Biological Engineering Study and Engineer Biology to … •develop molecular therapeutics (antibodies, peptides, small molecules, antivirals) •improve drug delivery (antibodies, peptides) •develop cellular therapeutics (tissue engineering, microbiome engineering) •improve chemical production (commodity chemicals and specialty chemicals) •enhance bioenergy production (biofuels, photosynthesis and carbon fixation) Biological Engineering at Multiple Scales
Molecules Reed Murphy Romero Palecek Shusta Pfleger Yin Cells/Viruses Palecek Shusta Pfleger Venturelli Reed Yin Cell Populations & Tissues Palecek Venturelli Shusta Yin Romero Lab: Data-driven protein engineering Molecular simulation Machine learning Protein design
Collaborators AI-driven biological discovery • Jim Dumesic • Brian Pfleger • Jennie Reed Technology development • Ophelia Venturelli • Statistics, Biochemistry Understand structure-function relationships Murphy Lab: Amyloid protein misfolding & aggregation
protein disease beta-amyloid Alzheimer disease alpha-synuclein Parkinson disease huntingtin Huntington disease transthyretin Senile systemic amyloidosis cystatin C Cerebral amyloid angiopathy
www.sheffield.ac.uk/ mbb/staff/staniforth Collaborators Starting polypeptides are diverse in sequence, structure and • Eric Shusta function but their aggregates share common structural features and cause disease • Reid van Lehn • What are the kinetics and mechanisms of aggregation? • Pharmacy • How do amyloid proteins exert biological effects on cells? • Can we design compounds or systems that interfere with aggregation? Brain Uptake of Retinol in Alzheimers
(a) AD# (b) (c) TTR-RBP v transfers re*nol# Aβ v retinol RBP# (Vitamin A) TTR# TTR$ RBP$ across cell re(nol$ membrane (d) (e) hRBP% aRBP% re(nol$ RDH$ CRBP$ Retinol regulates re(nal$ transcription of RALDH$ re(noic$acid$ RAR/RXR$ RARE$ other proteins CRABP$ LRAT% CRBP% How does Ab get metabolism$ from the brain into the blood? v v
How does retinol cross the BBB? What roles do TTR and RBP play? v v Pfleger Lab: Synthetic Biology
Collaborators • Jennie Reed What do we study? • Phil Romero • Gene discovery and characterization • Ophelia Venturelli • Gene expression, regulation • Jim Dumesic • Synthetic biology tool development • Metabolic engineering • George Huber • Functional genomics and systems biology • Thatcher Root • Bioreactor studies and bioprocess design • Christos Maravelias • Victor Zavala Applied • Microbiology, Genetics, Microbiology Biochemistry • PSU, UIUC (CABBI), UNL Vanderbilt Synthetic Systems Biology Biology Model-Guided Thioesterase Engineering
Goal: Create mutants of ‘TesA with specificity for octanoic (C8) acid and dodecanoic (C10) acid • Iterative Protein Redesign & Optimization (IPRO) based on crystal structure information. • Minimize interaction energy for C8:0 and maximize interaction energy for undesired substrates (C14:0).
Organism Escherichia coli of origin Native 53 % C14:0, 38 % selectivity C12:0 M. Grisewood C. Maranas J. Thoden H. Holden N. Hernández-Lozada B. Pfleger Crystal Available Computational Crystallography of Variant Construction structure (1U8U; 2FAE) enzyme design WT and top variant and Testing Usage Biodiesel – Steen et al., Nature Methyl-ketones – Goh et al., AEM
top 10 octanoic acid producing mutants* Reed Lab: Systems Biology • Discover enzyme functions
40-60% of genes have unknown function
• Engineer metabolic & regulatory Collaborators networks for chemical production • Brian Pfleger • Phil Romero • Understand microbe-microbe and microbe-host interactions • Christos Maravelias Image • Ophelia Venturelli courtesy of McFall-Ngai • Microbiology, ECE, ISYE, Lab Biochemistry, Vet School Optimize Expression Constructs to Maximize Valine Production
What combinations of RBSs and genes are needed to maximize valine yields?
