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E. Coli

Featured Image: A scanning electron microscopy image of the bacterium Escherichia coli. Many bacteria, including E. coli, "talk" to each other by secreting and perceiving small molecules, a process called quorum sensing. Flagella and appendages that extend out of the cell walls can be produced in response to this signaling. Nearest neighbors control group behavior. Disrupting this intercellular communication could prove to be a new way to fight infection or disease.

The Bentley Group uses metabolic engineering tools to understand the phenomenon of bacterial quorum sensing. Quorum sensing is a method of bacterial communication controlled by the release uptake and processing of small molecules called auto inducers. Quorum sensing brings about a population wide changes in bacterial cellular response and can lead to bioluminescence, biofilm formation and increased pathogenecity. Functional genomic tools such as genetic engineering, microarrays for understand gene expression on a global basis and quantitative real time PCR have been used to elucidate QS based mechanisms in E coli.

As part of the Maryland Biochip Collaborative, the group combines these metabolic engineering tools with the biofabrication interface chitosan to create quorum sensing modulating bio-modular devices. One of the first examples is biological nanofactories which are bio-inspired, nano-sized and comprised of multiple functional modules. When deployed, biological nanofactories bind to the surface of cells they target and alter the response of these cells by locally synthesizing and delivering molecules-of-interest there. Two types of biological nanofactories; magnetic and antibody nanofactories, for synthesis and delivery of the universal quorum sensing bacterial signaling molecule autoinducer-2 (AI-2), on the bacterial cell surface have been demonstrated (Fernandes etal, 2007, Fernandes etal 2008). Protein G a specifically binds the Fc region of antibodies. The tyrosine tagged protein G created as a part of this project enables attachment of the biological modules to MEM devices through antibodies enabling creation of (bioMEMS) networks for understanding the quorum sensing mechanism. The nanofactory is a proof of concept for targeted drug delivery and the (bioMEMS) device conceptualizes drug discovery microfluidic networks. Biological modules which can degrade the universal signaling molecule AI-2 are employed as tools for inhibiting QS based signaling and phenotypic responses.

William E. Bentley

William Bentley

William E. Bentley (Ph.D., University of Colorado at Boulder, 1989) is the Robert E. Fischell Distinguished Professor and Chair of the Fischell Department of Bioengineering at the University of Maryland, College Park. He holds joint appointments with the Maryland Technology Enterprise Institute (MTECH) and the Institute for Bioscience and Biotechnology Research (IBBR). He is a Fellow of the American Academy of Microbiology, American Association for the Advancement of Science, and the American Institute for Medical and Biological Engineering.

Dr. Bentley has an outstanding record of technical contributions in metabolic engineering, modeling of genetic circuits, cellular stress responses and E. coli protein expression, bioreactor design and optimization, and insect cell and Larvae/Baculovirus expression systems. His work is internationally recognized.

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Group Members' Areas of Research

Tanya Gordonov

Varnika Roy

Tanya Gordonov is a graduate student in the Bioengineering program at the University of Maryland.  She earned her BS in Biological Sciences from Rutgers University in 2009 and started working in Dr. Bentley’s lab in 2010.

Tanya’s project aims to bridge the communication gap between biology and electronics through the use of redox molecules. Redox molecules carry information in the form of electrons and can interact in meaningful ways with both electrodes and biological entities such as redox-sensitive proteins in cells. By controlling the state of these mediators at the electrode, one can control their interaction with the redox-sensitive transcription factors inside the cells which control gene expression. The development of an electrically-responsive genetic promoter system would allow for “programmable” and dynamic gene expression. This advancement would be an important addition to the growing repertoire of dynamically- and remotely-controlled promoters in synthetic biology, a field in which Tanya is greatly interested and to which she would like to make a contribution.

Modified AI-2

Scheme 1. Tanya’s aim is to develop systems that allow relevant information transfer from electronics to cells and from cells to electronics that retains dynamic signal characteristics and allow for amplification and synchronization of the signals.

