Craig R. Forest

Programming cells by multiplex genome engineering and accelerated evolution

The breadth of genomic diversity found among organisms in nature allows populations to adapt to diverse environments. However, genomic diversity is difficult to generate in the laboratory and new phenotypes do not easily arise on practical timescales. Although in vitro and directed evolution methods have created genetic variants with usefully altered phenotypes, these methods are limited to laborious and serial manipulation of single genes and are not used for parallel and continuous directed evolution of gene networks or genomes. Here, we describe multiplex automated genome engineering (MAGE) for large-scale programming and evolution of cells. MAGE simultaneously targets many locations on the chromosome for modification in a single cell or across a population of cells, thus producing combinatorial genomic diversity. Because the process is cyclical and scalable, we constructed prototype devices that automate the MAGE technology to facilitate rapid and continuous generation of a diverse set of genetic changes (mismatches, insertions, deletions). We applied MAGE to optimize the 1-deoxy-d-xylulose-5-phosphate (DXP) biosynthesis pathway in Escherichia coli to overproduce the industrially important isoprenoid lycopene. Twenty-four genetic components in the DXP pathway were modified simultaneously using a complex pool of synthetic DNA, creating over 4.3 billion combinatorial genomic variants per day. We isolated variants with more than fivefold increase in lycopene production within 3 days, a significant improvement over existing metabolic engineering techniques. Our multiplex approach embraces engineering in the context of evolution by expediting the design and evolution of organisms with new and improved properties.

Automated whole-cell patch-clamp electrophysiology of neurons in vivo

Whole-cell patch-clamp electrophysiology of neurons is a gold-standard technique for high-fidelity analysis of the biophysical mechanisms of neural computation and pathology, but it requires great skill to perform. We have developed a robot that automatically performs patch clamping in vivo, algorithmically detecting cells by analyzing the temporal sequence of electrode impedance changes. We demonstrate good yield, throughput and quality of automated intracellular recording in mouse cortex and hippocampus.

Monaco: fundamentals of molecular nano-communication networks

This article presents a branch of research where the use of molecules to encode and transmit information among nanoscale devices (nanomachines) is investigated as a bio-inspired viable solution to realize nano-communication networks. Unlike traditional technologies, molecular communication is a radically new paradigm, which demands novel solutions, including the identification of naturally existing molecular communication mechanisms, the establishment of the foundations of a molecular information theory, or the development of architectures and networking protocols for nanomachines. The tight connection of this cutting edge engineering research field with biology will ultimately enable both the bio-inspired study of molecular nanonetwork architectures and their realization with tools already available in nature. The testbed described in this article, which is based on a microfluidic device hosting intercommunicating populations of genetically engineered bacteria, is a clear example of this research direction.

Metrology of thin transparent optics using Shack-Hartmann wavefront sensing

The surface topography of thin, transparent materials is of interest in many areas. Some examples include glass substrates for computer hard disks, photomasks in the semiconductor industry, flat panel displays, and x-ray telescope optics. Some of these applications require individual foils to be manufactured with figure errors that are a small fraction of a micron over 10- to 200-mm lengths. Accurate surface metrology is essential to confirm the efficacy of manufacturing and sub- strate flattening processes. Assembly of these floppy optics is also facili- tated by such a metrology tool. We report on the design and perfor- mance of a novel deep-ultraviolet (deep-UV) Shack-Hartmann surface metrology tool developed for this purpose. The use of deep-UV wave- lengths is particularly advantageous for studying transparent substrates such as glass, which are virtually opaque to wavelengths below 260 nm. The system has a 1433143-mm 2 field of view at the object plane. Per- formance specifications include 350-mrad angular dynamic range and 0.5-mrad angular sensitivity. Surface maps over a 100 mm diam are accurate to ,17-nm rms and repeatable to 5 nm rms.

