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Upstream Processing: Microbial Metabolic Engineering: New Biomolecular Tools for Microbial Metabolic Engineering
10:30am - 12:30pm USA / Canada - Eastern - August 24, 2021 | Room: Zoom Room 03
Melisa Carpio, Organizer; Danielle Ercek, Organizer, Northwestern University; Nitya Jacobs, Organizer; Aditya Kunjapur, Presider, MIT; Josh Leonard, Presider, ‍ ; Tiffany Rau, Presider, ‍ ; Quinn Mitrovich, Presider, ‍
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
Division/Committee: [BIOT] Division of Biochemical Technology

Advances in synthetic biology have enabled the design and construction of cell factories for the cleaner production of chemicals and other biological products important to humans. Metabolic engineering aims to develop tools and strategies that can be used in the optimization of biochemical pathways and in the design and implementation of non-native pathways, leading to more efficient biocatalysts and access to novel products. For this session we welcome presentations on topics related to microbial metabolic engineering, including the design, construction and testing of whole-cell biocatalysts, the development of tools for metabolic modeling and profiling, and the incorporation of novel substrates and biochemical reactions within biological systems.

Tuesday
Engineering a global regulatory system potentiates rapid growth of Saccharomyces cerevisiae on numerous non-native substrates
10:30am - 10:50am USA / Canada - Eastern - August 24, 2021 | Room: Zoom Room 03
Sean Sullivan, Presenter, Tufts University; Vikas Trivedi, Tufts University; Venkatesh Gopinarayanan
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
The potential of biotechnology to replace fossil fuel-based processes for the production of value-added molecules will depend largely on its economic competitiveness. To this end, there have been decades of work to engineer the ability of the versatile industrial cellular factory Saccharomyces cerevisiae to grow on cheap and renewable sources of carbon and energy such as lignocellulosic biomass, glycerol, and various C1 compounds. However, the implementation of heterologous catabolic pathways to enable the assimilation of these non-native substrates has so far generally relied on constitutive gene expression without consideration for global regulatory systems that may enhance nutrient assimilation and cell growth. Previous work by our lab has demonstrated that activating the galactose (GAL) regulon (a REG approach), the regulatory metabolic structure responsible for coordinating growth on the native substrate galactose, during growth on the non-native sugar xylose results in higher growth rates and more efficient substrate utilization compared with traditional constitutive expression (a CONS approach). The present study seeks to build on this foundation by exploring whether the large-scale gene expression changes observed upon activation of the GAL regulon are broadly beneficial to growth on non-native substrates. We start by constructing and characterizing a self-activating variant of the sensor-protein (GAL3-SA) and find that expression of this mutant is sufficient to fully activate the GAL regulon without requiring an inducing substrate. We find that despite moving from substrate-specific activation (in the native GAL regulon) to non-specific activation, cells carrying GAL3-SA do not seem to incur serious fitness costs or sacrifice the previously observed benefits during growth. Finally, we compare CONS and REG approaches to growth on the renewable and structurally distinct substrates arabinose, cellobiose, and glycerol and observe that while a REG approach is superior in all cases, the magnitude of the benefit appears to be both substrate- and media-specific. Ongoing work seeks to identify the genes and gene expression patterns that underlie the observed phenotypic benefits across substrates. Taken together, these results both validate GAL regulon activation as a general approach to engineering more efficient catabolism of non-native substrates and provide a tool (GAL3-SA) for applying it to attractive substrates beyond those described here.
Tuesday
Dynamic metabolic control improves NADPH flux and xylitol biosynthesis in engineered E. coli
10:50am - 11:10am USA / Canada - Eastern - August 24, 2021 | Room: Zoom Room 03
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual

Metabolic engineering has broad applications in the biosynthesis of a wide variety of products, including biofuels, pharmaceuticals, and food chemicals. Xylitol is a sugar alcohol with a primary use as a sweetener and is produced at ~125,000 tons annually. Compared with the chemical synthesis method, biosynthetic production of xylitol has the potential to decrease costs and be more environmentally friendly. Xylitol can be synthesized from xylose via an NADPH dependent reductase. In our work, we reported improved NADPH flux and xylitol biosynthesis in engineered E. coli using 2-stage dynamic metabolic control, where products are made in a metabolically productive phosphate depleted stationary phase. The implementation of this approach relies on the combined use of controlled proteolysis and gene silencing, using degron tags and CRISPR interference, respectively. Strains with reduced levels of enoyl-ACP reductase and glucose-6-phosphate dehydrogenase, led to altered metabolite pools resulting in the activation of the membrane bound transhydrogenase and an NADPH generation pathway, consisting of pyruvate ferredoxin oxidoreductase coupled with NADPH dependent ferredoxin reductase, leading to increased NADPH fluxes, despite a reduction in NADPH pools. These strains produced titers of 200 g/L of xylitol from xylose at 86% of theoretical yield in instrumented bioreactors. We expect dynamic control over the regulation of the membrane bound transhydrogenase as well as NADPH production through pyruvate ferredoxin oxidoreductase to broadly enable improved NADPH dependent bioconversions or production via NADPH dependent metabolic pathways.