Modified AI-2

Scheme 2. A redox mediator’s state can be changed by passing current through an electrode with which it interacts. The mediator then activates a transcription factor TF, which in turn activates a promoter sequence and the transcription of reporter protein X. The goal is to produce reporter in amounts that correlate to the current input.

To facilitate cell-to-electrode communication, an enzymatic and electrochemically-detectable reporter system using the β-galactosidase enzyme is being improved and investigated as a means to obtain dynamic electrochemical information from cells.

Modified AI-2

Scheme 3. The inducer enters the cells and interacts with a transcription factor. This results in the LacZ gene transcription and ß-galactosidase production. Extracellular addition of the PAPG molecule results in its cleavage. PAP, one of the products, can be oxidized at the electrode. Thus different inducer amounts can result in different increases in current.

Bacterial quorum sensing has been previously used in order to amplify bacterial communication and synchronize the response of many cells. Here the intent is to use quorum sensing molecules to propagate, amplify, and synchronize the signal that is either coming from the electrode to the cells or from the cells to the electrode. This would allow for a lower detection limit and better signal integrity from colonies of cells.
The Biochip Collaborative has expertise in electrochemistry, genetic engineering, micro and bio fabrication and it is Tanya’s intent to capitalize on the collaborative knowledge and put to use as much of it as possible in order to achieve these goals. The Biochip Collaborative is uniquely positioned to solve the challenges that this project presents, and we believe that the resulting advancements will allow us to merge the capabilities of microelectronics and biology and bring us a step closer to utilizing and understanding biology’s repertoire of exquisite detection and response capabilities.

Sara Hooshangi

Dr. Sara Hooshangi specializes in the characterization of AI-2 uptake in E. coli quorum sensing circuitry. While the quorum sensing phenomenon has been studied in a large number of bacterial species, many questions remain unanswered. Previous models have addressed aspects of population dynamics in Pseudomonas aeruginosa and Vibrio fischeri but no comprehensive model of autoinducer AI-2 uptake has been proposed. The AI-2 signal transduction network comprises several important network topologies including a positive feedback loop, an autoregulation motif and, a number of negatively regulated modules which make it particularly interesting. The complex interaction of these network motifs are not yet fully understood and can be further analyzed using stochastic simulations. This study focuses on developing a computational model that captures the dynamics of quorum signal generation, receptor driven recognition, and AI-2 uptake. By combining the existing experimental data with comprehensive mathematical models, the relationship between basic cellular circuitry of quorum sensing and the phenotypic responses observed at the macroscopic scale can be elucidated.

Dr. Hooshangi (Ph.D., Princeton University, 2006) is a postdoctoral fellow. She earned her B.Eng. in electrical engineering from McGill University.


Hsuan-Chen Wu

Hsuan-Chen Wu

Hsuan-Chen has developed a generic strategy for the covalent assembly of proteins onto patterned surfaces, including sensor surfaces, by incorporating a tyrosine rich "pro-tag" at the C- terminus of a protein of interest. The tyrosyl residues of the pro-tag are enzymatically activated by tyrosinase and then covalently coupled to the primary amines of pH-stimuli responsive polysaccharide chitosan, which could be electrodeposited on conductive polymers or electrode surfaces. Protein G has a specific binding affinity to the heavy chain constant (Fc) region of immunoglobulin G (IgG). Taking advantage of this feature, we fused a pentatyrosine pro-tag to the C-terminus of the IgG binding domain of protein G, and then conjugated the fusion protein onto biofabricated aminopolysaccharide chitosan. The assembled complex could serve as a generic sensor for antigens or other proteins of interest.

Hsuan-Chen is a graduate student in the Fischell Department of Bioengineering. He earned a M.S. in life science from Tunghai University, Taiwan, in 2002 , and a B.S. in chemical engineering from National Taiwan University in 2000.


