Hok 'Em Sok 'Em Robiotics

Presenter: Jason D. Kahn                             Status: Faculty

Authors: UMaryland iGEM Team 2015


Abstract: The poster summarizes the activities of the 2015 UMaryland iGEM (International Genetically Engineered Machines) team. The team adapted a bacterial plasmid-borne suicide module as a synthetic biology tool for maintaining plasmids without antibiotics, and they characterized the time course for protein expression from a gene on the same plasmid. They also demonstrated a $60 DIY PCR thermal cycler and carried out a variety of community outreach activities. The team won a Gold medal at the 2015 iGEM Jamboree in September.


An Impedance Microsensor with Virus-like-particle Nanoreceptors for Accelarated ELISA-on-a-chip

Presenter: Faheng Zang                               Status: Graduate Student

Authors: Faheng Zang, Xiao Zhu Fan, Konstantinos Gerasopoulos, Lindsay Naves, James Culver, and Reza Ghodssi


Abstract: We report an impedimetric approach that monitors nano sensing probe functionalization in real-time to optimize on-chip enzyme-linked immunosorbent assays (ELISA). Using continuous measurement of the electrical impedance between interdigitated microelectrodes, the self-assembly of Tobacco mosaic virus-like particles (VLPs) as bioreceptor layers was monitored.  VLPs modified with cysteine groups and FLAG-tag peptides (VLP-FLAG) were found to completely saturate the sensor surface after 3 hours of assembly. ELISA performed with target anti-FLAG antibody for a 3 hour VLP-FLAG assembly time exhibited similar impedance change to experiments with 18 hours, which was previously the standard VLP assembly time for antibody sensing. When compared to VLPs without the peptide tags (VLP-1cys), the VLP-FLAG functionalized sensor showed a 64% higher impedance change. These results combined highlight the potential of genetically modified VLPs for rapid and selective immunoassays.


Autonomous Sensing and Translation of the Interspecies Signal Molecule AI-2 for Population Based Behavior

Presenter: Kristina Stephens                                  Status: Graduate Student

Authors: Kristina Stephens, Chen-Yu Tsao, Amin Zargar, Melissa Rhoads, William E. Bentley


Abstract: The level of the interspecies signal molecule autoinducer-2 (AI-2), recognized and produced by a wide range of microbial species, gives insight to the number of AI-2 producing cells in a culture. Here, we developed translator cells capable of translating the interspecies AI-2 signal in a mixed population to a species specific, high fidelity AI-1 signal. A third population of cells alters its own behavior in response to the AI-1 level, and hence the AI-2 level, without altering the original AI-2 signal. To engineer this system, the E. coli AI-2 and P. aeruginosa 3OC12 homoserine lactone AI-1 quorum sensing circuits were coupled to construct an E. coli translator strain that produces AI-1 in response to AI-2. Here, we show that the amount of AI-1 produced by the translator cells is dependent on the level of AI-2 in the culture and that reporter cell behavior changes based on the amount of AI-1 produced by the translator cells. The use of synthetic biology to regulate behavior based on characteristics of a consortia may have probiotic therapeutic potential.6


Effects of Astrocytes from Brain Microenvironments on Motilityand Morphology of Tumor Cells

Presenter: Marina Shumakovich                             Status: Graduate Student

Authors: Marina A. Shumakovich, Kenneth Wesley Bryant, Kimberly M. Stroka


Abstract: Cancer metastasis results in over 90% of cancer related deaths. It is unclear how cancer cells permeate the blood-brain-barrier during metastasis to the brain; however the significant involvement of astrocytes is suspected. Astrocytes surround blood vessels in the brain and regulate various brain processes. They are believed to express factors which (positively or negatively) influence invasion and survival of cancer cells. The blood-brain barrier consists of endothelial cells linked by tight junctions. Tight junctions are present through the expression of various proteins, such as ZO-1, which can be affected by cues from the microenvironment, including astrocyte signals and the composition and stiffness of the matrix. The goal is to determine how biochemical signals from astrocytes as well as substrate composition affect the migration and protein expression of metastatic cancer cells as well as examine cancer cell incorporation into human brain microvascular endothelial cell monolayers on hydrogel matrices. The treatment of breast cancer cells with astrocyte conditioned media has shown a decrease in their motility as well as a change in their expression of ZO-1. Human brain microvascular endothelial cells (HBMECs) formed successful monolayers on soft hyaluronic acid hydrogels and the invasion of such monolayers by cancer cells was observed. The interaction of cancer cells with HBMECs also resulted in a change in cancer cell ZO-1 expression. Hence, it is suggested that the behavior of cancer cells is affected by neural cells such as astrocytes, as well as their extracellular matrix and the barrier which they are crossing.