Tuesday
Two-stage dynamic control over redox state improves cytosolic expression of disulfide containing proteins in E. coli
11:10am - 11:30am USA / Canada - Eastern - August 24, 2021 | Room: Zoom Room 03
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
E. coli is a common expression host in both research and industry, however the reducing environment of the cytoplasm creates challenges for soluble expression of proteins with cysteine residues. Approximately 50% of cysteine residues in heterologous proteins form disulfide bonds required for proper folding and activity. These disulfide bonds are reduced in the E. coli cytoplasm resulting in misfolded inactive protein and aggregation in inclusion bodies. Previously strains have been engineered to constitutively increase the oxidative potential of E. coli’s cytoplasm by deleting key enzymes in reducing pathways including glutathione oxidoreductase (gor) and thioredoxin reductase (trxB). However, constitutive oxidative stress has a toxic effect, slowing growth and limiting conditions for expression. Additionally, overexpression of a disulfide bond isomerase (dsbC) and disulfide bond oxidase (evr1p) have been shown to improve soluble expression of proteins containing disulfide bonds. Specifically, evr1p catalyzes cysteine oxidation and dsbC isomerizes disulfide bonds to improve correct folding when multiple disulfide bonds are present. We report improved cytoplasmic expression of disulfide containing recombinant proteins in engineered E. coli with two-stage dynamic control over the redox state, disulfide oxidase and isomerase activities. Recombinant proteins are expressed in a phosphate limited stationary phase coincident with dynamic control over cytoplasmic reducing power. An oxidized environment is created in the cytosol by deleting gor and dynamic reductions trxB and glutamate-cysteine ligase (gshA) levels upon entry into stationary phase. Additionally, increases in correctly folded disulfide bonds are obtained through overexpression of dsbC and evr1p, again upon entry into the stationary phase. Tightly controlled expression and a reducing cytoplasm prior to phosphate depletion enables robust exponential growth and autoinduction of both an oxidative environment and heterologous protein expression upon phosphate depletion. Disulfide containing proteins with improved expression include single chain variable fragments, human hyaluronidase-I, and tissue plasminogen activator which contain 2-17 disulfide bonds. The host strains and plasmids offer a tightly controlled, robust and scalable approach for the expression and purification of disulfide containing proteins.
Tuesday
Investigating the impact of cofactor availability and enzyme composition on metabolic pathway performance in bacterial microcompartments
11:30am - 11:50am USA / Canada - Eastern - August 24, 2021 | Room: Zoom Room 03
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
Bacterial microcompartments (MCPs) are large, inducible, proteinaceous organelles that encapsulate the enzymes, substrates, and cofactors required for specific metabolic pathways in certain species of bacteria. MCPs allow for the spatial organization of pathways containing volatile or toxic intermediates that would be detrimental to the cell if produced at the same levels in the cytosol. One example of an MCP is the 1,2-propanediol utilization (Pdu) MCP found in Salmonella enterica, which sequesters the 1,2-propanediol degradation pathway, including the toxic intermediate propionaldehyde. This sequestration is desirable to repurpose for other heterologous pathways that may suffer from similar inefficiencies, but important questions about how exactly the compartments function to enhance pathway performance must be answered before compartmentalization can be widely used in metabolic engineering strategies. One function of MCPs that is hypothesized to improve pathway performance is the creation of a private encapsulated cofactor pool that is internally recycled. By purifying and then studying the MCPs in vitro using bacterial extracts, we evaluated how model pathways are impacted by controlling the compartment environment directly, including external presence or absence of required cofactors, and addition or removal of pathway and recycling enzymes internally. This method allowed us to study a suite of conditions that would normally not be accessible using in vivo methods. From these experiments we found that, surprisingly, redox recycling is not always required, and we gained unexpected insights into the permeability of the proteinaceous shell. We then used these insights to investigate the benefits of encapsulation on isoprenoid production by encapsulating three steps of the mevalonate pathway.
Tuesday
Biopolymer: Biobricks polyketide metabolic enginering of minimal polyketide synthases in streptomyces coelicolor
11:50am - 12:10pm USA / Canada - Eastern - August 24, 2021 | Room: Zoom Room 03
Prof. Eric Nybo, Ph.D., Presenter, Ferris State University College of Pharmacy; Jennifer Nguyen; Kennedy Riebschleger; Katelyn Brown
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
Soil bacteria from the actinomycetes called anthracyclinones (ACs), some of which have powerful therapeutic activity. ACs have complicated structures that are produced by enzymes found in the bacteria. This project will “mix-and-match” these enzymes to make novel ACs expected to have antibiotic and anticancer activity. The overall goal fo the project is to develop the BIOPOLYMER® BIOBricks POLYketide Metabolic EngineeRing platform In this work, we engineered early stage enzymes composing the “minimal polyketide synthase” (minPKS) from three different pathways: (1) the C-21 doxorubicin pathway (dpsABGCD genes) and the C-21 aclacinomycin pathway (aknBCDE2F genes) (2) the C-20 nogalamycin pathway (snoa123), and (3) the C-19N oxytetracycline pathway (oxyABCD). We expressed the minPKS enzymes from these three pathways in actinomycete “superhosts” that are genetically engineered for heterologous expression of biosynthetic pathways: S. lividans K4-114, Streptomyces coelicolor M1146, Streptomyces coelicolor M1152, and Streptomyces coelicolor M1154. In this work, we expressed both wildtype and codon-optimized versions of the constructs under the control of different promoters to compare the impact of codon adaptation on aromatic polyketide production titer. We discovered that S. coelicolor M1154 was the best chassis for production of aromatic polyketides and that expression of minPKS operons from the kasOp* promoter lead to significant polyketide accumulation in shake flasks. We also discovered that utilization of the native translational coupling between the dimeric KS subunits, and utilization of the wildtype codon preference, were significant for reconstitution of the minPKS enzyme complexes in vivo. These efforts resulted in S. coelicolor M1154 strains that produced greater than 120 mg/L SEK15 in shake flask cultures.
Tuesday
De novo biosynthesis and incorporation of a nitroaromatic amino acid
12:10pm - 12:30pm USA / Canada - Eastern - August 24, 2021 | Room: Zoom Room 03
Neil Butler, Presenter, University of Delaware; Lucas Brown; Aditya Kunjapur, MIT
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
Nitroaromatic compounds have broad application space and the development of biosynthetic pathways offers a green chemistry alternative to hazardous industrial synthesis methods. To this point, metabolic engineering efforts toward de novo nitro-product biosynthesis and investigation into nitro-forming enzymes has been limited. Here, we present an integrated heterologous pathway for the production of a stable, non-toxic nitroaromatic product in Escherichia coli. Expression of four exogenous enzymes redirects metabolic flux in the shikimate pathway, including a previously uncharacterized amine oxygenase, to a nitro-containing amino acid with titers above 0.5 mM. Through a fluorescence-based screen, we have identified an amino-acyl tRNA synthetase (AARS)/tRNA pair capable of selective incorporation of the nitro-containing nsAA. Integration of the metabolic pathway with the AARS/tRNA pair enabled biosynthesis and incorporation of a nitro-containing amino acid intro proteins from a supplemented precursor metabolite, and could enable in-situ production of nitro-containing proteins.
Case Studies in Tech Transfer, Scaleup, & Integrated Process Design: Case Studies in Tech Transfer, Scaleup, & Integrated Process Design
10:30am - 12:30pm USA / Canada - Eastern - August 24, 2021 | Room: Zoom Room 04
Daniel Bracewell, Organizer, UCL Dept Biochemical Engr; Wai Chung, Organizer, Biogen Inc; Elizabeth Goodrich, Organizer, MilliporeSigma; Arne Staby, Presider, Novo Nordisk A/S; Jim Neville, Presider, ‍
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
Division/Committee: [BIOT] Division of Biochemical Technology

When transferring biotechnological processes to manufacturing facilities, the scale-up and scale-down of downstream unit operations is not always obvious and straightforward. Assuring successful implementation in a facility can require new approaches to scaling, simulation, and/or modeling to gain additional insight in comparison to traditional approaches. These may include fundamental/engineering models used to anticipate large scale performance, as well as development of appropriate small-scale models used for process characterization and/or troubleshooting. We seek abstracts covering these scale-up and scale-down topics, particularly for cases where challenges related to scaling and facility fit were observed and novel solutions or technologies were required to drive successful implementation. Case studies covering adoption of new paradigms, including advances in integrated process designs that utilize closed non-classified process operations and related commercial robustness, are encouraged. This also includes approaches focusing on design space, as well as productivity increases and/or cost reduction through innovation. Abstracts that highlight recent trends and areas of key focus during health authority review are relevant.

