The complex interactions between humans and the rest of the biosphere have created some of our most challenging global problems in human history such as energy sustainability, severe pollutions, and emergence or re-emergence of old and new epidemics and diseases. Research in our laboratory is focused on the development of next generation synthetic biology tools in addressing these key global problems. Proteins are the most versatile among the various biological building blocks. However, the strength of proteins - their versatility and specific interactions - also complicates and hinders their systematic design and engineering. Our lab has been interested in exploiting the modular nature of protein domains to design synthetic complexes that can perform new biological functions across different length scales. By adding logical and stimuli responsive components into the design, smart protein complexes can be created to sense and adapt to the constantly changing cellular environments. We are currently working on several projects in connecting exchangeable protein domains into functional devices for synthetic biology applications in two major areas:


A. Dynamic modulation of cellular phenotypes


B. Nano protein scaffolds for biocatalysis, biosensing, and therapeutics


Current Research


A. Dynamic modulation of cellular phenotypes


In nature, dynamic interactions between proteins play a crucial role in defining many cellular functions such as metabolism, cell signaling, transcription regulation, apoptosis, cellular targeting, and protein degradation. By controlling the spatial and temporal organization of these supramolecular complexes, cellular functions can be modulated in a highly dynamic manner for optimum efficiency. Understanding how these proteins interact holds the key to deciphering their roles in native cellular function and in creating new cellular functions for synthetic biology applications. By adding logical components into our designs, our goal is to create new synthetic biology frameworks that are dynamic in nature in order to adapt to the constantly changing cellular environments for dynamic modulation of phenotypes on demand.




Project 1. Synthetic Control of Metabolism by Dynamic Metabolons (NSF)

The ability to optimize pathway flux is one of the most important factors toward maximizing product titers. The traditional approaches largely focused on the overexpression of rate-limiting enzymes, competing pathway deletion, and resource management. However, most of these approaches are static in nature and do not provide dynamic regulation of pathway fluxes based on substrate, precursor, and product availability. Although many genetic circuit designs have been implemented to provide dynamic control of gene expression and pathway fluxes, these dynamic strategies usually provide only excellent “up-regulation” control but down regulation is much slower as it requires the degradation of the associated regulatory components. In this proposal, we seek to investigate a new transformative strategies that provide faster on-and-off control based on enzyme proximity control inspired by natural dynamic metabolons. The goal to develop synthetic dynamic metabolons that will allow carbon flux to be redirected at will. The end result is the ability to provide dynamic optimization of microbial metabolism for optimal product formation. This will be achieved by the dynamic shifts between the assembly and disassembly of synthetic metabolons in order modulate the overall output function. Nature has already based on the use of RNA scaffold. The central framework is to design RNA- or protein-based dynamic metabolons that assemble in response to specific metabolic demands and to exploit the dynamic shift between the assembly and disassembly of enzyme complexes to coordinate metabolic pathways for optimal product titer. The tools developed here could be transferred to other organisms, and used to address fundamental questions about control and regulation of metabolism.


Project 2. Repurposing the CRISPR-Cas9 system for dynamic control of cellular metabolism (NSF) - with Prof. Papoutsakis

Cellular metabolism is capable of highly specific and efficient chemical synthesis at mild temperatures and pressures far beyond the capability of most synthetic chemical routes. Although pathway engineering can be used to further improve the range of compounds that could be synthesized, achieving commercially viable productivity remains challenging. An emerging strategy to combat these issues is to organize pathway enzymes into a sequential multi-enzyme complex in order to improve the overall pathway flux, to minimize cross-reactions, and to provide kinetic driving forces that redirect the carbon flux through essentially reversible steps. Although synthetic biologists have taken a more modular approach using biomolecular scaffolds to co-localize target enzymes, the current approaches either lack the required binding affinity or flexibility in tuning the cascade assembly. To address these shortcomings while providing a highly modular and flexible platform for assembling in vivo enzyme cascades, we propose here a new and potent approach that enables specific and high-affinity binding to DNA scaffolds using a special version of the CRISPR/Cas9 system. By combining the ability to provide site-specific, dCas9-guided enzyme assembly and the ability to provide disassembly by controlled dCas9 degradation, the overall goal is to develop a transformative framework to create highly efficient and dynamic enzyme cascades suitable for many synthetic-biology and metabolic engineering applications.




