Our technological ability to both rapidly sequence and synthesize DNA has transformed the way we study biological systems.  High-throughput technologies have led to a large-scale characterization and connectivity map of the genes involved in various biological processes in a field known as 'Systems Biology.' A complimentary approach, 'Synthetic Biology', is emerging to synthetically build small networks of genes and then systematically increase complexity. These approaches lead to an interesting and open question-- What are the underlying design principles that make a network of genes perform a particular function? And how best can we rewire gene networks to build applications that shape the environment, energy, and human health?  

My research interests span a diverse array of biological systems with particular emphasis on quantitative understanding and engineering of dynamic behavior. The approach I take combines microfluidic technologies, time-lapse fluorescence microscopy, molecular cloning, in vivo animal techniques, and quantitative modeling to both understand and engineer biological systems.  For further reading, abstracts from selected works are shown below.  The Video & Art+Outreach pages show related content.  



Programmable probiotics for detection of cancer in urine

Tal Danino, Arthur Prindle, Gabriel A. Kwong, Matthew Skalak, Howard Li, Kaitlin Allen, Jeff Hasty, and Sangeeta Bhatia.  " Programmable probiotics for detection of cancer in urine."  Science Translational Medicine Vol. 7, Issue 289, p. 289ra84

Summary:   Rapid advances in the forward engineering of genetic circuitry in living cells has positioned synthetic biology as a potential means to solve numerous biomedical problems, including disease diagnosis and therapy. One challenge in exploiting synthetic biology for translational applications is to engineer microbes that are well tolerated by patients and seamlessly integrate with existing clinical methods. We use the safe and widely used probiotic Escherichia coli Nissle 1917 to develop an orally administered diagnostic that can noninvasively indicate the presence of liver metastasis by producing easily detectable signals in urine. Our microbial diagnostic generated a high-contrast urine signal through selective expansion in liver metastases (106-fold enrichment) and high expression of a lacZ reporter maintained by engineering a stable plasmid system. The lacZ reporter cleaves a substrate to produce a small molecule that can be detected in urine. E. coli Nissle 1917 robustly colonized tumor tissue in rodent models of liver metastasis after oral delivery but did not colonize healthy organs or fibrotic liver tissue. We saw no deleterious health effects on the mice for more than 12 months after oral delivery. Our results demonstrate that probiotics can be programmed to safely and selectively deliver synthetic gene circuits to diseased tissue microenvironments in vivo.  [PDF Link]    

PROP-Z (Programmable Probiotics with lacZ) for noninvasive cancer detection.  The PROP-Z diagnostic platform is made up of probiotic EcN bacteria transformed with a dual-stabilized, high-expression lacZ vector as well as a genomically integrated luxCDABE cassette that allows for luminescent visualization without providing exogenous luciferin (blue). (1) PROP-Z is delivered orally. (2) Probiotics rapidly (within 24 hours) translocate across the gastrointestinal tract and (3) specifically amplify within metastatic tumors present in the liver. (4) PROP-Z expresses high levels of the enzyme lacZ (red), which can cleave systemically injected, cleavable substrates (green and yellow). Cleavage products of the substrates (yellow) filter through the renal system (5) into the urine for detection (6).  


PROP-Z (Programmable Probiotics with lacZ) for noninvasive cancer detection.  The PROP-Z diagnostic platform is made up of probiotic EcN bacteria transformed with a dual-stabilized, high-expression lacZ vector as well as a genomically integrated luxCDABE cassette that allows for luminescent visualization without providing exogenous luciferin (blue). (1) PROP-Z is delivered orally. (2) Probiotics rapidly (within 24 hours) translocate across the gastrointestinal tract and (3) specifically amplify within metastatic tumors present in the liver. (4) PROP-Z expresses high levels of the enzyme lacZ (red), which can cleave systemically injected, cleavable substrates (green and yellow). Cleavage products of the substrates (yellow) filter through the renal system (5) into the urine for detection (6).

 


 

A synchronized quorum of genetic clocks

Tal Danino, Octavio Mondragón-Palomino, Lev Tsimring, and Jeff Hasty. "A synchronized quorum of genetic clocks." Nature 463, no. 7279 (2010): 326-330. 