Presenter: Thomas Winkler                         Status: Graduate Student

Authors: Thomas E. Winkler, Hadar Ben-Yoav, Deanna L. Kelly, Reza Ghodssi


Abstract: We present a combined study of analytical and finite element models (AM and FEM), validated with experimental data, on the interplay of hydrodynamic focusing and impedance cytometry toward enhanced sensitivity in differential leukocyte counts. These counts are critical in diagnosis and monitoring of a wide range of medical conditions. In recent years, significant advances have been made toward small, portable lab-on-a-chip (LOC) impedance cytometers to address this clinical need. The label-free method probes changes in dielectric properties caused by particles passing between two electrodes, recorded in terms of changes in impedance |ΔZ|, allowing for multi-dimensional single-cell analysis. However, current implementations still suffer from limited resolution. By integrating hydrodynamic focusing, wherein the effective interaction volume is decreased by constricting the sample flow through a virtual aperture (VA) defined by deionized water, modeling predicts an up to 10-fold increase in sensitivity. This process guided our optimized device design, where we have experimentally validated the enhanced performance.:


Multi-Variate Modeling for Blood Measurement: Accounting for Population Variability

Presenter: George Banis                               Status: Graduate Student

Authors: Sheryl Chocron, Hadar Ben-Yoav, Deanna Kelly, Reza Ghodssi


Abstract: This work utilizes a partial least squares regression (PLSR) chemometric model to process the responses of an electrochemical sensor in order to enable the detection of low therapeutic drug levels in complex serum samples. From the hundreds of species present in serum some can interfere with point-of-care sensor measurement. In previous studies uric acid (UA) was identified as the dominant endogenous interfering species in electrochemical sensing of an antipsychotic clozapine (CLZ) in serum, where therapeutic drug monitoring is recommended by prescribing guidelines. Thus, the presence and physiological variation of UA concentrations can cause reliability issues. Here, coupling the sensor response to a PLSR chemometric model enables accounting for UA variation during the CLZ measurement. In essence, this model maps the sensor responses into a new coordinate system (principal components) defined by the eigenvectors of the data. This approach conditions the data such that challenges from the UA and CLZ response interference and colinearity are reduced, improving the sensor performance and enabling the transition of sensors to the point-of-care. The key to this approach of accounting for population variability in complex serum matrices is to couple the knowledge of specific interfering species with advanced models that are calibrated with expected composition changes.


Role of Valency of Transcytosis of ICAM-1-Targeted Nanocarriers into the Brain

Presenter: Rachel L. Manthe                                    Status: Graduate Student