Former Group Members' Areas of Research

Varnika Roy

Varnika Roy

The goal of Varnika's research is to metabolically engineer the sensing circuit used to modulate the bacterial communication mechanism known as quorum sensing. Quorum sensing is the phenomenon of bacterial communication mediated by production. secretion and uptake of small signaling molecules called autoinducers. AI-2 is the "universal" signaling molecule observed in many bacterial species. Bacteria coordinate their activities at a population level using quorum sensing and this leads to biofilm formation, secretion of virulence factors and increase in bacterial pathogenecity. This work aims to modulate, more specifically inhibit the pathogenic quorum sensing response by utilizing enzymatic tools of the native in vivo quorum sensing circuitry of Escherichia coli. Enzymes that process and cleave AI-2 inside the E. coli cell are delivered outside the cell in order to degrade AI-2 outside the cell itself and knock down the quorum sensing mediated signaling. The effect of the phosphorylation and consequent degradation of the signaling molecule by these enzyme modules, on the quorum sensing circuitry genes and phenotypes such as bioluminescence and biofilm formation are monitored. This work envisions development of a new generation of anti microbials based on disabling a population of cells by modulating quorum sensing responses.

Modified AI-2

a) Native response of E. coli cells to AI-2 outside the cells. b) Engineered response: AI-2 phosphorylated outside the cells by delivering a kinase and change in cellular response is measured.

Varnika earned a B.S. from the University of Westminster London in 2006. She is currently a Ph.D. candidate in the Department of Molecular and Cell Biology. She has been with the Bentley Group since 2007.


Dr. Rohan Fernandes

Rohan Fernandes

The overall goal of Dr. Fernandes' research was to demonstrate biological nanofactories as an alternative approach to achieving targeted delivery by locally synthesizing and delivering small molecules at surface of the targeted cells. Biological nanofactories are nano-dimensioned and are comprised of multiple functional modules. In its most basic form, biological nanofactories consist of a cell targeting module and a synthesis module. When deployed, a biological nanofactory binds to the targeted cell surface and locally synthesizes and delivers small molecules at the surface of the cell, thus altering the response of the targeted cells. Dr. Fernandes' research dealt with biological nanofactories for the localized synthesis and delivery of the "universal" quorum sensing signaling molecule autoinducer-2. Two types of biological nanofactories were investigated: magnetic nanofactories and antibody nanofactories. When added to cultures of quorum sensing bacteria, the nanofactories bind to the surface of the targeted cells via the targeting module and locally synthesize and deliver AI-2 there via the synthesis module. The cells sense and uptake the AI-2 and alter their natural response. Prospects of using biological nanofactories to alter the native response of targeted cells to a "desired" state, especially with respect to down-regulating undesirable co-ordinated bacterial response such as bacterial pathogenicity, biofilm formation, bioluminescence are envisioned.

Nanofactory Scematic

Schematic of the method used to create magnetic nanofactories for altering bacterial response via localized synthesis and delivery of AI-2.

Nanofactory Scematic

a) Scanning electron microscope (SEM) image of magnetic nanofactories attached to targeted E. coli cells. b) Transmission electron microscope (TEM) image of magnetic nanofactories attached to the same bacterial cells.

Enhanced Nanofactory Using Fusion Protein

Improved Magnetic Nanofactory Using Fusion Protein
a) Schematic of biosynthesis of AI-2 and its role in signaling b) Construction of a plasmid that expresses the AI-2 pathway enzymes on a fusion protein c) Assembly of magnetic nanofactories: fusion protein onto magnetic nanoparticles d) Use of magnetic nanofactories to alter bacterial response e) Amount of AI-2 delivered to targeted bacteria via different techniques f) Cellular response to AI-2 delivered in (e).

Dr. Fernandes (Ph.D., University of Maryland, 2008) orginally worked for the Bentley Group as a student, then became a Faculty Research Associate at the Center for Biosystems Research, University of Maryland Biotechnology Institute. He holds an M.S. in chemical engineering from the University of Maryland and a B.S. in chemical engineering from the Mumbai University Institute of Chemical Technology.

