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Full-Text Articles in Biological Engineering

Determining The Effects Of Glycocalyx Modifications On The Electrophysical Properties Of Human Mesenchymal Stem Cells, Rominna E. Valentine Ico Dec 2023

Determining The Effects Of Glycocalyx Modifications On The Electrophysical Properties Of Human Mesenchymal Stem Cells, Rominna E. Valentine Ico

Electronic Theses, Projects, and Dissertations

Human mesenchymal stem cells (hMSCs) have gained popularity in clinical trials due to their multipotent differentiation characteristics, ability to secrete bioactive molecules, migrate into diseased or damaged tissues, and their immunosuppressive properties. HMSC cultures are heterogeneous, containing stem cells, partially differentiated progenitor cells, and fully differentiated cells. One of the major challenges with hMSCs therapeutic potential is the inability to select specific cell subpopulations due to an insufficient number of biomarkers. Often the biomarkers used, like those for fluorescence-activated cell sorting, are not sufficient to define hMSCs because they overlap with other cell types. Consequently, there is a need to …


A Comparison Of Optical Measurement Methods For The Growth Of S. Cerevisiae, Jackson Black May 2023

A Comparison Of Optical Measurement Methods For The Growth Of S. Cerevisiae, Jackson Black

Chemical Engineering Undergraduate Honors Theses

Genetic engineering of living organisms provides the opportunity to express and harvest different proteins from cell surfaces. Yeast (S. cerevisiae) is one such organism and is capable of being grown on an industrial scale. Cellular concentration is an important parameter to monitor while fermentation processes are underway, in order to control the environment inside the growth medium and maximize yields. Spectrophotometry is a conventional method for measuring concentration, but is limited by a narrow absorbance range, and the need for on-site periodic sampling. A continuous method of measurement, as provided by Bug Labs BE2100 non-invasive biomass monitor, would …


Microalgae Immobilization With Filamentous Fungi: Process Development For Sustainable Food Systems, Suvro Talukdar Jan 2023

Microalgae Immobilization With Filamentous Fungi: Process Development For Sustainable Food Systems, Suvro Talukdar

Theses and Dissertations--Biosystems and Agricultural Engineering

Demand for sustainable food sources has increased because of the rapid growth of the world's population.  In this study, microalgae cells of Haematococcus pluvialis were immobilized using the edible fungal strain Aspergillus awamori for potential food applications. The study investigated the impact of fungal loading, pellet geometry, and initial microalgae cell concentration on the immobilization performance and product characteristics. It was found that higher fungal loading and larger fungal pellets contributed to increased immobilization performance while increased initial microalgae concentration inhibited the process. Larger fungal pellets had decreased biomass density, which led to decreased surface concentration of immobilized microalgae but …


Frontiers In The Self-Assembly Of Charged Macromolecules, Khatcher O. Margossian Oct 2022

Frontiers In The Self-Assembly Of Charged Macromolecules, Khatcher O. Margossian

Doctoral Dissertations

The self-assembly of charged macromolecules forms the basis of all life on earth. From the synthesis and replication of nucleic acids, to the association of DNA to chromatin, to the targeting of RNA to various cellular compartments, to the astonishingly consistent folding of proteins, all life depends on the physics of the organization and dynamics of charged polymers. In this dissertation, I address several of the newest challenges in the assembly of these types of materials. First, I describe the exciting new physics of the complexation between polyzwitterions and polyelectrolytes. These materials open new questions and possibilities within the context …


Extraction Of Melanin From Black Knot Fungus: Optimization And Applications, Amara Davis Jan 2022

Extraction Of Melanin From Black Knot Fungus: Optimization And Applications, Amara Davis

Williams Honors College, Honors Research Projects

The goal of this research project was to optimize the extraction of melanin from black knot fungus and to analyze possible applications of the extracted melanin. An extraction method that was previously created was examined for possible improvements. Improvements that were studied include the elimination of steps in the extraction process while obtaining the same quality of product, examining options for machinery that could be used to increase production rate when moved to a production plant setting, and optimize the concentrations of the raw materials that are used in the extraction for possible reduction of cost without compromising yield or …


Real-Time Monitoring Of Cell Death Progress Using Capacitance Spectroscopy, Suyang Wu Dec 2021

Real-Time Monitoring Of Cell Death Progress Using Capacitance Spectroscopy, Suyang Wu

Electronic Theses and Dissertations

Biologics, including the monoclonal antibody (mAb), has experienced rapid development in the last decade. However, the price of biologics is often prohibitively high because of the low process efficiency. Delaying the inevitable cell death improves the productivity of upstream bioprocessing, whose success relies on monitoring the cell death onset that indicates the timing for preventive actions.

