(6es) Microfluidic Approaches Towards Metabolic and Genetic Engineering

Authors: 
Zeitoun, R. I., University of Colorado Boulder

Microbes provide a promising platform for broad and sustainable chemical production because of their potential to produce a wide range of molecules with high specificity from renewable feedstocks. Although metabolic engineering has existed for several decades, leaps in progress have been driven in part by tangential technological advancements in DNA synthesis, sequencing and automation.   These advancements have streamlined editing genomes towards desirable phenotypes and have spurred the development of seminal technologies like trackable multiplexed recombineering (TRMR), multiplexed automated genome engineering (MAGE) and phage-assisted continuous evolution (PACE). Even entire genomes are now possible to synthesize de novo.  The ever-improving simplicity of manipulating organisms and the demonstrable effectiveness of automation technologies towards this end, suggest that that the future of metabolic engineering and synthetic biology will soon require a drastically different focus, shifting from the basics manipulating and editing genomes to how to effectively engineer and optimize organisms from interdisciplinary approaches.  My research effort will utilize an interdisciplinary approach from a fundamental chemical engineering perspective, combining microfluidic and automation technology development to address the needs of metabolic engineering and synthetic biology.  This approach will enable more efficient and effective methods to optimize organisms thereby drastically improving the scope, throughput and basic understanding of genetic and metabolic engineering. My planned research efforts are as follows:

Proposed Research Projects

Parallelization and miniaturization of chemical processes as a broad screening approach: Correlating the phenotype of an engineered cell to its genotype by screening limits throughput in the design-build-test phase of genetic engineering. Identifying extracellular products produced, like biofuels, can involve chemical processes in series (production, separation, detection) which reduces the throughput of genotypes that can be tested and thereby limits the effectiveness of engineering organisms.  The research proposed here is to develop a broad approach toward parallelizing the production, separation and detection of extracellular products using multiple emulsions in a microfluidic system.  This idea can be used to screen for a variety of chemicals and capable of throughputs up to 103-fold greater than conventional approaches. By coupling separations and colorimetric reactions in a multiple emulsion format, a method could be developed to perform processes in parallel enabling high-throughput analysis single-genotype analysis.  With high-throughput screening, genome-wide search technologies and methodologies will be applied to understand the metabolic engineering of strains whose investigation is currently limited by screening technologies.

Miniaturized, long-write DNA synthesis methodology and development: Many genetic engineering tools and technologies use massive quantities of diverse synthetic oligonucleotides and as such have relied on advancements in DNA synthesis. But, the high and somewhat stable costs of gene synthesis, caused by harsh chemistry and inefficient synthesis, have limited the further applicability of DNA synthesis. The research proposed here is developing a concurrent approach of enzyme and chemistry development with microfluidic system engineering to develop a novel and superior approach to DNA synthesis with respect to the current industry standard. Specifically an enzyme-based DNA synthesis chemistry will be developed in an electrophoresis-driven microfluidic platform allowing for millisecond transport and reaction times, precise temperature control and low reagent use. By coupling molecular biology and protein engineering along microfluidic system development, I envision a bench top system could be developed to improve de novo oligonucleotide and gene synthesis.

Automation of functional pathway discovery: Integrating heterologous genes in non-native hosts can allow for the production of a wide-range of chemicals by either adding a new metabolic pathway or bypassing the host cell’s native regulation scheme.  However, this is largely a trial and error process and heterologous gene expression typically can result in incompatible enzyme products.  To overcome this, I propose an automated system of chemostats, coupled with novel microfluidic transformation technologies in series to continuously create combinations of different pathways of heterologous genes in E.coli. This system will be use to rapidly construct entire pathways which can be screened for chemical production. By integrating an automation approach to recursive postivite/negative selection I will develop an open-sourced continuous flow directed evolution system capable of constructing pathways.

Previous Research

I studied under Professor Mark Burns in the Ph.D. program at the University of Michigan to develop technologies enabling active control of flow, particles and chemicals in microfluidic systems.  There, I integrated chemical engineering concepts governing microfluidic design (transport and fluid dynamics), system engineering and microfabrication.  My Ph.D. resulted in three first author research publications, three contributing author publications and a book chapter contribution. I was awarded the competitive Microfluidics Training Program Fellowship, an NSF graduate honorable mention, and doctoral qualifying exam distinction having the top research and grades for my Ph.D. qualifying exam.  After completing my Ph.D. I worked at Becton Dickinson on their BD Max platform - an open-source automated molecular diagnostic robotic system which utilized microfluidic technology developed from the Burns lab.

Currently, I am a research associate in Professor Ryan Gill’s research group at the University of Colorado studying methods to relieve throughput bottlenecks in the design-build-test cycle which is core to synthetic biology and metabolic engineering. My most notable work has been developing a method to track populations of combinatorial mutants by utilizing core chemical engineering concepts along with next-generation sequencing technologies.  This work has improved the throughput of analyzing combinatorial engineered populations by over 104-fold. Another of my publications uncovered implications of a fluid phenomena in microchannels - an effect that arose while developing microfluidic systems for genetic engineering.  I have also been involved in authoring several successful grants including the DARPA living foundries grant and DoE biosystems design grant.

Teaching and Professional Activities

Teaching is an important focus of mine and as a professor I will aim to impart students with the knowledge, skills and attitudes to ensure successful transition from education to application.  Aside from positively contributing to the undergraduate and graduate educational structure through the ABET requirements, I would also like to apply concepts and ideas published in educational literature like, Chemical Engineering Education. I have experience teaching undergraduate discussion classes in Material and Energy Balances and Transport Phenomena.  Through these experiences, and other instructional experiences outside of the classroom setting, I’ve worked closely with a variety of learning styles giving me a good perspective on how best to instruct and interact with students.  In addition, I was able to work with many B.S. and M.S. engineers at Becton Dickinson, and was able to observe the skills that I saw promoted success from an engineering perspective. I would like to use my opportunity as a professor to support rich undergraduate extracurricular work. I have had extremely positive experiences in the past with undergraduates and REU students and hope to continue giving opportunities for independent and directed hands-on learning for students.  I would also like to support undergraduate students by making them aware of the opportunities available to them for industry, graduate school and various internships (SURF, REU, etc).  Finally, I was trained on the Microfluidics in Biomedical Sciences Training Program at Michigan and saw it as a fantastic experience.  I would like to support programs like this and initiate the development of collaborative training programs.