Summary:   The engineering of genetic circuits with predictive functionality in living cells represents a defining focus of the expanding field of synthetic biology. This focus was elegantly set in motion a decade ago with the design and construction of a genetic toggle switch and an oscillator, with subsequent highlights that have included circuits capable of pattern generation, noise shaping, edge detection and event counting. Here we describe an engineered gene network with global intercellular coupling that is capable of generating synchronized oscillations in a growing population of cells. Using microfluidic devices tailored for cellular populations at differing length scales, we investigate the collective synchronization properties along with spatiotemporal waves occurring at millimetre scales. We use computational modelling to describe quantitatively the observed dependence of the period and amplitude of the bulk oscillations on the flow rate. The synchronized genetic clock sets the stage for the use of microbes in the creation of a macroscopic biosensor with an oscillatory output. Furthermore, it provides a specific model system for the generation of a mechanistic description of emergent coordinated behaviour at the colony level.  [PDF Link]    

Synchronized genetic clocks. (A) Network diagram. The luxI promoter drives production of the luxI, aiiA and yemGFP genes in three identical transcriptional modules. LuxI enzymatically produces a small molecule AHL, which can diffuse outside of the cell membrane and into neighbouring cells, activating the luxI promoter. AiiA negatively regulates the circuit by acting as an effective protease for AHL. (B) The "Supernova". Fluorescence microscopy overlay of a growing colony in a microfluidic device at the time it reaches a quorum.  (C) Fluorescence slices of a typical experimental run demonstrate synchronization of oscillations in a population of E. coli residing in the microfluidic device (Movie in Videos page). Inset in the first snapshot is a ×100 magnification of cells.  

Synchronized genetic clocks. (A) Network diagram. The luxI promoter drives production of the luxI, aiiA and yemGFP genes in three identical transcriptional modules. LuxI enzymatically produces a small molecule AHL, which can diffuse outside of the cell membrane and into neighbouring cells, activating the luxI promoter. AiiA negatively regulates the circuit by acting as an effective protease for AHL. (B) The "Supernova". Fluorescence microscopy overlay of a growing colony in a microfluidic device at the time it reaches a quorum.  (C) Fluorescence slices of a typical experimental run demonstrate synchronization of oscillations in a population of E. coli residing in the microfluidic device (Movie in Videos page). Inset in the first snapshot is a ×100 magnification of cells.

 


 

Entrainment of a population of synthetic genetic oscillators

Mondragón-Palomino, Octavio, Tal Danino, Jangir Selimkhanov, Lev Tsimring, and Jeff Hasty. "Entrainment of a population of synthetic genetic oscillators." Science 333, no. 6047 (2011): 1315-1319.

Summary:   Biological clocks are self-sustained oscillators that adjust their phase to the daily environmental cycles in a process known as entrainment. Molecular dissection and mathematical modeling of biological oscillators have progressed quite far, but quantitative insights on the entrainment of clocks are relatively sparse. We simultaneously tracked the phases of hundreds of synthetic genetic oscillators relative to a common external stimulus to map the entrainment regions predicted by a detailed model of the clock. Synthetic oscillators were frequency-locked in wide intervals of the external period and showed higher-order resonance. Computational simulations indicated that natural oscillators may contain a positive-feedback loop to robustly adapt to environmental cycles.  [PDF Link]

Entrainment of genetic oscillators.  (A) Architectures of eukaryotic circadian clocks and bacterial synthetic oscillators contain positive- and negative-feedback loops that are sensitive to external stimuli. (B) Fluorescence images from a time-lapse experiment show coherent GFP oscillations (green) in a colony of single-cell oscillators subject to a 30-min cycle of arabinose (red) (movie S1). (C) Fluorescence time series of a single-cell oscillator (green). The concentration of arabinose (red) changes sinusoidally according to [ara](t) = 0.3 + Asin(2πt/Tf) [percent weight/volume (% w/v)], with A = 0.15% and Tf = 30 min. The intensity plot above the graph corresponds to the cell trace. a.u., arbitrary units. (D) Fluorescence intensity plots of free-running and forced oscillators. Each row in the two panels represents a single-cell trace. The top row of the forced set represents the modulated concentration of arabinose (A = 0.15%). (E) Entrainment regions indicate which forcing periods (Tf) and amplitudes (A) result in locking of the oscillator according to a deterministic model (SOM text). Entrainment of order 2:1 means that two oscillation peaks are observed for one peak of arabinose. Tn is the natural period of the oscillator. Images and cell traces shown in (B), (C) and [(D), forced oscillations] correspond to point 4. Points 1 to 3 signal some parameter values explored experimentally.  