This study proposes to develop a real-time monitoring model that quantifies the dying cell percentage in lab-scale bioreactors using capacitance spectroscopy. The capacitance spectroscopy contains cell death-related information due to various physical properties changes during the cell death process, e.g., cytoplasmic conductivity change. The …


Greenhouse Tomatoes: Process Simulation, Juan Gabriel Marin Jr. Dec 2021

Greenhouse Tomatoes: Process Simulation, Juan Gabriel Marin Jr.

Graduate Theses and Dissertations

Growing population demand and challenges brought on by climate change have spurred the need for more resilient fruit and vegetable supply chains. One agricultural technology of significant interest is the use of greenhouses for food production. Greenhouses create a stable and adaptable environment for crops such as tomatoes to grow year-round. Fresh tomatoes are the second most consumed vegetable per capita in U.S. diets, currently averaging 20.7 pounds. The growing consumption of fresh tomatoes has been the result of increasing cultural diversity in the United States.

To meet the growing demand, Venlo-type greenhouses have been frequently used by growers. It …


A Portable Point-Of-Care Device Using Joule Heating And Latent Energy Storage For The Temperature Regulation Of Isothermal Nucleic Acid Amplification Tests, Aubrey Lynn Schultz Jul 2021

A Portable Point-Of-Care Device Using Joule Heating And Latent Energy Storage For The Temperature Regulation Of Isothermal Nucleic Acid Amplification Tests, Aubrey Lynn Schultz

Graduate Theses and Dissertations

Accurate and early diagnosis of infectious diseases extremely important. Rapid diagnosis allows for effective treatment and increases the chance for recovery without complications. Additionally, the ability to test the populace frequently, swiftly, and affordably significantly aids in containing wide-scale outbreaks. In terms of specificity and sensitivity, nucleic acid amplification tests (NAAT) are one of the best options for diagnosing infectious diseases. Isothermal NAATS present a unique opportunity to create diagnostic tests deployed at a Point-of-Care (POC) level. Specifically, loop-mediated isothermal amplification (LAMP) and recombinase polymerase amplification (RPA) have the potential to deliver reliable POC diagnostics in low-resource settings. When designing …


Design Of An Undergraduate Laboratory Experiment Utilizing A Stirred-Tank, Jacketed Bioreactor, Brennen Middleton May 2021

Design Of An Undergraduate Laboratory Experiment Utilizing A Stirred-Tank, Jacketed Bioreactor, Brennen Middleton

Honors Theses

Bioreactors are utilized in many industries, such as the food, alternative fuel, and pharmaceutical industries, to design and manufacture products. Unlike the similar chemical batch reactors utilized in many chemical processing facilities, bioreactor utilization requires engineers to consider a wider range of operating conditions and parameters. This results in a more complex reaction system and controls network. Due to this, it is imperative for chemical and biomedical engineering students to not only understand the theory surrounding these reactor systems, but also understand how to properly design and perform operating procedures with these systems. Thus, it was determined to create an …


Engineered Switch Protein Inspired By Novel Protein Affinity Transition Mode, Liang Fang May 2021

Engineered Switch Protein Inspired By Novel Protein Affinity Transition Mode, Liang Fang

Doctoral Dissertations

Many natural proteins involved in complex biological processes such as ligand binding and protein folding demonstrate multiple, allosterically-regulated conformational states, with protein activity regulated by effector molecules. The alpha L integrin and its inserted domain (I domain) is one example of such a protein. The binding of the effector molecule such as talin or filamin to the cytoplasmic domain of the integrin increases the binding affinity between I- domain and its ligand intercellular adhesion molecule-1, known as ICAM-1.There are multiple models attempting to describe the mechanism responsible for the change in binding affinity. According to research conducted by our lab, …


Development Of Light Actuated Chemical Delivery Platform On A 2-D Array Of Micropore Structure, Hojjat Rostami Azmand, Hojjat Rostami Azmand Jan 2021