Bentley Group Presentations

  1. “Enzymatic assembly and protein engineering for advancing molecular detection techniques”. Tanya Gordonov, Jessica Terrell, Hsuan-Chen Wu, Chen-Yu Tsao, Darryll Sampey, Xiaolong Luo, Yi Cheng, Yi Liu, Gary Rubloff, Gregory Payne, and William Bentley.  ACS 241st National Meeting. March 2011. Talk.
  2. “Biofabrication for interrogating cell signaling”. AVS 58th International Symposium & Exhibition. Oct 2011. Invited talk.
  3. “Electric control of enzymatic activity through redox mediators”. Tanya Gordonov, Eunkyoung Kim, Gregory Payne, and William Bentley. ACS 243rd National Meeting. March 2012. Poster.
  4. “Electric control of enzymatic activity through redox mediators”. Tanya Gordonov, Eunkyoung Kim, Gregory Payne, and William Bentley. BMES 2012 Annual Meeting. October 2012. Poster.
  5. “Bio-based Redox-Capacitor to Intercede in Microbe-Electrode Electron Flow”. Eunkyoung Kim, Tanya Gordonov, Yi Liu, Yossef A. Elabd, William E. Bentley and Gregory F. Payne. AIChE 2012 Annual Meeting. November 2012. Talk
  6. Deciphering bacterial communication using multi-modular biological nanofactories in a microfluidics device (Oral Presentation). Rohan Fernandes, Xiaolong Luo, Gary W. Rubloff and William E. Bentley. American Chemical Society 236th National Meeting & Exposition. August 17-21, 2008, Philadelphia.
  7. In vitro LsrK: toward an AI-2 phosphorylation nanofactory that modulates bacterial talk (Poster), Varnika Roy, Rohan Fernandes, Chen Yu Tsao, William Bentley, American Chemical Society 236th National Meeting& Exposition. August 17-21, 2008, Philadelphia
  8. Vesicle Nanofactories: tuning bacterial response via localized synthesis and delivery of AI-2 (Poster). Rohan Fernandes, Matthew Dowling, Srinivasa Raghavan and William E. Bentley. Annual Biomedical Engineering Society Conference. September 26-29, 2007, Los Angeles.
  9. Nanofactories for synthesis and delivery of signaling molecules: A tool for engineering metabolism (Oral Presentation). Rohan Fernandes, Chen-Yu Tsao, Chong Yung and William E. Bentley. American Chemical Society 234th National Meeting & Exposition. August 19-23, 2007, Boston.
  10. Magnetic Nanofactories for synthesis and delivery of signal molecule AI-2 to bacterial cell surfaces (Oral Presentation). Rohan Fernandes, C. Tsao, Y. Hashimoto, L. Wang, T. K. Wood, G. F. Payne and W. E. Bentley. Annual Biomedical Engineering Society Conference. October 11-14, 2006, Chicago.
  11. Magnetic Nanofactories: Localized Synthesis and Delivery of Quorum Sensing Signal Molecule Autoinducer-2 to Bacterial Cell Surfaces (Poster). Rohan Fernandes, Chen- Yu Tsao, Yoshifumi Hashimoto, Gregory F. Payne and William E. Bentley. Presented by Angela T. Lewandowski. Metabolic Engineering VI: From rec DNA Towards Engineering Biological Systems. October 1-5, 2006, Leeuwenhorst, The Netherlands.
  12. Nano-Scale Engineering at the Cell Surface: Synthesis and Delivery of a Quorum Sensing Autoinducer at the Cell Surface Using Magnetic "˜Nanofactories' (Poster). Rohan Fernandes and William E. Bentley. Third International Nanomedicine and Drug Delivery Symposium. September 26-27, 2005, Baltimore.