Authors: Rachel L. Manthe, Janet Hsu, and Silvia Muro


Abstract: Drug delivery across the endothelium and into the brain can be achieved by targeting nanocarriers (NCs) to receptors involved in transcytosis. Despite knowledge on transcytosis of natural ligands, how NC parameters impact this process is unknown. As receptor binding induces uptake and trafficking, we examined how NC valency (number of targeting molecules/NC) affects this process. We targeted NCs to intercellular adhesion molecule-1 (ICAM-1; an endothelial marker that mediates transcytosis-like leukocyte extravasation), which has shown promise for drug delivery into and across the endothelium. Model polystyrene or PLGA NCs were coated with varying ratios of antibodies (Ab) against ICAM-1 and control IgG, resulting in 180nm-diameter NCs with similar total Ab coating (~240), but varying targeting valency: high (245 Ab/NC), intermediate (125 Ab/NC), or low valency (64 Ab/NC). NC binding, uptake, and lysosomal trafficking were assessed in brain endothelial cells by fluorescence microscopy, and transcytosis and NC distribution in mice were examined using radiotracing. Increasing valency accelerated NC binding (Kd 84, 146, 397min for high-low valency), due to greater NC avidity (100, 112, 223pM). Yet, NC uptake remained similar (Kd 19, 32, 39min for high-low valency), suggesting that while uptake requires binding, it is independently regulated. Valency also had little impact on lysosomal trafficking (Kd 65, 82, 57min). Unexpectedly, lower valency increased the rate of NC transcytosis (Kd 220 vs. 342min for low and high valency). Perhaps, higher valency NCs establish enough receptor anchoring points that it hinders receptor recycling, resulting in intracellular entrapment. In accord, low valency NCs enhanced mouse brain delivery of ASM, the enzyme used for Niemann-Pick Disease therapy: high valency NCs enhanced ASM uptake 4-fold over free enzyme, while low valency NCs improved brain uptake by 10-fold. Thus, NC valency can be tuned to optimize delivery across the endothelium.


Controlled Delivery of Immunomodulatory nucleic acid via Polyelectrolyte Multilayers for the Treatment of Multiple Sclerosis

Presenter: Boyan Xia                                    Status: Undergraduate Student

Authors: Boyan Xia, Lisa Tostanoski, Christopher Jewell


Abstract: Multiple Sclerosis (MS) is an autoimmune disease where inflammation and demyelination occur due to immune cells recognizing naturally occurring myelin protein as foreign. Recent studies have demonstrated that treatment with soluble immunomodulatory nucleic acids (e.g., GpG) can partially suppress the inflammatory response triggered by autoimmune diseases such as MS. However, these therapies are limited by the fact that they do not target a specific region in the body which could lead to undesired systemic effects, such as broad immunosuppression. Biomaterials can help address these limitations by enabling targeted and controlled release in mice, and eventually, in patients. We hypothesize that the formation of multilayers using GpG and biodegradable polymers, poly(β-amino ester) (PBAEs), will generate tightly controlled drug release with tunable degradation rates. This idea could offer an improved mechanism for more specific kinetics of drug release, while simultaneously allowing for interaction with primary immune cells to promote tolerance.


Engineering Yeast Cells to Overexpress Isoamyl Acetate

Presenter: Rahul Raiker                               Status: Undergraduate Student

Authors: Rahul Raiker, Charlie Liu, Ben Woodard


Abstract: Isoamyl Acetate is an ester that creates a banana odor/flavor and it can be produced by certain genes that allow its creation. This project's aim is to engineer Saccharomyces Cerevisiae to overexpress isoamyl acetate than what is found naturally in order to have applications in the food industry.  BAT2, THI3, and ATF1 are the genes necessary to be incorporated into a yeast cell in order to allow the over expression of banana odor. This engineered Saccharomyces Cerevisiae would be a self-contained biosynthetic system that creates the banana odor/flavor without the use of any additional additives.


Functionalized Surfactant Vesicles.   A Platform for a Francisella Vaccine.  

Presenter: Philip DeShong                           Status: Faculty

Authors: K. Richard, B.J. Mann, L. Stocker, E.M. Barry, A. Qin, L.E. Cole, S.N. Vogel, R.K. Ernst, S.M. Michalek, D.C. Stein, P. DeShong


Abstract: Functionalized surfactant vesicles were used to present cell surface antigens from either Francisella tularensis type B live vaccine strain (Ft-LVS) or Ft type A Schu S4 strain (Ft-Schu4).   Immunization of mice with either vaccine produced a strong immune response that was protective in challenge studies.   In addition, anti-sera from Ft-LVS immunized mice conferred passive protection against a challenge by Ft-LVS.   This vaccine platform can be used to prepare vaccines against a wide variety of Gram-negative and Gram-positive pathogens.  