Development Of Light Actuated Chemical Delivery Platform On A 2-D Array Of Micropore Structure, Hojjat Rostami Azmand, Hojjat Rostami Azmand

Dissertations and Theses

Localized chemical delivery plays an essential role in the fundamental information transfers within biological systems. Thus, the ability to mimic the natural chemical signal modulation would provide significant contributions to understand the functional signaling pathway of biological cells and develop new prosthetic devices for neurological disorders. In this paper, we demonstrate a light-controlled hydrogel platform that can be used for localized chemical delivery in a high spatial resolution. By utilizing the photothermal behavior of graphene-hydrogel composites confined within micron-sized fluidic channels, patterned light illumination creates the parallel and independent actuation of chemical release in a group of fluidic ports. The …


Micro-Physiological Models To Mimic Mucosal Barrier Complexity Of The Human Intestine In Vitro, Abhinav Sharma Dec 2020

Micro-Physiological Models To Mimic Mucosal Barrier Complexity Of The Human Intestine In Vitro, Abhinav Sharma

Doctoral Dissertations

The mucosal barrier in the intestine is vital to maintain selective absorption of nutrients while protecting internal tissues and maintaining symbiotic relationship with luminal microbiota. This bio-barrier consists of a cellular epithelial barrier and an acellular mucus barrier. Secreted mucus regulates barrier function via in situ biochemical and biophysical interaction with luminal content that continually evolves during digestion and absorption. Increasing evidence suggests that a mucus barrier is indispensable to maintain homeostasis in the gastrointestinal tract. However, the importance of mucus barrier is largely underrated for in vitro mucosal tissue modeling. The major gap is the lack of experimental material …


The Effects Of Solution Condition On Virus Filtration Performance, Fnu Namila Dec 2020

The Effects Of Solution Condition On Virus Filtration Performance, Fnu Namila

Graduate Theses and Dissertations

Virus filtration is an integral part of the downstream purification of mammalian cell culture-derived biotherapeutics to assure the viral safety of the products. Virus filtration membranes remove viruses based on a size-exclusion mechanism. Commercial parvovirus filers possess unique membrane structure and are designed to remove smaller non-enveloped parvoviruses with size 18-26 nm. However, some filters face issues, such as pre-mature fouling, the decline of filtrate flux, and reduction in virus retention. This doctoral dissertation focused on identifying the factors that influence the filtrate flux and the virus retention capability of commercial virus filters. The effects of solution pH and ionic …


Incorporation Of Lignin In Natural And Synthetic Biomaterials To Alter Mechanical And Biochemical Properties For Enhanced Wound Healing, Jorge Alfonso Belgodere Nov 2020

Incorporation Of Lignin In Natural And Synthetic Biomaterials To Alter Mechanical And Biochemical Properties For Enhanced Wound Healing, Jorge Alfonso Belgodere

LSU Doctoral Dissertations

It is estimated that chronic, non-healing wounds affect more than 6.5 million Americans annually, with an estimated healthcare cost beyond $14 billion. Here, we attempted to create composites of natural (collagen type I or gelatin-methacrylate) or synthetic (poly(ethylene glycol) polymers incorporating a natural plant component, lignin, to combat the costs and limitations current wound healing methods face. Three-dimensional matrices of collagen type I (Col I) are widely used in tissue engineering applications for its abundance in many tissues, bioactivity with many cell types, and excellent biocompatibility. Inspired by the structural role of lignin in plant tissue, we found that sodium …


Experimental And Computational Tools For Single Cell Analysis In Cancer Diagnostics, Manibarathi Vaithiyanathan Jan 2020

Experimental And Computational Tools For Single Cell Analysis In Cancer Diagnostics, Manibarathi Vaithiyanathan

LSU Doctoral Dissertations

Substantial evidence shows that cellular heterogeneity commonly exists within an isogenic or clonal population. Whether in isolation or caused through a combination of the above events, cellular heterogeneity can dramatically influence cellular decision making and cell fate, however, this can be masked by the average response from a population. One approach to solve this issue is to analyze a population at the individual cell level. The goal of this work is to develop high-throughput experimental and computational platforms to screen and quantify single cancer cells for specific intracellular enzyme activities. An interdisciplinary approach was taken to 1) better understand the …