Project 3. Redirecting cellular metabolism via synthetic toehold-gated dCas9 regulators (NSF)

Cellular metabolism is capable of highly specific and efficient chemical synthesis at mild temperatures and pressures far beyond the capability of most synthetic chemical routes. Engineering specific pathways can be used to further improve the range of compounds that can be synthesized but it is a major challenge to achieve commercially viable productivity. To maximize productivity, it is crucial to fine-tuning pathway fluxes. The goal of this project is to develop a new transformative approach to modulate cell metabolism based on endogenous cellular information. An emerging strategy is the use of regulators that provide dynamic control of pathway fluxes. A recently discovered modified CRISPR based tool offers a unique approach for DNA targeting and transcriptional regulation. These new generation of dCas9 regulators can be used for dynamic gene repression and activation for many synthetic-biology and metabolic engineering applications. A new generation of toehold-gated dCas9 regulators governed by conditional sgRNA structures that are activated by toehold-mediated strand displacement will be created to provide simultaneous, orthogonal, and autonomous control of cellular metabolism. This new framework to design toehold-gated dCas9 regulators responsive to any endogenous mRNA will lay the foundation as a new transformative approach for implementing dynamic control of cellular metabolism.




Project 4. Dynamic modulation of cellular functions by controlled protein degradation (NSF)

Aberrant protein levels and activities contribute to numerous diseases, including cancers, cardiovascular disease, diabetes, and neurodegenerative diseases. No current therapy dynamically degrades excess protein(s) to treat diseases. The ability to tightly coordinate the levels of cellular proteins is critical for normal healthy cells to maintain homeostasis. We are developing new strategies that elicit dynamic and reversible protein degradation, which would overcome the challenges associated with targeting elevated levels of essential proteins.




B. Nano protein scaffolds for biocatalysis, biosensing, and therapeutics


Proteins are the most versatile among the various biological building blocks and efforts in protein engineering have resulted in many industrial and biomedical applications. Our approach is to exploit the modular nature of different protein domains in order to design synthetic protein scaffolds that can perform completely new biological functions. We are developing new strategies in designing exchangeable protein domains for predicative engineering applications in (1) biocatalysis, (2) biosensing, and (3) disease therapeutics.




Project 1. Synthetic multilayer targeting DNA devices for detection of specific cancer indicators and programmed assembly of split yCD for prodrug activation (NSF)

One of the most pressing needs in cancer treatment is to distinguish and treat cancer vs healthy cells. Active targeting of surface markers alone is inadequate and must be merged with additional layers of intracellular signals to provide a higher level of specificity. Advances in synthetic biology have enabled the engineering of new cellular sensors, actuators, and amplifiers toward the creation of clinically relevant “smart drugs” that sense the disease state in a complex cellular environment and actuate an appropriate, localized therapeutic response for treatment. Prior efforts have primarily been focused on implementing therapeutic gene circuits for targeted cancer therapies. However, practical utility of these synthetic devices is limited as multiple genes must be delivered and expressed within the target cell to produce needed detection components. Less complex DNA-based logic devices can potentially bypass this stringent delivery hurdle but the few successful examples reported so far can only be executed in vitro due to the need for a restriction enzyme as part of the activation circuit. Dynamic DNA-gated lock and key devices based on toehold-mediated strand displacement is a new powerful method to create intracellular logic circuits that can be triggered by cancer-specific biomarkers. By combining these dynamic DNA devices with active extracellular targeting and exogenously applied, inactive prodrugs to trigger cell death, a higher degree of specificity for cancer cells can be achieved by creating these multi-layer targeting, sensing, and responsive DNA-based devices. In this proposal, we seek to develop a new generation of synthetic DNA devices based on toehold-mediated strand displacement that can be used for programmable intracellular reconstitution of the yeast cytosine deaminase (yCD), a split suicide enzyme capable of activating the prodrug deaminate 5-fluorocytosine (5-FC) into the toxic product 5-fluorouracil (5-FU). Although the simplicity of the design allows highly adaptable, multi-input capabilities suitable for a range of cancer targets, the feasibility of the approach will be illustrated with a two-input device using two microRNAs (miRNAs) targets, miR-720 and miR-1380, associated with highly invasive inflammatory breast cancer (IBC) cells, as inputs.




Project 2. Design of Multi-Functional SplitCore HBV Capsids for Precisely Controlled Multi-siRNA Delivery in Cancer Therapeutics (NSF) - with Prof. Sullivan

Virus-like particles are perfectly monodisperse self-assemblies of protein-only subunits that are ideal for therapeutic delivery applications because both capsid surfaces and interiors can be stoichiometrically decorated with moieties for cell targeting and siRNA capture/release. We will exploit the newly discovered SplitCore strategy for the formation of HBV capsids using fragment complementation of the separately expressed N- and C-cores to provide a streamlined approach to attach four unique decorations (3 exterior, 1 interior) to each HBV monomer into a highly modular nanoplatform suitable for customizable delivery and siRNA release. This significant advance can enable key increases in both cell specificity and therapeutic efficacy through incorporation of well-defined combinations of targeting ligands as well as siRNAs, ultimately creating hybrid structures that fuse the delivery efficiency of viruses to the design versatility of non-viral vehicles.