A Strategy to Avoid Phagocytosis of Drug Nanocarriers by Macrophages Without Affecting Receptor-Mediated Endocytosis by Specifically Targeted Cells

Presenter: Joshua Kim                                 Status: Undergraduate Student

Authors: Joshua Kim, Sauradeep Sinha, Janet Hsu, Silvia Muro


Abstract: Nanocarriers (NCs) are being extensively explored for targeting and delivering drugs to specific tissues in the body to improve their therapeutic outcome. However, macrophages and similar cells in the spleen and liver clear drug NCs out of the bloodstream via phagocytosis, reducing their therapeutic potential. In nature, erythrocytes express CD47 on their surface to avoid phagocytosis when they circulate through these organs. Similarly, coating CD47 on drug NCs reduces phagocytosis in the liver and spleen. Yet, these studies used NCs not aimed at uptake within tissue cells, and it is unknown whether NCs with CD47 can avoid macrophage phagocytosis without affecting targeted endocytosis to tissue cells. We have investigated this issue using fluorescent polymer NCs co-coated with CD47 and an antibody to intercellular adhesion molecule (ICAM-1), which our laboratory uses to enable endocytosis within endothelial and other cells. Anti-ICAM/CD47 NCs were compared to control anti-ICAM NCs, IgG NCs, as well as IgG/CD47 NCs, whereby their phagocytosis by peritoneal macrophages or endocytosis by endothelial cells was investigated using fluorescence microscopy. Our results show that the presence of CD47 reduced uptake of both anti-ICAM and IgG NCs by macrophages (~50% vs. absence of CD47). Yet, anti-ICAM/CD47 NCs targeted ICAM-1 on endothelial cells similarly to control anti-ICAM NCs (57 and 50 NCs/cell by .5hr, respectively). Not only was targeting possible in the presence of CD47, but uptake was also similar to control anti-ICAM NCs (99.5% and 99.9% of all targeted NCs at 5h) and via the same pathway, CAM-mediated endocytosis (~40% inhibition by amiloride). Also intracellular trafficking was similar in the presence vs. absence of CD47: ~75% of NCs co-localized in lysosomes by 8 h. Therefore, by co-coating of CD47 with a targeting antibody, we were able to markedly reduce NC phagocytosis by macrophages without affecting specific receptor-mediated targeting, endocytosis, and intracellular transport in tissue (endothelial) cells. This strategy could have a major impact in improving the efficacy of targeted NCs for drug delivery.


Multiple siRNA Delivery Using Bioreducible Polymeric Nanoparticles for Treatment of Primary Human Brain Cancer Cells

Presenter: Casey Vantucci                           Status: Undergraduate Student

Authors: Casey Vantucci, Marissa Gionet-Gonzales, Kristen L. Kozielski, and Jordan J. Green


Abstract: The use of short interfering RNA (siRNA) allows for gene knockdown through RNA interference (RNAi), and it has exciting potential for the treatment of primary human brain cancer cells. Aberrant gene expression in these cancer cells allow them to proliferate rapidly, migrate away from the bulk tumor site, and invade healthy tissue. When surgical resection or targeted therapies, like radiotherapy, are used on the primary tumor, these cells can escape treatment and allow for tumor recurrence. Targeting one overexpressed gene for knockdown has not proved as effective at preventing these behaviors because the cancer cells overcompensate by upregulating a different gene. The motivation of this work is to reduce tumor recurrence by preventing the cancer cells from overcompensating in this way by knocking down multiple genes at once using multiple siRNA delivery. A preliminary study determined that siRNA has the same knockdown efficiency at percent concentrations as low as 10% within a nanoparticle. At this concentration, there is the potential to deliver up to 10 siRNAs at once. In this work, we are investigating the effectiveness of several isoforms of siRNA for the following genes that are upregulated in primary brain cancer cells: NKCC1, CDK4, EGFR, YAP1, ROBO1, and PDGFR. Once the most effective siRNA isoform for each gene has been determined, these can then be combined into a single nanoparticle. The ability for multiple aberrant genes to be targeted and knocked down at once highlights the potential for siRNA-based nanomedicine.