Kumar, Adamczyk et al. To be submitted Venturelli Lab: Engineering Gut Microbiome
Computational modeling Microbiome engineering
Negative interaction Positive interaction BV CA BT
CH BH DP BU
BO PC Actinobacteria Bacteroidetes Firmicutes FP Proteobacteria ER EL Collaborators • Brian Pfleger • Jennie Reed • Phil Romero Microfluidic technologies • Victor Zavala • Microbiology Rapid inference of microbial interaction networks in microfluidic droplets
Venturelli awarded the 2017 ARO Young Investigator
Provisional patent filed
Hsu RH et al. In review Palecek Lab: Stem Cell Biomanufacturing
Processes to generate clinical grade cells from human pluripotent stem cells
Collaborators • Dave Lynn • Eric Shusta • Medical School (Cell & Regenerative Cardiac aggregates ‘omics discovery of critical Biology, Medicine), quality attributes Biochemistry, to treat heart disease Chemistry, BME • Georgia Tech, U. Puerto Rico Mayaguez, UCSF Human Pluripotent Stem Cells (hPSCs) Properties of hPSCs: 1. Pluripotent – ability to generate all cell types found in the adult 2. Potential for infinite self-renewal
A system to study early developmental processes
A source of cells and tissues for screening effects of drugs and other chemical compounds
A clonal source of vast amounts of desired cells and tissues for therapeutic applications
Lutolf et al. Nature, 2009 Shusta Lab: Antibody Engineering & BBB
Antibody Discovery iPSC Differentiation Technology Protein Engineering
PK/Biodistribution Blood-Brain Barrier Modeling
“Blood-Brain Barrier” Nanoparticles/ Modeling the Diseased NVU controlled release Neurovascular Unit
Collaborators • Regina Murphy • Sean Palecek Therapeutic Development • Dave Lynn • Medical School (Neurosurgery, Pathology), Biomedical Engineering • Cedars Sinai/UCSD Getting therapeutics across the blood-brain barrier
Mouse injected with radiolabeled Histamine (111 Da) Pardridge, Brain Drug Targeting, 2001
BBB controls transport of substances into and out of the brain: -Provides the brain with nutrients -Transports metabolites out of the brain -Maintains brain homeostasis -Regulates immune function -Implicated in a number of diseases -Serves as a significant bottleneck for brain drug delivery -98% of small molecule pharmaceuticals do not cross BBB -Only lipophilic, small molecules diffuse through Using hPSC to a Build Blood Brain Barrier Model Collaboration between Prof. Palecek and Prof. Shusta Differentiation
(Lippmann, Azarin et al., Nature Biotech, 2012) (Wilson et al., FBCNS, 2015) (Qian et al., Science Advances, 2017
• Human BBB endothelial cells Human pluripotent • Scalable stem cells (hPSC) • Substantial barrier
(Lippmann et al., Scientific Reports, 2014) (Canfield et al., J Neurochem, 2017) (Stebbins et al., Science Advances, 2019) Differentiation Astrocytes & Neurons Neural stem cells Yin Lab: Systems Approaches to Biology Chemistry Virus input Q: How can reactions of simple chemicals (e.g., amino acids) begin to show behaviors* of living systems? * store information, self replicate, adapt host cell
Drying- induced de novo Virus output peptide Q: How can material and energy flows synthesis during virus growth give insights into how diseases spread and persist? How bad is your cold? Identify biomarkers and potentially drug targets for human rhinovirus infections (common cold).
Human rhinovirus Palmenberg, Biochemistry Pediatric immunology Gern, Sch. Med. Pub. Health Biostatistics/machine learning . Roy, Biostat. Med. Info. Student Experiences • Patent Applications • Internships • WARF Ambassadors Ø Cargill Ø Merck • UG Research Mentors Ø Genencor • DELTA Interns Ø Genentech • K-12 Outreach Ø Dupont Ø Dow Agrosciences (Corteva) • Training Programs Ø Intrexon • Campus Seminars Ø LS9 • Conferences Ø AbbVie Ø USDA • International Research Ø Kimberly-Clark • Funded Grants • Great Lakes Bioenergy Research Center (GLBRC) • Metabolomics and Proteomics • Fermentation Facilities • JGI Sequencing and DNA Synthesis • Bigger Picture of Overall Challenges • Communication with Broader Science Community Involvement in Campus Life Science Initiatives Cross-Disciplinary Training Programs • Biotechnology Training Program • Computation and Informatics in Biology and Medicine • Genomic Sciences Training Program • Clinical Neuroengineering Training Program • Chemistry-Biology Interface Training Program • Cellular and Molecular Pathology Training Program • Neuroscience Training Program • Virology Training Program
Cross-Disciplinary Graduate Colleagues • Cellular and Molecular Biology Training Program • Microbiology Doctoral Training Program • Biomedical Engineering • Material Science • Chemistry • Biophysics • Food Science
Centers • UW Stem Cell and Regenerative Medicine Center • UW Comprehensive Cancer Center • Great Lakes Bioenergy Research Center • NSF ERC Cell Manufacturing Technologies (Georgia Tech, U Georgia, U Puerto Rico)