Tuesday
Manufacturing innovation as a must to enabling accessibility and affordability of biologics
10:30am - 11:10am USA / Canada - Eastern - August 24, 2021 | Room: Zoom Room 04
Jorg Thommes, Presenter
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
The core promise of CMC is the supply of products to patients with zero supply shortages. This promise creates a certain risk adversity and conservative attitude towards innovative change. Currently, access and affordability to biotherapeutics is in part limited exactly for that reason. Current manufacturing processes are focused on uninterrupted supply rather than on reducing cost to serve. To broaden the access to biotherapeutics to the world’s most vulnerable populations, we need to expand our approach to CMC development. This presentation will present a “two and done” and approach to CMC development for biologics with a special emphasis on access and affordability to Low and Middle Income Countries. While speed to FIH studies is the focus of early stage development and manufacturing, at risk investment in late stage process development is proposed as soon as FIH studies have commenced. Introducing innovative manufacturing concepts that break with the traditional mammalian cell based fed batch processes will be essential to transform biologics from a solution only available to high income country populations to products that are equitably accessible. The presentation will discuss a vision to redesign biologics manufacturing without the burden of “this is how we’ve always done it”.
Tuesday
Scale up based on volume basis for flow-through mode chromatography: A case study
11:10am - 11:30am USA / Canada - Eastern - August 24, 2021 | Room: Zoom Room 04
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
In order to produce cGMP materials at manufacturing scale for clinical trials and commercial applications, generally lab-scale development is performed first and then scaled up to pilot scale and then manufacturing scale. In a typical scale-up process, both chromatographic and non-chromatographic factors need to be taken into consideration, including process constraints with facility fit limitations such as pressure, flow rate and available column dimensions. Although scale-up by constant bed height is a simple and most common scale-up technique used for column chromatography, sometimes this may not be feasible due to lack of commercially available column dimensions for the intended scale, given that the columns are only provided in discrete diameters. Keeping a constant residence time, which is also referred to as scale up based on volume basis, offers more flexibility. However, there has not been data published to take a comprehensive examination on the limits and applicable ranges for this procedure, such as the ranges of bed heights and residence time.
Here we present our findings of scale-up with constant residence time. Results for AEX, HIC and MMC are shown comparing a wide range of bed heights for small scale, pilot scale as well as large scale operations. Residence time was also investigated by looking into a range that is applicable in large scale manufacturing. Our results suggest that for flow-through mode chromatography, scale-up based on constant residence time resulted in comparable performance in aspects of process performance as well as product PQ, including yield, HMW, charge profile, purity, DNA and HCP. We will also discuss the reasonable ranges for bed heights and residence time that could be applied.

Tuesday
From development to manufacturing: How does freezing of mAb process intermediates impact critical quality attributes
11:30am - 11:50am USA / Canada - Eastern - August 24, 2021 | Room: Zoom Room 04
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
The platform process for production of monoclonal antibodies can be divided into three main sections: upstream, downstream and formulation & fill. These sections are typically handled by individual departments and thus require transport of process intermediates between them. While final fill of the biological drug is often performed off-site, up- and downstream processes work hand-in-hand on the production site and liquid batches are pumped from one department to the other for drug manufacturing. In process development, however, clarified harvest from the upstream process can not be evaluated instantly by the downstream process development department due to subsequently applied screenings for suitable purification methods. Therefore, the clarified harvest is often frozen to improve its shelf-life. Freezing of protein solutions reduces degradation processes by host cell proteins but also exerts stresses on the drug such as cold denaturation, surface interaction and freeze concentration. Although the platform process is well established throughout the pharmaceutical industry, a study on the impact of freeze-thaw processes on process intermediates is missing yet.

The presented case study evaluates the impact of freeze-thaw processes on monoclonal antibody process intermediate and the subsequent purification. Therefore, cell culture supernatant from Chinese Hamster Ovary Cells containing monoclonal antibody was frozen at different temperatures, thawed and purified by protein A affinity chromatography. Critical quality attributes such as host cell protein concentration and dimer content were measured by automated ELISA and size exclusion chromatography throughout the process. Furthermore, the frozen monoclonal antibody and host cell protein concentration in the bulk harvest were evaluated by capillary electrophoresis. A protein size dependent freeze concentration as well as a resulting correlation between freezing temperature and critical process attributes were found in the study.

Tuesday
Investigation of low concentration of EDTA excipient in diafiltration buffer used in a monoclonal antibody purification process
11:50am - 12:10pm USA / Canada - Eastern - August 24, 2021 | Room: Zoom Room 04
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
EDTA (ethylenediaminetetraacetic acid) is used in biopharmaceutical processes to remove metal impurities that may be bound to the protein. It is a water-soluble hexadentate chelating agent which is widely used to bind to metal ions such as iron and calcium. It is one of several excipients added to the buffer used in the ultrafiltration-diafiltration step. The excipient concentration in the diafiltration buffer is particularly important because its composition will be used for the protein formulation. During diafiltration, the buffer is exchanged with the protein solution for more than 7 diavolumes. Excipient concentrations between the buffer and final protein concentration can change because of the attractive charge between EDTA and the monoclonal antibody (Gibbs-Donnan effect) as the protein is filtered across a 30 kDa membrane. This causes the EDTA concentration to increase over the ultrafiltration-diafiltration step. Based on the calculation and estimation, the proposed EDTA concentration was used in the buffer for the first intent. However, we observed the EDTA concentration was consistently low in the buffer which resulted in a low concentration in the final bulk drug substance across small- and large-scale batches. A root-cause analysis was performed to investigate process and analytical causes. The analytical platform EDTA assay method was adjusted based on the findings and additional process development work was performed, and eventually a project-specific approach was implemented to achieve the accurate detection of EDTA in formulation buffer, in-process and BDS samples.
Tuesday
Non-GLP viral clearance studies as a risk mitigation strategy in the development of biologics
12:10pm - 12:30pm USA / Canada - Eastern - August 24, 2021 | Room: Zoom Room 04
Alice R Butler, Presenter, Asahi Kasei Bioprocess America; Brian Buesing; Aesha Shah; Takayuki Sohka; Daniel Strauss
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
During the development of biotherapeutics, regulatory agencies require demonstrated capability for removing and inactivating viruses throughout the manufacturing process. While viral clearance validation studies must be performed in compliance with good laboratory practices (GLP), the cost and resource requirements of such studies make them prohibitive for use as preliminary studies. There certainly are many advantages to troubleshooting conditions throughout the drug development process prior to the required GLP studies. To facilitate the inclusion of preliminary viral clearance studies in your development process, we have established a non-GLP viral clearance laboratory that we operate in the spirit of GLP but without the cost and scheduling burdens of contracting with a CRO. These cost-effective studies produce high quality data that can be used to inform many decisions throughout process development and manufacturing. Results from our in-house non-GLP studies have been used in preliminary stages to evaluate novel process conditions and characterize processes, during scale up to inform process platforming decisions and during commercial manufacturing to provide support for deviations. One such study performed in 2019 evaluated parvovirus removal from a monoclonal antibody by Planova BioEX filters at two different operating pressures with multiple process pauses during the filtration. Filtrations at both operating pressures met the target throughput of 500 L/m2 with parvovirus logarithmic reduction values of ≥4.7. The non-GLP study provided data to support the use of a viable alternative to a previously platformed virus filter. This ability to confirm successful filtration results prior to required validation is a valuable tool for developing robust downstream processes.
Biomolecular & Biophysical Processes: Protein Structure & Function: Protein Structure & Function
10:30am - 12:30pm USA / Canada - Eastern - August 24, 2021 | Room: Zoom Room 05
Cesar Calero Rubio, Organizer, Sanofi Genzyme; Mary Krause, Organizer, Bristol Myers Squibb; Krishna Mallela, Organizer, Univ of Colorado Denver; Surinder Singh, Presider, ‍ ; Chiwook Park, Presider, ‍
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
Division/Committee: [BIOT] Division of Biochemical Technology