Higher Tensile Forces Across Cellular Junctions And An Intact Nuclear Linc Complex Is Required For Epithelial Function And Stability, Fnu Vani Narayanan Jan 2020

Higher Tensile Forces Across Cellular Junctions And An Intact Nuclear Linc Complex Is Required For Epithelial Function And Stability, Fnu Vani Narayanan

Theses and Dissertations

Recent advances in three-dimensional (3D) cell culture systems have provided key insights into the understanding of biochemical and physiological states of native tissue. A significant progress in the field of mechanobiology involves measuring cellular traction forces in a more native 3D environment. However, the effects of mechanical forces exerted across cellular junctions and the nuclear LINC complex, in an organized 3D system has not been investigated thus far. Epithelial cells spontaneously form acini (also known as cysts or spheroids) with a single, fluid-filled central lumen, when grown in 3D matrices. The size of the lumen is dependent on apical secretion …


Lipemia Levels Analysis From Human Blood Samples, Zainab Ibrahim Alshoug Jan 2020

Lipemia Levels Analysis From Human Blood Samples, Zainab Ibrahim Alshoug

Dissertations, Master's Theses and Master's Reports

Worldwide, an estimated 31% of people who die each year have a cardiovascular disease (CVD), an all-encompassing group of diseases and conditions of the heart or blood vasculature. Access to portable, user-friendly tools to test lipid levels accurately and efficiently, without requiring large volume blood draws, will help improve access to wellness management by reducing costs and facilitating early screening and monitoring of CVD thus improving outcomes. Point of care micro or millifluidic chips to test biofluids that are integrated into detection units are an attractive measurement tool because of their potential for ease of use by patients at home …


A Study Of Protein And Peptide-Directed Nanoparticle Synthesis For Catalytic Materials, Abdollah Mosleh Aug 2019

A Study Of Protein And Peptide-Directed Nanoparticle Synthesis For Catalytic Materials, Abdollah Mosleh

Graduate Theses and Dissertations

Nanoparticles have received much attentions due to their unique properties that makes them suitable candidates for a broad range of applications. As the size of particles decreases, their surface area-to-volume ratio would increase which is the main cause of much attention. In addition to the size, their morphologies and compositions may also play important roles for defining unique properties. Nanoparticle synthesis include both bottom-up and top-down strategies. To control the process of inorganic nanoparticles synthesis one could follow the bottom-up approach to have atom-level control over their compositions, morphologies, phases, and sizes which is the subject of this work. Due …


Maximizing And Modeling Malonyl-Coa Production In Escherichia Coli, Tatiana Thompson Silveira Mello Jun 2019

Maximizing And Modeling Malonyl-Coa Production In Escherichia Coli, Tatiana Thompson Silveira Mello

LSU Master's Theses

In E. coli, fatty acid synthesis is catalyzed by the enzyme acetyl-CoA carboxylase (ACC), which converts acetyl-CoA into malonyl-CoA. Malonyl-CoA is a major building block for numerous of bioproducts. Multiple parameters regulate the homeostatic cellular concentration of malonyl-CoA, keeping it at a very low level. Understanding how these parameters affect the bacterial production of malonyl-CoA is fundamental to maximizing it and its bioproducts. To this end, competing pathways consuming malonyl-CoA can be eliminated, and optimal nutritional and environmental conditions can be provided to the fermentation broth. Most previous studies utilized genetic modifications, expensive consumables, and high-cost quantification methods, making …


Stability Analysis Of A More General Class Of Systems With Delay-Dependent Coefficients, Chi Jin, Keqin Gu, Islam Boussaada, Silviu-Iulian Niculescu May 2019

Stability Analysis Of A More General Class Of Systems With Delay-Dependent Coefficients, Chi Jin, Keqin Gu, Islam Boussaada, Silviu-Iulian Niculescu

SIUE Faculty Research, Scholarship, and Creative Activity

This paper presents a systematic method to analyse the stability of systems with single delay in which the coefficient polynomials of the characteristic equation depend on the delay. Such systems often arise in, for example, life science and engineering systems. A method to analyze such systems was presented by Beretta and Kuang in a 2002 paper, but with some very restrictive assumptions. This work extends their results to the general case with the exception of some degenerate cases. It is found that a much richer behavior is possible when the restrictive assumptions are removed. The interval of interest for the …