Entrainment of genetic oscillators.  (A) Architectures of eukaryotic circadian clocks and bacterial synthetic oscillators contain positive- and negative-feedback loops that are sensitive to external stimuli. (B) Fluorescence images from a time-lapse experiment show coherent GFP oscillations (green) in a colony of single-cell oscillators subject to a 30-min cycle of arabinose (red) (movie S1). (C) Fluorescence time series of a single-cell oscillator (green). The concentration of arabinose (red) changes sinusoidally according to [ara](t) = 0.3 + Asin(2πt/Tf) [percent weight/volume (% w/v)], with A = 0.15% and Tf = 30 min. The intensity plot above the graph corresponds to the cell trace. a.u., arbitrary units. (D) Fluorescence intensity plots of free-running and forced oscillators. Each row in the two panels represents a single-cell trace. The top row of the forced set represents the modulated concentration of arabinose (A = 0.15%). (E) Entrainment regions indicate which forcing periods (Tf) and amplitudes (A) result in locking of the oscillator according to a deterministic model (SOM text). Entrainment of order 2:1 means that two oscillation peaks are observed for one peak of arabinose. Tn is the natural period of the oscillator. Images and cell traces shown in (B), (C) and [(D), forced oscillations] correspond to point 4. Points 1 to 3 signal some parameter values explored experimentally.

 



A sensing array of radically coupled genetic "biopixels."

Prindle, Arthur, Phillip Samayoa, Ivan Razinkov, Tal Danino, Lev S. Tsimring, and Jeff Hasty. "A sensing array of radically coupled genetic "biopixels." Nature 481, no. 7379 (2012): 39-44.

Summary: Although there has been considerable progress in the development of engineering principles for synthetic biology, a substantial challenge is the construction of robust circuits in a noisy cellular environment. Such an environment leads to considerable intercellular variability in circuit behaviour, which can hinder functionality at the colony level. Here we engineer the synchronization of thousands of oscillating colony ‘biopixels’ over centimetre-length scales through the use of synergistic intercellular coupling involving quorum sensing within a colony and gas-phase redox signalling between colonies. We use this platform to construct a liquid crystal display (LCD)-like macroscopic clock that can be used to sense arsenic via modulation of the oscillatory period. Given the repertoire of sensing capabilities of bacteria such as Escherichia coli, the ability to coordinate their behaviour over large length scales sets the stage for the construction of low cost genetic biosensors that are capable of detecting heavy metals and pathogens in the field. 
[PDF Link]

Bacteria "biopixels".  (A) Network diagram. The luxI promoter drives expression of luxI, aiiA, ndh and sfGFP (superfolder variant of GFP) in four identical transcription modules. The quorum-sensing genes luxI and aiiA generate synchronized oscillations within a colony via AHL. The ndh gene codes for NDH-2, an enzyme that generates H2O2 vapour, which is an additional activator of the luxI promoter. H2O2 is capable of migrating between colonies and synchronizing them. (B) Conceptual design of the sensing array. AHL diffuses within colonies while H2O2 migrates between adjacent colonies through the PDMS. Arsenite-containing media is passed in through the parallel feeding channels. (C) Fluorescent image of an array of 500 E. coli biopixels containing about 2.5 million cells. Inset, bright-field and fluorescent images display a biopixel of 5,000 cells. (D) Heat map and trajectories depicting time-lapse output of 500 individual biopixels undergoing rapid synchronization. Sampling time is 2 min.  

Bacteria "biopixels".  (A) Network diagram. The luxI promoter drives expression of luxIaiiAndh and sfGFP (superfolder variant of GFP) in four identical transcription modules. The quorum-sensing genes luxI and aiiA generate synchronized oscillations within a colony via AHL. The ndh gene codes for NDH-2, an enzyme that generates H2O2 vapour, which is an additional activator of the luxI promoter. H2O2 is capable of migrating between colonies and synchronizing them. (B) Conceptual design of the sensing array. AHL diffuses within colonies while H2O2 migrates between adjacent colonies through the PDMS. Arsenite-containing media is passed in through the parallel feeding channels. (C) Fluorescent image of an array of 500 E. coli biopixels containing about 2.5 million cells. Inset, bright-field and fluorescent images display a biopixel of 5,000 cells. (D) Heat map and trajectories depicting time-lapse output of 500 individual biopixels undergoing rapid synchronization. Sampling time is 2 min.

 



In vivo gene expression dynamics of tumor-targeted bacteria

Danino, Tal, Justin Lo, Arthur Prindle, Jeff Hasty, and Sangeeta N. Bhatia. "In vivo gene expression dynamics of tumor-targeted bacteria." ACS synthetic biology 1, no. 10 (2012): 465-470.