Gene silencing therapy based on siRNAs offer unique promise for cancer treatment by providing highly potent and target-specific silencing of genes dysregulated during cancer progression. However, delivery of siRNA remains extremely challenging because of its negative charge and tendency to degrade rapidly under physiological conditions. Furthermore, the unusual genetic and phenotypic signatures in aggressive cancers pose additional barriers for siRNA nanomedicines, as the invasive behaviors in these cells typically originate from simultaneous alterations in multiple cell surface receptors and gene expression profiles. Accordingly, there is an unmet need for therapeutic strategies able to more accurately target these cells by recognition of their specific balance in surface receptor expression, with the subsequent triggered release of appropriate siRNA cocktails. Design of such approaches could provide significant improvements in therapeutic efficacy relevant to metastatic cancers and a variety of other diseases. In this proposal, our objective is to design highly tunable, multi-functional Hepatitis B Virus (HBV) capsids suitable for cell-specific siRNA delivery in inflammatory breast cancer (IBC) cells, a canonical aggressive cancer whose invasive behavior is defined by multifactorial changes in gene expression. 




Project 3. Design of RNA-triggered Disassembly Mechanisms in Multi-responsive Polymer Nanocapsules for Personalized Physiological Profiling and Tailored Therapeutics (NSF)

This research addresses the National Academy of Engineering’s Grand Challenge to engineer better medicines by developing new approaches that will ultimately allow rapid assessment of the genetic profiles in patients and the release of personalized drug cocktails.  Our approach is to incorporate DNA “strand displacement” biosensing designs within polymer nanocarriers such that the DNA interface controls nanocarrier stability.  Recognition of specific RNA or DNA sequences will result in a strand displacement reaction that destabilizes the nanocarrier and allows the release of encapsulated drugs or diagnostics.  The major milestones in this work include the design of DNA strand displacement designs sensitive to breast cancer-specific RNAs; incorporation of these DNA duplexes within block polymer micelles; establishment of the specificity and efficacy of RNA-mediated nanocarrier disassembly; addition of cancer cell-targeting peptides on the surface of the nanocarriers; and establishment of selective cellular uptake and efficient cargo (dye) release in breast cancer cells. 




Project 4. Engineering protein nanostructures to illuminate protein delivery and cellular processing (NSF) - with Prof. Sullivan

This project will develop new approaches to create nanoparticles using therapeutic proteins as building blocks. The protein building blocks will be designed so that they can assemble together spontaneously to form flexible, elongated nanoparticles; additionally, the protein building blocks will be designed to incorporate clusters of ‘targeting’ molecules that will bind strongly and specifically to diseased cells. These approaches will enable control of the size, shape, and chemistry of the resulting protein nanoparticles, which in turn will determine where the nanoparticles accumulate in the human body after injection .





Project 5. Advanced Biomanufacturing of Functional Bionanoparticles for Bioimaging (NSF)

Research on nanomaterials in the past decades has undergone explosive growth. Although recent progress in nanomanufacturing technologies led to successfully large-scale manufacturing of a range of inorganic and organic nanostructured materials, the extension of those manufacturing processes to the production of advanced functional bio-nanoparticles (bio-NPs) is currently an unmet challenge. This is mainly due to the sensitivity of bio-NPs to the harsh conditions of current manufacturing processes. The goal of this proposal is to design an advanced biomanufacturing process to manufacture genetically engineered multi-functional bio-NPs. The local and global structure of the advanced bio-NPs will be monitored and characterized, and their functionality and utility will be examined and validated in bioimaging of brain tumor in mouse model.


Our hypothesis is that by employing genetically engineered vesicle-forming bacteria as microbial cell factories, we will reliably produce uniform bio-NPs with precisely controlled biological functionality in a continuous and scalable way. To accomplish it, recombinant DNA technology will first be used to design novel genetically engineered protein multi-functional bio-NPs for capture and detection functions in bioimaging. The bio-NPs are lipid-based outer membrane vesicles (OMVs) with a uniform diameter of ~50 nm and the outer leaflet of the bilayer is decorated with novel engineered protein fusion, endowing multi-functionality. The OMVs, co-displaying multiple copies (~50, each) of super-active NanoLuc luciferase enzyme (~150-fold more active than other luciferases), will contain (i) an antibody-binding domain (ZZ domain) for anchoring antibodies of interest, and (ii) a thermo-responsive elastin-like protein domain (ELP, soluble at room temperature and insoluble/aggregated at 42 °C) for simple purification of the OMVs via size filtration. A fermentation process integrated with two-stage size filtration will then be designed for continuous, sustainable production of multi-functional OMVs at a large quantity.