Coatings On Mammalian Cells: Interfacing Cells With Their Environment, Kara A. Davis, Pei-Jung Wu, Calvin F. Cahall, Cong Li, Anuhya Gottipati, Brad J. Berron Jan 2019

Coatings On Mammalian Cells: Interfacing Cells With Their Environment, Kara A. Davis, Pei-Jung Wu, Calvin F. Cahall, Cong Li, Anuhya Gottipati, Brad J. Berron

Chemical and Materials Engineering Faculty Publications

The research community is intent on harnessing increasingly complex biological building blocks. At present, cells represent a highly functional component for integration into higher order systems. In this review, we discuss the current application space for cellular coating technologies and emphasize the relationship between the target application and coating design. We also discuss how the cell and the coating interact in common analytical techniques, and where caution must be exercised in the interpretation of results. Finally, we look ahead at emerging application areas that are ideal for innovation in cellular coatings. In all, cellular coatings leverage the machinery unique to …


Feasibility Of Integrating Microfluidics Into Point-Of-Care Bio-Diagnostics, Swetha Manoharan Jan 2019

Feasibility Of Integrating Microfluidics Into Point-Of-Care Bio-Diagnostics, Swetha Manoharan

Theses

Potassium in the body needs to be maintained in proper levels for the normal functioning of heart and kidneys. The aim of this work is to develop a simple technique to measure the concentration of Potassium present in a bodily fluid sample, which can be used to assess the health condition of extremely sick patients, especially when the patient's condition needs to be diagnosed and results needs to be obtained rapidly under Point-of-Care settings. Microfluidics is one of the platforms used in bio-diagnostics because of several advantages such as low sample volume requirement and integration into high-throughput screening. In this …


Modification Of Lipid Microenvironments On Solid Support Structures For Use In Transmembrane Protein Assays, William J. Houlihan Jan 2019

Modification Of Lipid Microenvironments On Solid Support Structures For Use In Transmembrane Protein Assays, William J. Houlihan

Dissertations and Theses

Gamma-Secretase (γ-secretase) is a transmembrane protease of increasing interest, which has been shown to have significant connections to both cancer and Alzheimer’s disease. γ-secretase cleaves both Notch-1, a transmembrane signaling protein, and Amyloid precursor protein (APP), a transmembrane protein whose cleavage may result in the formation of β-amyloid plaques in the brain. Notch-1 and APP are widely studied proteins that have substantial impacts on the development and proliferation of cancer and Alzheimer’s disease, respectively. Notch-1 partakes in the signaling of apoptosis in damaged and mutated cells, thus its cleavage by γ-secretase within the plasma membrane has ramifications on cell growth …


Biophysical Features Of The Extracellular Matrix Direct Breast Cancer Metastasis, Alyssa Schwartz Nov 2018

Biophysical Features Of The Extracellular Matrix Direct Breast Cancer Metastasis, Alyssa Schwartz

Doctoral Dissertations

Breast cancer is plagued by two key clinical challenges; drug resistance and metastasis. Most work to date probes these events on an extremely rigid plastic surface, which recapitulates few aspects of these processes in humans. A malignant cell first resides in breast tissue, then likely travels to the bone, brain, liver, or lung, each of which has a distinct mechanical and biochemical profile. Cells transmit mechanical forces into intracellular tension and biochemical signaling events, and here we hypothesize that this mechanotransduction influences drug response, growth, and migration. To probe the impact of extracellular matrix on drug resistance, we defined a …


Radical Social Ecology As Deep Pragmatism: A Call To The Abolition Of Systemic Dissonance And The Minimization Of Entropic Chaos, Arielle Brender May 2018

Radical Social Ecology As Deep Pragmatism: A Call To The Abolition Of Systemic Dissonance And The Minimization Of Entropic Chaos, Arielle Brender

Student Theses 2015-Present

This paper aims to shed light on the dissonance caused by the superimposition of Dominant Human Systems on Natural Systems. I highlight the synthetic nature of Dominant Human Systems as egoic and linguistic phenomenon manufactured by a mere portion of the human population, which renders them inherently oppressive unto peoples and landscapes whose wisdom were barred from the design process. In pursuing a radical pragmatic approach to mending the simultaneous oppression and destruction of the human being and the earth, I highlight the necessity of minimizing entropic chaos caused by excess energy expenditure, an essential feature of systems that aim …


