Synthetic Reconstitution of Complex Cellular Behavior

Can we build biological systems that recapitulate complex cellular functions like those seen in nature? Answering this question is the central goal of our research.

This question also forms the basis of reconstitution, an established experimental approach that reimagines how a biological process can be recapitulated outside of its natural context (e.g. outside of the cell and in a test tube) using a reduced set of molecular components. Biochemical reconstitution has been successfully applied to recreate many processes, enabling precise control over molecular parameters and a powerful way to test mechanistic models and establish sufficiency. Our vision is to implement the power and precision afforded by biochemical reconstitution within the complex environment of a living cell. If we can achieve this, then we can understand and predictably control complex cellular functions that have eluded our understanding, such as those that regulate how cells make decisions, establish memories, actuate responses, and develop into multicellular organisms.

To do this, our laboratory is developing novel tools at the intersection of synthetic & systems biology, protein & cell engineering, laboratory evolution, genomics, and computation that enable us to recapitulate and control cellular behavior with synthetic circuitry. This enables us to effectively replace biochemistry with genetics. Taking this leap forward is fundamentally important for basic biology, to discover how cellular behaviors and diseases arise from complex networks of interacting molecules. It is also important for engineering and medicine, offering the potential to precisely control cellular function for next-generation therapies and to “teach” cells and organisms to solve the greatest health, climate, and engineering challenges of today.

Specifically, our laboratory develops tools of synthetic biology that allow us to construct regulatory circuits inside living cells. We are using our tools to dissect the molecular circuits that control gene regulation in eukaryotes, toward addressing the grand challenge of understanding their organization across scale and how they function to generate diverse cellular phenotypes. The basic insights we generate inform the development of platforms to program therapeutically-useful cellular functions for emerging gene and cell-based therapies, such as CAR-T cells for cancer. In addition, our team is developing novel continuous evolution technologies that are automated and scalable, and applying these to generate biomolecules with radically altered or new functions to address unmet needs in biology, medicine, and biotechnology. To broaden the impact of our basic science and medical discoveries, we make the technologies we develop widely usable and accessible to the scientific community. Overall, by learning how to build biological systems from scratch, our broad goal is to connect the molecular building blocks of life to a comprehensive understanding of cellular behavior and ultimately to clinical and other applications.

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A Multiplex MoClo Toolkit for Extensive and Flexible Engineering of Saccharomyces cerevisiae
William M. Shaw, Ahmad S. Khalil and Tom Ellis
ACS Synthetic Biology, 12: 3393-3405 (2023)

Cooperative Assembly Confers Regulatory Specificity and Long-Term Genetic Circuit Stability
Meghan D. J. Bragdon*, Nikit Patel*, James Chuang, Ethan Levien, Caleb J. Bashor* and Ahmad S. Khalil*
Cell, 186: 3810-3825 (2023)

Mapping the Dynamics of Epigenetic Adaptation
Ajay Larkin*, Colin Kunze*, Melissa Seman, Alexander Levashkevich, Justin Curran, Ahmad S. Khalil and Kaushik Ragunathan
bioRxiv, doi: 10.1101/2023.07.10.548368 (2023)

Deep Neural Networks for Predicting Single-Cell Responses and Probability Landscapes
Heidi E. Klumpe*, Jean-Baptiste Lugagne*, Ahmad S. Khalil and Mary J. Dunlop
ACS Synthetic Biology, 12: 2367-2381 (2023)

Long-Term Evolution of Proliferating Yeast Cells Using the eVOLVER Platform
Daniel Garcia-Ruano, Akanksha Jain, Zachary J. Heins, Brandon G. Wong, Ezira Yimer Wolle, Ahmad S. Khalil and Damien Coudreuse
Open Biology, 13: 230118 (2023)

Transcriptional Kinetic Synergy: A Complex Landscape Revealed by Integrating Modelling and Synthetic Biology
Rosa Martinez-Corral, Minhee Park, Kelly M. Biette, Dhana Friedrich, Clarissa Scholes, Ahmad S. Khalil, Jeremy Gunawardena and Angela H. DePace
Cell Systems, 14: 324-339 (2023)

High-Throughput Continuous Evolution of Compact Cas9 Variants Targeting Single-Nucleotide-Pyrimidine PAMs
Tony P. Huang*, Zachary J. Heins*, Shannon M. Miller, Brandon G. Wong, Pallavi A. Balivada, Tina Wang, Ahmad S. Khalil and David R. Liu
Nature Biotechnology, 41: 96-107 (2023)

Multidimensional Control of Therapeutic Human Cell Function with Synthetic Gene Circuits
Hui-Shan Li*, Divya V. Israni*, Keith A. Gagnon, Kok Ann Gan, Michael H. Raymond, Jeffry D. Sander, Kole T. Roybal, J. Keith Joung, Wilson W. Wong and Ahmad S. Khalil
Science, 378: 1227-1234 (2022)

A Toolkit for Precise, Multi-Gene Control in Saccharomyces cerevisiae
Adam Sanford, Szilvia Kiriakov and Ahmad S. Khalil
ACS Synthetic Biology, 11: 3912-3920 (2022)

High-Performance Multiplex Drug-Gated CAR Circuits
Hui-Shan Li*, Nicole M. Wong*, Elliot Tague, John T. Ngo, Ahmad S. Khalil and Wilson W. Wong
Cancer Cell, 40: 1-12 (2022)

Modular Design of Synthetic Receptors for Programmed Gene Regulation in Cell Therapies
Iowis Zhu*, Raymond Liu*, Julie M. Garcia, Axel Hyrenius-Wittsten, Dan I. Piraner, Josef Alavi, Divya V. Israni, Bin Liu, Ahmad S. Khalil and Kole T. Roybal
Cell, 185: 1431-1443 (2022)

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