Microfluidic Thrombosis under Multiple Shear Rates and Antiplatelet Therapy Doses

The mainstay of treatment for thrombosis, the formation of occlusive platelet aggregates that often lead to heart attack and stroke, is antiplatelet therapy. Antiplatelet therapy dosing and resistance are poorly understood, leading to potential incorrect and ineffective dosing. Shear rate is also suspected to play a major role in thrombosis, but instrumentation to measure its influence has been limited by flow conditions, agonist use, and non-systematic and/or non-quantitative studies. In this work we measured occlusion times and thrombus detachment for a range of initial shear rates (500, 1500, 4000, and 10000 s−1) and therapy concentrations (0–2.4 µM for eptifibatide, 0–2 mM for acetyl-salicylic acid (ASA), 3.5–40 Units/L for heparin) using a microfluidic device. We also measured complete blood counts (CBC) and platelet activity using whole blood impedance aggregometry. Effects of shear rate and dose were analyzed using general linear models, logistic regressions, and Cox proportional hazards models. Shear rates have significant effects on thrombosis/dose-response curves for all tested therapies. ASA has little effect on high shear occlusion times, even at very high doses (up to 20 times the recommended dose). Under ASA therapy, thrombi formed at high shear rates were 4 times more prone to detachment compared to those formed under control conditions. Eptifibatide reduced occlusion when controlling for shear rate and its efficacy increased with dose concentration. In contrast, the hazard of occlusion from ASA was several orders of magnitude higher than that of eptifibatide. Our results show similar dose efficacy to our low shear measurements using whole blood aggregometry. This quantitative and statistically validated study of the effects of a wide range of shear rate and antiplatelet therapy doses on occlusive thrombosis contributes to more accurate understanding of thrombosis and to models for optimizing patient treatment.

Time-Elapse Communication: Bacterial Communication on a Microfluidic Chip

Bacterial populations housed in microfluidic environments can serve as transceivers for molecular communication, but the data-rates are extremely low (e.g., 10-5 bits per second.). In this work, genetically engineered Escherichia coli bacteria were maintained in a microfluidic device where their response to a chemical stimulus was examined over time. The bacteria serve as a communication receiver where a simple modulation such as on-off keying (OOK) is achievable, although it suffers from very poor data-rates. We explore an alternative communication strategy called time-elapse communication (TEC) that uses the time period between signals to encode information. We identify the limitations of TEC under practical non-zero error conditions and propose an advanced communication strategy called smart time-elapse communication (TEC-SMART) that achieves over a 10x improvement in data-rate over OOK. We derive the capacity of TEC and provide a theoretical maximum data-rate that can be achieved.

Stress Enables Reinforcement-Elicited Serotonergic Consolidation of Fear Memory

BACKGROUND Prior exposure to stress is a risk factor for developing posttraumatic stress disorder (PTSD) in response to trauma, yet the mechanisms by which this occurs are unclear. Using a rodent model of stress-based susceptibility to PTSD, we investigated the role of serotonin in this phenomenon. METHODS Adult mice were exposed to repeated immobilization stress or handling, and the role of serotonin in subsequent fear learning was assessed using pharmacologic manipulation and western blot detection of serotonin receptors, measurements of serotonin, high-speed optogenetic silencing, and behavior. RESULTS Both dorsal raphe serotonergic activity during aversive reinforcement and amygdala serotonin 2C receptor (5-HT2CR) activity during memory consolidation were necessary for stress enhancement of fear memory, but neither process affected fear memory in unstressed mice. Additionally, prior stress increased amygdala sensitivity to serotonin by promoting surface expression of 5-HT2CR without affecting tissue levels of serotonin in the amygdala. We also showed that the serotonin that drives stress enhancement of associative cued fear memory can arise from paired or unpaired footshock, an effect not predicted by theoretical models of associative learning. CONCLUSIONS Stress bolsters the consequences of aversive reinforcement, not by simply enhancing the neurobiological signals used to encode fear in unstressed animals, but rather by engaging distinct mechanistic pathways. These results reveal that predictions from classical associative learning models do not always hold for stressed animals and suggest that 5-HT2CR blockade may represent a promising therapeutic target for psychiatric disorders characterized by excessive fear responses such as that observed in PTSD.