Elucidation of the protein structure and function relationship is crucial for gaining deeper insights into the protein(s) molecular mechanism of action. Understanding protein structure and function is a key driver in devising an effective control strategy for developing various biotechnological applications of proteins, such as safe and efficacious protein-based drugs in the bio-pharmaceutical industry. This session seeks presentations focused on scientific approaches to understand and decipher the fundamental principles connecting the primary and higher-order structures of proteins, post translational modifications, and protein-protein interactions to their function and behavior in vitro and in vivo.

Tuesday
Identification of CQAs for biotherapeutic products
10:30am - 10:50am USA / Canada - Eastern - August 24, 2021 | Room: Zoom Room 05
Dr. Anurag S Rathore, Presenter, Indian Institute of Technology Delhi
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
The ever-increasing cost of healthcare together with our improving understanding of biotech therapeutic drugs have fuelled the rise of biosimilars. Discussion and resolution of the various scientific and regulatory factors that play a role in approval of biosimilars is arguably one of the most significant events over the last decade for biotechnology. Key scientific factors include the complexity of biotech products and processes, use of complex raw materials that are not always well characterized, and our relatively limited understanding of how the numerous quality attributes that define a biotherapeutic impact the product’s safety and/or efficacy in the clinic. This talk will present a summary of our attempts towards investigation of CQAs for a variety of biotherapeutic products, both mAbs and non-mAbs. Case studies will illustrate the complexity of making these assessments as well as key findings. Case studies include impact of methionine oxidation in GCSF, aggregation in mAbs, and charge variants of mAbs.
Tuesday
Photophysical characterization of previously unidentified intrinsic charge transfer states in proteins
10:50am - 11:10am USA / Canada - Eastern - August 24, 2021 | Room: Zoom Room 05
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
The intrinsic optical properties of proteins (i.e., absorbance at 280 nm and fluorescence around 340 nm) are known to originate from the electronic transitions of aromatic amino acids, such as tryptophan. As aromatic amino acids are present in nearly all proteins, intrinsic absorbance and fluorescence have limited use in protein characterization. Recently however, a new weak intrinsic absorbance signal has been uncovered in the visible region, even in proteins which lack aromatic amino acids. This previously overlooked visible absorbance signal has been theorized to arise from charge transfer states which form between charged amino acids within the protein. The unique interactions between charged amino acids likely affect this new intrinsic absorbance signal, and could be used to identify specific protein structures or sequences. Despite experimental observation of this new optical state, the exact photophysical mechanism has not yet been elucidated spectroscopically. Here, we characterize this newly uncovered optical state using advanced spectroscopy techniques in three highly charged proteins. The absorbance spectra of each protein was carefully measured using an integrating sphere to minimize contributions from scattering. We then used transient absorption spectroscopy to fully characterize the energy transfer within each protein, uncovering states with charge transfer character, consistent with theoretical simulations. By fully understanding this intriguing new charge transfer state, protein absorbance could eventually find use as an intrinsic probe for in vivo optical microscopy, or as a new method for the identification of specific protein conformations.
Tuesday
Investigating macromolecular transport through tunable collagen and hyaluronic acid tissue matrix
11:10am - 11:30am USA / Canada - Eastern - August 24, 2021 | Room: Zoom Room 05
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
Hyaluronic acid (HA) is an extracellular matrix (ECM) component that is classically involved in inhibiting delivery of therapeutics due to its high viscosity and negative charge. For example, increased HA near subcutaneous lymphatic ducts and pancreatic tumors is responsible for decreased transport of drugs to their respective targets, however the degradation of HA via hyaluronidase has been shown to improve bioavailability. Though this effect has been previously studied, the parameterization of molecular transport as a function of known macromolecule and ECM properties for understanding drug/tissue interactions and preclinical drug efficacy has not yet been described. To address this gap, we utilized a transwell chamber with non-invasive, label-free UV spectroscopy to measure the mass recovery of macromolecules across various in vitro ECM barriers. Here, we show the effect of increasing HA concentration within collagen-hyaluronic acid matrices (ColHA) on the mass recovery of macromolecules – bovine serum albumin (BSA), β-Lactoglobulin (BLg), lysozyme (Lys), dextran (Dex), and bovine immunoglobulin G (IgG). We found that as HA concentration, and subsequent viscosity and negative charge density, increased in ColHA matrices, mass recovery decreased. However, against pure HA with no collagen fiber network, the negative charge of BSA, BLg, and IgG played a large role in increasing transport compared to ColHA matrices, despite the higher viscosity. In pure HA, positively charged Lys had the lowest transport due to the highly negative matrix. These results demonstrate that electrostatic and viscous effects from HA modulate macromolecule transport in ColHA ECMs, that HA sequesters and increases the residence time of oppositely charged macromolecules, and that the presence of collagen as a majority substituent of the ECM plays an important role. Furthermore, the development of empirical predictive models relating properties of ECM and macromolecules with the resultant molecular transport may have the potential to establish the preclinical data required to avoid possible drug failures during costly clinical trials, aiding in the development of cost-effective and successful therapeutics.
Tuesday
Tissue-specific dystrophin isoforms have different stabilities and functional roles
11:30am - 11:50am USA / Canada - Eastern - August 24, 2021 | Room: Zoom Room 05
Vaibhav Upadhyay, Presenter; Sudipta Panja; Swati Bandi; Krishna Mallela, Univ of Colorado Denver
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
Mutations in Duchenne muscular dystrophy (DMD) gene that affect the expression of dystrophin protein lead to a number of disorders collectively called dystrophinopathies characterized by muscle degeneration and loss. Dystrophin is a large protein (427 KDa) made up of N-terminal calponin homology (CH) domains, 24 spectrin repeats, cystein rich (CR) domain and C-terminal (CT) domain. Numerous isoforms of dystrophin have been shown to exist due to alternative splicing and usage of alternate promoters. These variations occur in the functionally important N-terminal and the C-terminal domains. The N-terminal domain mediates interaction with actin, whereas the C-terminal domain interacts with cytosolic proteins like dystrobrevin and syntrophin. The impact of these isoform variations on the structure and function of dystrophin is unexplored. Dystrophin isoforms have differing tissue specificity and thus the differences in their structure-function become important in understanding tissue-specific symptoms of dystrophinopathies. We probed the impact of these variations on structure-function relationship of dystrophin. We found that minor N-terminal variations in the CH domain of dystrophin isoforms modulate their thermodynamic stability and actin-binding function, thus leading to specificity in dystrophin-actin interactions in various tissues. We also characterized the yet unexplored CT domain of dystrophin, and the variations in the CT domain of dystrophin lead to differences in the stability and binding specificity towards its binding partners. These results show that sequence variations in tissue specific isoforms do govern thermodynamic stabilities, thus affecting tissue-specific dystrophin expression and dystrophin interaction networks. These differences should be considered before designing effective therapeutic strategies for dystrophinopathies that specifically affect different tissues with a myriad of symptoms.