Summary: The engineering of bacteria to controllably deliver therapeutics is an attractive application for synthetic biology. While most synthetic gene networks have been explored within microbes, there is a need for further characterization of in vivo circuit behavior in the context of applications where the host microbes are actively being investigated for efficacy and safety, such as tumor drug delivery. One major hurdle is that culture-based selective pressures are absent in vivo, leading to strain-dependent instability of plasmid-based networks over time. Here, we experimentally characterize the dynamics of in vivo plasmid instability using attenuated strains of S. typhimurium and real-time monitoring of luminescent reporters. Computational modeling described the effects of growth rate and dosage on live-imaging signals generated by internal bacterial populations. This understanding will allow us to harness the transient nature of plasmid-based networks to create tunable temporal release profiles that reduce dosage requirements and increase the safety of bacterial therapies.  [PDF Link]

Dynamics of Tumor-Targeted Bacteria. (A) S. typhimurium are injected via tail vein into nude mice and localize to subcutaneous tumors where they replicate. (B) Sequence of IVIS images for an S.Typhimurium strain with a luminescent plasmid at 10^6 dosage over the course of 60 h.  The IVIS signal rises due to rapid bacterial growth and then decays due to plasmid loss and luciferase instability.  

Dynamics of Tumor-Targeted Bacteria. (A) S. typhimurium are injected via tail vein into nude mice and localize to subcutaneous tumors where they replicate. (B) Sequence of IVIS images for an S.Typhimurium strain with a luminescent plasmid at 10^6 dosage over the course of 60 h.  The IVIS signal rises due to rapid bacterial growth and then decays due to plasmid loss and luciferase instability.

 


In-silico patterning of vascular mesenchymal cells in three dimensions

Danino, Tal, Dmitri Volfson, Sangeeta N. Bhatia, Lev Tsimring, and Jeff Hasty. "In-silico patterning of vascular mesenchymal cells in three dimensions." PloS one 6, no. 5 (2011): e20182.

Summary: Cells organize in complex three-dimensional patterns by interacting with proteins along with the surrounding extracellular matrix. This organization provides the mechanical and chemical cues that ultimately influence a cell's differentiation and function. Here, we computationally investigate the pattern formation process of vascular mesenchymal cells arising from their interaction with Bone Morphogenic Protein-2 (BMP-2) and its inhibitor, Matrix Gla Protein (MGP). Using a first-principles approach, we derive a reaction-diffusion model based on the biochemical interactions of BMP-2, MGP and cells. Simulations of the model exhibit a wide variety of three-dimensional patterns not observed in a two-dimensional analysis. We demonstrate the emergence of three types of patterns: spheres, tubes, and sheets, and show that the patterns can be tuned by modifying parameters in the model such as the degradation rates of proteins and chemotactic coefficient of cells. Our model may be useful for improved engineering of three-dimensional tissue structures as well as for understanding three dimensional microenvironments in developmental processes. [PDF Link]

3d pattern formation of vascular mesenchymal cells.  (A) Interactions between BMP-2, MGP, and cells in culture.  The binding of a BMP-2 dimer to receptors R and S stimulates production of BMP-2 and MGP, while the inding of MGP to BMP-2 outside of the cell prevents this process. (B) The derived model shows spherical spots (k = 0.2,c = 0.12), tubes (k = 0.2,c = 0.04), and sheet-like structures (k = 0.8,c = 0.04) by varying k and c. The parameters used were D = 0.005, q = 0.003, K = 0.25, B = 1.1, γ = 600 and the box length of the simulation is equivalent to 1 cm. The lowest values were made transparent for clarity while red color indicates higher cell densitywhile blue indicates low.

3d pattern formation of vascular mesenchymal cells.  (A) Interactions between BMP-2, MGP, and cells in culture.  The binding of a BMP-2 dimer to receptors R and S stimulates production of BMP-2 and MGP, while the inding of MGP to BMP-2 outside of the cell prevents this process. (B) The derived model shows spherical spots (k = 0.2,c = 0.12), tubes (k = 0.2,c = 0.04), and sheet-like structures (k = 0.8,c = 0.04) by varying k and c. The parameters used were D = 0.005, q = 0.003, K = 0.25, B = 1.1, γ = 600 and the box length of the simulation is equivalent to 1 cm. The lowest values were made transparent for clarity while red color indicates higher cell densitywhile blue indicates low.

 


Full List of Publications  CV Here