters that are not individually SI, as shown in the accompanying figure example. COMBOS was developed for facile instructional as well as research use. It includes several built-in examples,with a 4th-order unidentifiable HIV dynamics model shown in the figure. COMBOS has been validated for models of moderate-dimension and is currently being optimized and tested for the largest models it can handle. The behind-the-scenes app symbolic differential algebra algorithm* is based on computing Gröbner bases of model attributes established after some algebraic transformations and is currently run inMaxima on our lab server. *Meshkat N, Anderson C, DiStefano III, JJ. Finding identifiable parameter combinations in nonlinear ODE models and the rational reparameterization of their input-output equations. Math Biosci 233:19-31 (2011).
Engineering Robust Control of Two-Component System Phosphotransfer Using Modular Scaffolds
Weston R. Whitaker, Stephanie A. Davis, Adam P. Arkin and John E. Dueber
The field of synthetic biology seeks to predictably forward engineer sophisticated biological systems. Remarkable successes towards this goal have been demonstrated with genetic circuits, aided by the clear modularity between transcription control elements and genes allowing new input control to be reliably achieved for a given gene. We demonstrate the feasibility of achieving similar modular rewiring in post-translational circuits, an underutilized layer of control in prokaryotes. We synthetically control enzyme-substrate co-assembly to direct phosphotransfer of two component systems. Scaffold proteins built from modular protein-protein interaction domains specifically co-target proteins tagged with corresponding interaction ligands. Flux can be directed from a histidine kinase to either of two non- cognate response regulators depending on the scaffold expressed. Finally, since this effect was highly sensitive to the balance of histidine kinase and response regulator concentrations, robustness to kinase concentration was gained by designing an allosterically-regulated kinase switch such that the ligand on the scaffold serves the dual functions of directing assembly and switching activation. These results are first steps towards a generalizable strategy for designing modular prokaryotic signal transduction.
Sparse linear modeling of next-generation mRNA sequencing (RNA-Seq) data for isoform discovery and abundance estimation
Jingyi Jessica Li, Ci-Ren Jiang, James B. Brown, Haiyan Huang, and Peter J. Bickel
Since the inception of next-generation mRNA sequencing (RNA-Seq) technology, various attempts have been made to utilize RNA-Seq data in assembling full-length mRNA isoforms de novo and estimating abundance of isoforms. However, for genes with more than a few exons, the problem tends to be challenging and often involves identifiability issues in statistical modeling. We have developed a statistical method called “sparse linear modeling of RNA-Seq data for isoform discovery and abundance estimation” (SLIDE) that takes exon boundaries and RNA-Seq data as input to discern the set of mRNA isoforms that are most likely to present in an RNA-Seq sample. SLIDE is based on a linear model with a design matrix that models the sampling probability of RNA-Seq reads from different mRNA isoforms. To tackle the model unidentifiability issue, SLIDE uses a modified Lasso procedure for parameter estimation. Compared with deterministic isoform assembly algorithms (e.g., Cufflinks), SLIDE considers the stochastic aspects of RNA-Seq reads in exons from different isoforms and thus has increased power in detecting more novel isoforms. Another advantage of SLIDE is its flexibility of incorporating other transcriptomic data such as RACE, CAGE, and EST into its model to further increase isoform discovery accuracy. SLIDE can also work downstream of other RNA-Seq assembly algorithms to integrate newly discovered genes and exons. Besides isoform discovery, SLIDE sequentially uses the same linear model to estimate the abundance of discovered isoforms. Simulation and real data studies show that SLIDE performs as well as or better than major competitors in both isoform discovery and abundance estimation. The SLIDE software package is available at https://sites.google.com/site/jingyijli/SLIDE.zip.