Tuesday
X-ray structure of fructosamine 6-kinase, a future biorecognition molecule for glycated protein sensing for the glycemic control in diabetic patients
11:50am - 12:10pm USA / Canada - Eastern - August 24, 2021 | Room: Zoom Room 05
Dr Mika Hatada, Presenter, The University of North Carolina at Chapel Hill; Keita Suzuki; Hiromi Yoshida; Ryutaro Asano; Wakako Tsugawa; Koji Sode
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
The x-ray structure of fructosamine 6-kinase (FN6K) derived from Escherichia coli (EcFN6K), which is expected as the sensing molecule for the glycated albumin (GA) measurement was determined in this study.
Glycated proteins, such as hemoglobin A1c (HbA1c) or GA are the important long-term glycemic control markers for diabetes patient. FN6K catalyzes the phosphorylation of fructosyl amino acids using ATP and produces fructosyl amino acid 6-phosphate and ADP. Extensive studies in E. coli have led to an understanding of the physiological role of FN6K as the key enzyme in the catabolic pathway of naturally occurring fructosyl amino acids, which is supported by the reports that growth on Amadori products as sole carbon and nitrogen source is possible for E. coli. Fructosyl amino acid/peptide oxidase (FAOx/FPOx) are the current standard enzyme for the enzymatic glycated protein measurement. FN6K does not react with any sugars or free amino acids, and are thus specific for fructosyl amino acid. FN6Ks show lower Km values than FAOXs/FPOXs, while FN6K and FAOXs/FPOXs have comparable Vmax values. These properties together with its robustness in the recombinant production, FN6K will provide alternative detection principles and its utilization as novel molecular recognition elements for glycated protein sensing. In this study, x-ray structure of EcFN6K was determined. To confirm the roles of elucidated residues which are predicted to serve significant roles in catalytic reaction and substrate recogition, the mutational studies were also carried out.
As the result, the x-ray structure of EcFN6K as the complex with ATP analog, Adenylyl-imidodiphosphate (AMPPNP) was obtained. The overall structure revealed that FN6K belongs to ribokinase family. Comparing the cavity for substrate entrance, that of EcFN6K was observed to be remarkably wider than those of FAOx/FPOxs. This observation suggested that the large substrate entrance and pocket in FN6K make it possible to react with large substrate such as intact GA. The mutational studies suggested that the conserved residues among in the other ribokinase family enzymes in FN6K are identified as the catalytic residue and residues recognizing the phosphate group of ATP.

Tuesday
Molecular dynamics investigation of metal coordination in a structural jaw protein from a marine sandworm
12:10pm - 12:30pm USA / Canada - Eastern - August 24, 2021 | Room: Zoom Room 05
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
There is a growing interest in biomaterials with spatially-defined and reconfigurable mechanical properties. For example, a gradient of elastic properties is essential in applications where soft materials join hard substrates. Materials with tunable mechanical properties can be engineered using proteins as structural components, and many of the functional properties of biological materials have been attributed to the presence of metal centers. One such biological structure that takes advantage of non-mineralized, proteinaceous material containing metal centers is the Nereis virens worm jaw which is used for grasping, piercing, and tearing. The hardness and stiffness of the Nereis jaw are comparable to mineralized biomaterials, such as dentin. The jaw protein, Nvjp-1 is primarily composed of glycine, histidine, acidic residues, and tyrosine. In this study, metal coordination of Nvjp-1 in a salt solution in the presence of Zn2+ is investigated by all-atom molecular dynamics simulations that employ a polarizable force field. The protonation state of the protein determines which types of residues capture zinc ions from the solution. At pH = 5 in ZnCl2, polar residues, such as tyrosine, predominantly interact with Zn2+, forming coordination bonds with at most two residues interacting with a single zinc ion. At this pH, negatively charged residues form salt bridges with protonated histidines and are effectively blocked from interacting with zinc ions. Changing the protonation state of the Nvjp-1 to reflect a pH of 8, and removing all unbound zinc ions, leads to the formation of new coordinate bonding with histidine and acidic residues where up to four residues interact with a single zinc ion. An accompanying reduction in the radius of gyration is taken as evidence of protein condensation and sclerotization. This study also looks at the role of the anion partner of zinc in terms of zinc coordination and the effect on the Nvjp-1 radius of gyration. Together, these simulations indicate that pH and anions strongly influence metal loading and crosslinking of the Nvjp-1 protein through modulation of amino acid side chain interactions. Additionally, these studies show how a pH-driven, metal-binding hierarchy can be functionally important in regulating the mechanical properties of an intrinsically disordered structural protein.
New Technologies in Cell & Microbiome Engineering and Stem Cell Therapy: New Technologies in Cell & Microbiome Engineering and Stem Cell Therapy
10:30am - 12:30pm USA / Canada - Eastern - August 24, 2021 | Room: Zoom Room 06
Yonghyun Kim, Organizer, The University of Alabama; Maryam Raeeszadeh Sarmazdeh, Organizer, University of Nevada, Reno; Samira Azarin, Presider; Thomas Mansell, Ph.D., Presider, Iowa State University; Marjan Rafat, Presider, ‍ ; Nicholas Sandoval, Presider, Tulane University
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
Division/Committee: [BIOT] Division of Biochemical Technology

This session will focus on emerging technologies in engineering host or microbial cells and their interactions in the context of a natural or synthetic microbial community. Talks are welcome on a broad range of topics including, but not limited to, host cell engineering to improve protein production, genetic stability, post-translational modification including glycosylation, engineered probiotics, genetic circuit design, signal transduction, cell-cell communication, host-microbe interaction, synthetic consortia, and evolution of such systems. Of particular interest are efforts in studying and designing microbiomes to achieve biotechnological or biomedical goals.