Molded Features In Pdms For Fabricating Bacterial Cellulose For Various Geometries, Mitchell Habegger Jan 2018

Molded Features In Pdms For Fabricating Bacterial Cellulose For Various Geometries, Mitchell Habegger

Williams Honors College, Honors Research Projects

The purpose of producing features on bacterial cellulose (BC) is to facilitate the elongation and alignment for cells, in this case Normal Human Dermal Fibroblast (NHDF) cells. The elongated cells have applications in wound healing, tissue engineering, disease diagnostics, and many other fields. Experiments were run to test the effectiveness of transferring features to BC sheets from features induced by fracturing on polydimethylsiloxane (PDMS) and those duplicated from molds with existing features. The features were duplicated to BC sheets by either air drying or Guided Assembly-Based Biolithography (GAB). The research results showed that fracture inducing on PDMS produced very small …


An Application Of M-Matrices To Preserve Bounded Positive Solutions To The Evolution Equations Of Biofilm Models, Richard S. Landry Jr. Dec 2017

An Application Of M-Matrices To Preserve Bounded Positive Solutions To The Evolution Equations Of Biofilm Models, Richard S. Landry Jr.

University of New Orleans Theses and Dissertations

In this work, we design a linear, two step implicit finite difference method to approximate the solutions of a biological system that describes the interaction between a microbial colony and a surrounding substrate. Three separate models are analyzed, all of which can be described as systems of partial differential equations (PDE)s with nonlinear diffusion and reaction, where the biological colony grows and decays based on the substrate bioavailability. The systems under investigation are all complex models describing the dynamics of biological films. In view of the difficulties to calculate analytical solutions of the models, we design here a numerical technique …


Gender And Participation In An Engineering Problem-Based Learning Environment, Laura Hirshfield, Milo D. Koretsky Nov 2017

Gender And Participation In An Engineering Problem-Based Learning Environment, Laura Hirshfield, Milo D. Koretsky

Interdisciplinary Journal of Problem-Based Learning

The use of problem-based learning (PBL) is gaining attention in the engineering classroom as a way to help students synthesize foundational knowledge and to better prepare students for practice. In this work, we study the discourse interactions between 27 student teams and two instructors in an engineering PBL environment to analyze how participation is distributed among team members, paying particular attention to the differences between male and female students. There were no statistically significant differences between the amount that male and female students spoke; however, stereotypical gender roles and traditional gendered behavior did manifest in the discussion. Also, regardless of …


Epitaxially Grown Collagen Fibrils Reveal Diversity In Contact Guidance Behavior Among Cancer Cells, Juan Wang, Joseph W. Petefish, Andrew C. Hillier, Ian C. Schneider May 2017

Epitaxially Grown Collagen Fibrils Reveal Diversity In Contact Guidance Behavior Among Cancer Cells, Juan Wang, Joseph W. Petefish, Andrew C. Hillier, Ian C. Schneider

Andrew C. Hillier

Invasion of cancer cells into the surrounding tissue is an important step during cancer progression and is driven by cell migration. Cell migration can be random, but often it is directed by various cues such as aligned fibers composed of extracellular matrix (ECM), a process called contact guidance. During contact guidance, aligned fibers bias migration along the long axis of the fibers. These aligned fibers of ECM are commonly composed of type I collagen, an abundant structural protein around tumors. In this paper, we epitaxially grew several different patterns of organized type I collagen on mica and compared the morphology …


Molecular Modeling Of Antibody-Antigen Binding Near Solid Surfaces, Derek Bush, Thomas Knotts Feb 2017

Molecular Modeling Of Antibody-Antigen Binding Near Solid Surfaces, Derek Bush, Thomas Knotts

Biomedical Engineering Western Regional Conference

Antibody microarrays are biosensors that have the potential to revolutionize molecular detection in medicine, scientific research, and national defense. However, current microarrays are not widely used due to problems including poor reproducibility and signal quality, unbalanced antibody performance, and cross-reactivity. Prior work in the area focused mainly on the stability of the antibody alone and not its affinity for its antigen. This presentation shows results of using molecular simulation to determine how different types of surfaces affect antigen binding to surface-tethered antibodies. The results offer an unprecedented, molecular-level view into these protein-protein-surface interactions and how to drive binding to occur.