Disposable platform provides visual and color-based point-of-care anemia self-testing

BACKGROUND Anemia, or low blood hemoglobin (Hgb) levels, afflicts 2 billion people worldwide. Currently, Hgb levels are typically measured from blood samples using hematology analyzers, which are housed in hospitals, clinics, or commercial laboratories and require skilled technicians to operate. A reliable, inexpensive point-of-care (POC) Hgb test would enable cost-effective anemia screening and chronically anemic patients to self-monitor their disease. We present a rapid, stand-alone, and disposable POC anemia test that, via a single drop of blood, outputs color-based visual results that correlate with Hgb levels. METHODS We tested blood from 238 pediatric and adult patients with anemia of varying degrees and etiologies and compared hematology analyzer Hgb levels with POC Hgb levels, which were estimated via visual interpretation using a color scale and an optional smartphone app for automated analysis. RESULTS POC Hgb levels correlated with hematology analyzer Hgb levels (r = 0.864 and r = 0.856 for visual interpretation and smartphone app, respectively), and both POC test methods yielded comparable sensitivity and specificity for detecting any anemia (n = 178) (<11 g/dl) (sensitivity: 90.2% and 91.1%, specificity: 83.7% and 79.2%, respectively) and severe anemia (n = 10) (<7 g/dl) (sensitivity: 90.0% and 100%, specificity: 94.6% and 93.9%, respectively). CONCLUSIONS These results demonstrate the feasibility of this POC color-based diagnostic test for self-screening/self-monitoring of anemia. TRIAL REGISTRATION Not applicable. FUNDING This work was funded by the FDA-funded Atlantic Pediatric Device Consortium, the Georgia Research Alliance, Childrens Healthcare of Atlanta, the Georgia Center of Innovation for Manufacturing, and the InVenture Prize and Ideas to Serve competitions at the Georgia Institute of Technology.

Rapid, quantitative, reverse transcription PCR in a polymer microfluidicchip

Quantitative PCR (qPCR) techniques have become invaluable, high-throughput tools to study gene expression. However, the need to measure gene expression patterns quickly and affordably, useful for applications such as stem cell biomanufacturing requiring real-time observation and control, has not been adequately met by rapid qPCR instrumentation to date. We report a reverse transcription, microfluidic qPCR system and its application to DNA and RNA amplification measurement. In the system, an environmental control fixture provides mechanical and thermal repeatability for an infrared laser to achieve both accurate and precise open-loop temperature control of 1 μl reaction volumes in a low-cost polymer microfluidic chip with concurrent fluorescence imaging. We have used this system to amplify serial dilutions of λ-phage DNA (10(5)-10(7) starting copies) and RNA transcripts from the GAPDH housekeeping gene (5.45 ng total mouse embryonic stem cell RNA) and measured associated standard curves, efficiency (57%), repeatability (~1 cycle threshold), melting curves, and specificity. This microfluidic qRT-PCR system offers a practical approach to rapid analysis (~1 h), combining the cost benefits of small reagent volumes with the simplicity of disposable polymer microchips and easy setup.

Modeling and validation of autoinducer-mediated bacterial gene expression in microfluidic environments

Biosensors exploiting communication within genetically engineered bacteria are becoming increasingly important for monitoring environmental changes. Currently, there are a variety of mathematical models for understanding and predicting how genetically engineered bacteria respond to molecular stimuli in these environments, but as sensors have miniaturized towards microfluidics and are subjected to complex time-varying inputs, the shortcomings of these models have become apparent. The effects of microfluidic environments such as low oxygen concentration, increased biofilm encapsulation, diffusion limited molecular distribution, and higher population densities strongly affect rate constants for gene expression not accounted for in previous models. We report a mathematical model that accurately predicts the biological response of the autoinducer N-acyl homoserine lactone-mediated green fluorescent protein expression in reporter bacteria in microfluidic environments by accommodating these rate constants. This generalized mass action model considers a chain of biomolecular events from input autoinducer chemical to fluorescent protein expression through a series of six chemical species. We have validated this model against experimental data from our own apparatus as well as prior published experimental results. Results indicate accurate prediction of dynamics (e.g., 14% peak time error from a pulse input) and with reduced mean-squared error with pulse or step inputs for a range of concentrations (10 μM-30 μM). This model can help advance the design of genetically engineered bacteria sensors and molecular communication devices.