Tuesday
Three-dimensional in vitro culture model of breast tumor spheroids: A DNA directed approach
10:30am - 10:50am USA / Canada - Eastern - August 24, 2021 | Room: Zoom Room 06
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
The study presented here describes the generation of single large spheroids by cell surface modification by using DNA interactions. In order to achieve this, DNA oligonucleotides were attached to cell surface using an Affinity Mediated Covalent Photoconjugation (AMCP) approach recently developed by our group.Specifically, these studies were focused using the human breast cancer cell line MDA-MB-468 which has been found difficult to culture into cohesive and manageable 3D spheroids using conventional methods. In this work, we first show the production of fusion proteins composed of an affibody domain that is known to bind the Epidermal Growth Factor Receptor (EGFR) and streptavidin. These EGFR binding affibodies were previously engineered to be photocrosslinked via a benzophenone unit to EGFR using long UV light or upconverting nanoparticles. In this study, the covalent ligation of the affibody-streptavidin into EGFR enabled the tagging of specific cells such as MDA-MD-468 with DNA oligonucleotides without the need for genetic engineering or depending on lipid lifetime and dynamics in the membrane bilayer.The DNA modified cells were then placed into poly HEMA coated wells in presence of the complementary DNA linker strands to promote cell cell assembly. As will be shown in the talk, these DNA assembled methods enabled the production of close packed 3D cellular spheroids within 72-96 h of culture. The tumor spheroids formed were found to be extremely uniform in nature and did not easily disassemble when handled during commonly used procedure such as media washing allowing for various imaging techniques and other analyses and for future studies such as evaluation against various anticancer drugs.
Tuesday
HiPSC encapsulation geometry impacts engineered cardiac tissue homogeneity and functionality
10:50am - 11:10am USA / Canada - Eastern - August 24, 2021 | Room: Zoom Room 06
Morgan Ellis; B. Justin Harvell, Presenter, Auburn University; Elizabeth Lipke
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
Cardiac tissue engineering strives to produce cardiac tissue that accurately recapitulates the native heart. Due to the heart’s low regenerative potential and limited donor availability, there is a large need for engineered cardiac tissue for use in regenerative medicine and drug development. Previously, we showed the ability to produce 3D engineered cardiac tissue (3D-ECTs) by encapsulating human induced pluripotent stem cells (hiPSCs) in poly(ethylene glycol) fibrinogen (PF) in a microisland (disc-like) geometry. Resulting 3D-ECTs were successful in recapitulating key features of maturing cardiac tissue including appropriate responses to drug exposure and presence of T-tubules; however, the microisland geometry resulted in tissue with dense outer bands containing areas of low viable cell density in the tissue center and radial contractions along the outer edges of the microisland, which is not typical of human myocardium. To improve cardiac tissue homogeneity and enhance resulting cardiac tissue functionality, here we examined two additional initial hiPSC encapsulation geometries, square and rectangle, utilizing our established direct cardiac differentiation platform and custom PDMS molds to produce 3D-ECTs that more closely recapitulate native human cardiac tissue. Resulting 3D-ECTs showed similar temporal changes gene expression and cardiomyocyte (CM) populations (~65%), indicating that initial encapsulation geometry did not impact whether or not cardiac differentiation occurred. Rather, initial encapsulation geometry played a role in the overall cardiac functionality and maturation. Rectangular 3D-ECTs were more homogeneous and displayed anisotropic contractions (MI 0.28 ± 0.03, SQ 0.35 ± 0.05, RT 0.79 ± 0.04), unlike microisland and square 3D-ECTs. Additionally, rectangular 3D-ECTs presented with enhanced myofibrillar alignment and Z-line formation, which are attributes of more mature CMs. This study establishes a simple, improved method for creating more mature 3D-ECTs without the use of external pacing and can be used for drug testing and disease modeling.
Tuesday
Multi-input CRISPR-based kill-switches for engineered probiotics
11:10am - 11:30am USA / Canada - Eastern - August 24, 2021 | Room: Zoom Room 06
Austin Rottinghaus; Aura Ferreiro; Gautam Dantas; Prof. Tae Seok Moon, Presenter, Washington University in St. Louis
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
Probiotics are effective chassis for diagnostic and therapeutic applications. However, there are safety concerns associated with using genetically engineered organisms for medical applications. To address these issues, we engineered the probiotic Escherichia coli Nissle to survive only when and where it is needed using CRISPR-based kill-switches (CRISPRks). We first designed a CRISPRks that induces cell death by expressing Cas9 and genome-targeting guide RNAs (gRNAs) in response to a chemical inducer. This design allows cell killing to occur while the microbe is in the gut in response to oral administration of the chemical. We optimized the efficiency and stability of the CRISPRks, achieved more than a 9-log reduction in cell number, and demonstrated genetic stability for up to 28 days of continuous growth. This high killing efficiency was maintained in vivo, where we achieved complete elimination of the probiotic after oral administration of the inducer. To our knowledge, this is the first time on-demand elimination of an engineered microbe that has been demonstrated in vivo. We next modified our chemically inducible-CRISPRks to also induce cell death in response to ambient temperatures below 33’C. This design induces cell killing either in response to oral administration of the chemical or when the microbe is excreted from the body in response to the reduced environmental temperature. This circuit achieved more than a 9-log or 7-log reduction in cell number after exposure to the chemical inducer or temperature downshift, respectively. Our CRISPRks strategy provides a benchmark for future microbial biocontainment circuits. The sensor and killing mechanism are well characterized and functional in many microbes, allowing the CRISPRks design to be broadly applicable. In addition, the temperature-sensing module can be easily replaced with sensors that recognize alternative signals, enabling generalizable kill-switches for applications beyond engineered probiotics.
Tuesday
Continuous evolution of Escherichia coli Nissle 1917 for biomedical applications
11:30am - 11:50am USA / Canada - Eastern - August 24, 2021 | Room: Zoom Room 06
Qiuge Zhang, Presenter, UNIVERSITY OF MINNESOTA; Samira Azarin; Casim Sarkar
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
E. coli strain Nissle 1917 (EcN) is widely taken orally as a probiotic and is also amenable to standard genetic engineering techniques, so it is a potentially attractive chassis for bacterial therapeutics. However, the low pH in the stomach and high bile salt concentration in the bile ducts and gallbladder dramatically reduce EcN cell viability, thus hindering therapeutic applications in the intestine. To improve tolerance to acid and bile salt, we performed continuous adaptive evolution on EcN and recovered mutated strains with greater tolerance to low pH or high bile salt. Unexpectedly, evolved EcN also exhibited greater adhesivity to an intestinal cell line in vitro, which may be an additional beneficial property in therapeutic applications. We will also discuss the mutational basis for pH and bile tolerance and their implications for biomedical applications such as oral delivery of therapeutic bacteria.
Tuesday
Directing the composition and function of an Escherichia coli consortia using electrogenetics
11:50am - 12:10pm USA / Canada - Eastern - August 24, 2021 | Room: Zoom Room 06
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
Microbial consortia play central roles in a variety of natural environments, including the soil rhizosphere and the gut microbiome. Within these systems, each subpopulation of a consortium needs to exist within a specific range of composition to fulfill a particular task or risk endangering the community. As synthetic biology has advanced to the potential of generating synthetic consortia for both remediation and therapeutic endeavors, further methodologies are needed to properly structure consortium composition to include the proper amounts of each member bacterium. In this work, we demonstrate the use of electrogenetics to alter the composition of a synthetic consortia of E. coli. We show that by altering electrochemical inputs, such as voltage and time, we can differentially activate the expression of OxyR-based gene circuits through the 2-electron oxygen reduction reaction known to generate the signaling molecule hydrogen peroxide. This molecule, in turn, activates populations local to the electrode to transmit acylhomoserine lactones (AHLs) before being rapidly degraded. These signals, in turn, are capable of activating specific populations to increase in growth rate, ultimately increasing their final composition within a batch culture. We demonstrate that by altering synthetic biology components, such as promoters, repressors and AHL-synthases, we can tune the response of subpopulations based upon device-imposed inputs such as voltage and time. Conversely, we also demonstrate the ability to alter device-imposed inputs to target specific populations when significant microbial engineering is not an option. Together, these features allow us to restructure a microbial community without significantly altering seed density or culture conditions.
Tuesday
Targeting infected host cells in vivo via responsive azido-sugar mediated metabolic cell labeling followed by click reaction
12:10pm - 12:30pm USA / Canada - Eastern - August 24, 2021 | Room: Zoom Room 06
Yang Bo, Presenter, University of illinois at Urbana-Champaign; Jianjun Cheng
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
The early in vivo diagnosis of infectious disease foci is largely hindered by invasion and concealment of pathogens in the host cells, making it difficult for conventional probes to detect and analyse intracellular pathogens. Taking advantage of the excessively produced reactive oxygen species (ROS) within host cells, herein we report the design of thiol-hemiketal blocked N-azidoacetyl galactosamine (Ac3GalNAzSP), an azido unnatural sugar bearing an unprecedent designed ROS-responsive moiety for targeted labelling of infected host cells. Ac3GalNAzSP showed great stability under physiological conditions, specifically released active unnatural sugar in host cells overproducing ROS, metabolically labelled infected host cells with azido groups, and enabled targeting the in vivo infection site by subsequent Click Chemistry reactions, substantiating an unprecedented approach for targeting infected host cells. This technique could be a powerful tool for early in vivo diagnosis and targeted treatment of infectious disease.

Upstream High-Throughput Screening & Automation : High-Throughput Screening & Automation
02:00pm - 03:20pm USA / Canada - Eastern - August 24, 2021 | Room: Zoom Room 03
Melisa Carpio, Organizer; Danielle Ercek, Organizer, Northwestern University; Nitya Jacobs, Organizer; Doug Densmore, Presider, ‍ ; Samuel Oliveira, Presider, ‍ ; Grant Murphy, Presider
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
Division/Committee: [BIOT] Division of Biochemical Technology

The discovery and development of biotherapeutics and biotechnologies depends on successful upstream process development to identify the ideal protein variant and cell line construct, culture media, bioreactor, and fermentation process. Enhancements in laboratory automation, DNA synthesis, and Synthetic Biology have led to rapid advances in the scale and approaches taken in upstream process development. The application of technologies such as cell-free expression, microfluidics, and automated bioreactors enables the rapid design and testing of increasingly large numbers of variant constructs and production conditions in reasonable timelines. Advances in machine learning and other computational methods are being combined with the multidimensional data sets generated during upstream process development to drive process decision-making and generate cross-program learning. This session will focus on developing and applying experimental and computational tools, workflows, and processes that increase our ability to perform construct discovery and development in a high-throughput, automated fashion. This session will highlight the benefits of laboratory automation workflows as robust data screening and preparation methods and automate genetic construct data analytics. This session will explore advances in functional AI algorithms for accurate sampling of property landscape, analytical characterization, and the rational design of biological materials and functions. Finally, of particular interest, this session will foster discussion on the use of community standards and initiatives to improve these processes as new ways to distribute and share data across the community.

Tuesday
Development of a high-throughput assay for measuring secreted protein titer based on a de novo fluorescent reporter
02:00pm - 02:20pm USA / Canada - Eastern - August 24, 2021 | Room: Zoom Room 03
Samuel Leach, Presenter, Northwestern University; Danielle Ercek, Northwestern University
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
Production of heterologous proteins in a microbial host can be advantageous due to their genetic tractability, fast growth, and robustness in large cultures. However, these hosts are limited because they retain the protein product intracellularly, requiring a complicated and expensive downstream purification scheme. A promising solution is to engineer the bacteria to secrete the protein product to the extracellular space, which would greatly ease downstream purification requirements. Many research groups have focused on secreting heterologous protein products through a variety of secretion pathways native to bacteria, but the titer of secreted protein is often too low for commercial interests. While many microbial engineering techniques could be used to improve secretion titer, studies are often limited by a lack of fast, quantitative, and high throughput assays for measuring secreted protein. For this purpose, we have utilized the mini fluorescence activating protein (mFAP), a small, de novo protein consisting of a single beta barrel, which reversibly binds to the substrate DFHBI to produce a fluorescent signal. In this talk, we show that this protein is readily secreted through multiple secretory pathways when fused to the relevant secretion tag, and it produces a measurable fluorescent signal in media when its substrate is added exogenously. A high-throughput plate based fluorescence assay for measuring secreted protein using this reporter can develop a full signal in minutes, can measure secreted protein amounts across three orders of magnitude, and can be used in undefined media which typically cause low signal to noise ratios. Using the Type 3 Secretion System of Salmonella typhimurium as an example, we show that this assay can accurately measure how engineering changes affect secretion titer, and we show how this assay can enable new studies which enable further improvements to secretion titer.
Tuesday
Open-source near-infrared fluorimetry tools for translation of single-walled carbon nanotubes for protein screening
02:20pm - 02:40pm USA / Canada - Eastern - August 24, 2021 | Room: Zoom Room 03
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
While high-throughput screening tools, such as plate readers and cell-sorters, have become increasingly ubiquitous for assaying reporter proteins and other fluorescent probes, their utility quickly diminishes for any reporter or probe that operates outside the sensitivity range of the silicon photodetector. A spectral region of considerable interest is the near-infrared, useful for both its capacity to penetrate deeper into biological media and for its function with novel, biocompatible fluorescent biosensors, such as single walled carbon nanotubes (SWCNT) and quantum dots. Use of novel near-infrared fluorophores and probes, such as our SWCNT probes, which effectively transduce enzyme activity in complex matrices on actual targe substrate, requires custom-built fluorimetry tools which can require significant time and capital to implement. To address this bottleneck and enable more research groups to use these novel probes in their screening efforts, we have developed and demonstrated several fluorimetry tools including a low-cost well plate reader and a portable fluorimeter that require minimal programming or machining expertise to reproduce and operate with an open-source programming language. Herein, we describe the design of these tools and demonstrate their functionality with sensors that utilize SWCNT as fluorescent transducers. We demonstrate the portable reader’s functionality with field-side soil enzyme activity measurement, and we demonstrate the plate reader through a range of high-throughput tests spanning enzyme screening, antimicrobial screening, and soil sample testing.
Tuesday
Screening potential therapeutic targets with an in vitro 3D familial Alzheimer’s disease model using a high-throughput platform
02:40pm - 03:00pm USA / Canada - Eastern - August 24, 2021 | Room: Zoom Room 03
Vibha Narayanan, Presenter, Rensselaer Polytechnic Institute; Andre Rodrigues; Jing Zhao; Chunyu Wang; Jonathan Dordick
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
The lack of a preventive therapy for neurodegenerative diseases, such as Alzheimer’s Disease (AD) has increased the urgency for gaining an understanding of the mechanisms that lead to cognitive decline and eventually death. The identification of several potential targets including the amyloid plaque, hyperphosphorylated tau tangles, microglial activation, reactive oxidative stress and neuroinflammation has led to the generation of various in vitro and in vivo models. In particular, the development of in vitro models of AD has been crucial to the understanding of heretofore poorly understood disease pathology. Such in vitro models have been able to partly recapitulate key pathological hallmarks of the disease using various neuronal cell lines including induced pluripotent stem cells, immortalized neural progenitor cells and neuroblastoma cells in 2D and 3D. While amyloid aggregation previously has been observed in 3D models, the use of a high throughput 3D platform as a screening tool to quantify changes in amyloid aggregation using high content imaging could have a wide range of applications. The primary focus of this research is to establish a stable cell line with mutations in the Amyloid Precursor Protein (APP) associated with Familial AD such as the Swedish double mutation (KM670/671NL) and the Indiana mutation (V717F) and use a high-throughput 384-well pillar plate system to understand the complexities of targeting aggregated amyloid beta in a 3D Familial AD model. To this end, we established a stable cell line by producing lentiviruses harboring the APP gene with the Swe/Ind mutation and transducing a HEK293T cell line to generate a stable cell population by the use of an antibiotic selection marker. A 50-fold increase in the level of Aβ42 was secreted by the cells in comparison to levels secreted by a wildtype APP-containing cell line, such as the BE (2)-M17 neuroblastoma cell line. Upon drug treatment with common gamma-secretase inhibitors such as gamma secretase inhibitor XXI and DAPT as well as other compounds inhibiting amyloid beta secretion, a dose dependent reduction in Aβ42 was observed in both 2D and 3D. This work provides a screening tool that can be used to mimic key pathological disease hallmarks that serve as drug targets for the applications of drug discovery.
Tuesday
Additive manufacturing and iterative design of biological detection devices
03:00pm - 03:20pm USA / Canada - Eastern - August 24, 2021 | Room: Zoom Room 03
Dr. Daniel Phillips, Presenter, ORISE / U.S. Army DEVCOM Chemical Biological Center; Dr. Marilyn Lee, US Army DEVCOM CBC; Steven Blum; Stephanie Cole; Casey Bernhards; Alexander Green; Keith Pardee; Patricia Buckley; Matthew Lux; Dr. Aleksandr Miklos, US Army DEVCOM Chemical Biological Center
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
The lateral flow immunoassay (LFI) provides an unpowered and low burden solution to biological detection, but the detection targets are fixed for each device leading to slower validation of a new LFI. Conversely, polymerase chain reaction (PCR)-based detection strategies are highly sensitive, specific, and PCR equipment can detect a new target by using new, easily-developed primer sets. However, these solutions carry a higher burden; specifically, they require powered equipment and specialized training. Here we present our “Dial-a-Threat" (DaT) assay concept, combining the speed and ease-of-use of an LFI with the specificity, sensitivity, and flexibility of PCR in a single-use, disposable, eye-readable colorimetric device. Our first generation DaT assay utilizes RNA toehold switches to detect the target RNA through RNA:RNA hybridization, resulting in the translation of a reporter gene by lyophilized cell-free protein synthesis reagents embedded within a paperfluidic device. Toehold switches can be swapped at the point of need to change the intended target organism. Devices are constructed from a series of wax printed paperfluidic channels in chromatography paper and assembled with a 3D printed cassette. The design is amenable to portable use in the field or a multiplexed setup for iterative testing. The handheld device is designed to perform sample preparation steps, precluding the need for specialized training with the system. The DaT device utilizes hydrogels to control directional diffusion and clustering of reaction components at specific sites on the paperfluidic tickets. Automated detection, segmentation, and image processing of colorimetric reaction outputs streamline data collection and optimization. Information gathered from each device iteration directly informs subsequent versions for improving signal output and shortening time-to-detection. Each iteration advances the goal of developing a highly specific, sensitive assay with ultra-low burden on the end user.
Tuesday
Three proteins play critical roles in the self-assembly and function of bacterial microcompartments
03:20pm - 03:20pm USA / Canada - Eastern - August 24, 2021 | Room: Zoom Room 03
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
Bacterial microcompartments (MCPs) are protein-based bacterial organelles that enclose metabolic pathways. MCPs help improve metabolic efficiency, providing an advantage for microbes in resource-limited environments. Metabolic engineers have attempted to re-engineer MCPs to improve yields of industrially relevant pathways of interest. However, the mechanisms for MCP assembly and function remain unclear, limiting engineering efforts. Here, we describe the role of the three primary protein components comprising the MCP shells from Salmonella enterica serovar Typhimurium LT2, providing critical insight into the assembly mechanism of a model MCP system. First, we demonstrate that two paralogous proteins, PduA and PduJ, have a unique propensity towards self-assembly. Using genomic manipulation techniques, we show that this property allows these two proteins to stimulate and drive MCP biogenesis. We also developed a novel, high-throughput method that enables rapid parallel analysis of MCP shell protein variants, which yielded exquisite detail on the role of these proteins in MCP shell assembly. These results provided the first demonstration of the essential yet partially redundant role that PduA and PduJ play in MCP shell formation. Furthermore, we demonstrated that a third MCP shell protein, PduB, is not necessary for the formation of MCP shells, a finding that contradicts prior research in the MCP field. Our work demonstrates that MCP shells lacking PduB are smaller and empty, lacking the encapsulated enzymatic core. This implies that while PduB is not essential for MCP shell assembly, it is important for interaction with the enzymatic core and in determining MCP morphology. Overall, these findings support a mechanism where PduA and PduJ drive MCP shell assembly. PduB then bridges the gap between the MCP shell and enzymatic core via interactions with PduA and PduJ. These are critical findings for those seeking to re-engineer MCPs for non-native functions, as an understanding of the essential components of MCPs, as well as their function in the MCP assembly process, will be necessary to repurpose or design compartments with novel functionalities.