Design of insulating devices for in vitro synthetic circuits

This paper describes a synthetic in vitro genetic circuit programmed to work as an insulating device. This circuit is composed of nucleic acids, which can be designed to interact according to user defined rules, and of few proteins that perform catalytic functions. A model of the circuit is derived from first principle biochemical laws. This model is shown to exhibit time-scale separation that makes its output insensitive to downstream time varying loads. Simulation results show the circuit effectiveness and represent the starting point for future experimental testing of the device

Design of insulating devices for in vitro synthetic circuits

This paper describes a synthetic in vitro genetic circuit programmed to work as an insulating device. This circuit is composed of nucleic acids, which can be designed to interact according to user defined rules, and of few proteins that perform catalytic functions. A model of the circuit is derived from first principle biochemical laws. This model is shown to exhibit time-scale separation that makes its output insensitive to downstream time varying loads. Simulation results show the circuit effectiveness and represent the starting point for future experimental testing of the device

Timing molecular motion and production with a synthetic transcriptional clock

The realization of artificial biochemical reaction networks with unique functionality is one of the main challenges for the development of synthetic biology. Due to the reduced number of components, biochemical circuits constructed in vitro promise to be more amenable to systematic design and quantitative assessment than circuits embedded within living organisms. To make good on that promise, effective methods for composing subsystems into larger systems are needed. Here we used an artificial biochemical oscillator based on in vitro transcription and RNA degradation reactions to drive a variety of “load” processes such as the operation of a DNA-based nanomechanical device (“DNA tweezers”) or the production of a functional RNA molecule (an aptamer for malachite green). We implemented several mechanisms for coupling the load processes to the oscillator circuit and compared them based on how much the load affected the frequency and amplitude of the core oscillator, and how much of the load was effectively driven. Based on heuristic insights and computational modeling, an “insulator circuit” was developed, which strongly reduced the detrimental influence of the load on the oscillator circuit. Understanding how to design effective insulation between biochemical subsystems will be critical for the synthesis of larger and more complex systems

Reliable gene expression and assembly for synthetic biological devices in E. coli through customized promoter insulator elements and automated DNA assembly

Building reliable genetic devices in synthetic biology is still a major challenge despite the various advances that have been made in the field since its inception. In principle, genetic devices with matching input and output expression levels can be assembled from well-characterized genetic parts. In practice, a priori genetic circuit design continues to be difficult in synthetic biology due to the lack of foundational work in this area. Currently, a successful genetic device is typically created by manually building and testing many combinatorial variants of the target device and then picking the best one. While this process is slow and error-prone, as synthetic genetic devices grow in complexity, this approach also becomes unmanageable and impractical. Fluctuations in genetic context have been identified as a major cause of rational genetic circuit design failures. Promoter elements often behave unpredictably as they are moved from the context in which they were originally characterized. Thus, the ordered location of parts in a synthetic device impacts expected performance. Synthetic spacer DNA sequences have been reported to successfully buffer promoters from their neighboring DNA sequence but design rules for these sequences are lacking. I address this problem with a novel method based on a randomized insulator library. I have developed a high-throughput, flow cytometry-based screen that randomly samples from a library of 4^36 potential insulators created in a single cloning step. This method provides precise control over genetic circuit expression. I further show that insulating the promoters in a genetic NOT-gate improves circuit performance and nearly eliminates the effect of the order in which the promoters are organized in the device. This foundational work will help improve the design of reliable genetic devices in E. coli. Finally, automated DNA assembly using liquid-handling robots can help increase the speed at which combinatorial synthetic device variants are assembled. However, these systems require significant investment in optimizing the handling parameters for handling very small volumes of the various liquids in DNA assembly protocols. I have optimized and validated these liquid-handling parameters on the Tecan EVO liquid handling robotic platform. These materials have been made available to the larger community.2017-12-03T00:00:00

Fully Integrated Biochip Platforms for Advanced Healthcare

Recent advances in microelectronics and biosensors are enabling developments of innovative biochips for advanced healthcare by providing fully integrated platforms for continuous monitoring of a large set of human disease biomarkers. Continuous monitoring of several human metabolites can be addressed by using fully integrated and minimally invasive devices located in the sub-cutis, typically in the peritoneal region. This extends the techniques of continuous monitoring of glucose currently being pursued with diabetic patients. However, several issues have to be considered in order to succeed in developing fully integrated and minimally invasive implantable devices. These innovative devices require a high-degree of integration, minimal invasive surgery, long-term biocompatibility, security and privacy in data transmission, high reliability, high reproducibility, high specificity, low detection limit and high sensitivity. Recent advances in the field have already proposed possible solutions for several of these issues. The aim of the present paper is to present a broad spectrum of recent results and to propose future directions of development in order to obtain fully implantable systems for the continuous monitoring of the human metabolism in advanced healthcare applications

Tunable genetic devices through simultaneous control of transcription and translation

Abstract Synthetic genetic circuits allow us to modify the behavior of living cells. However, changes in environmental conditions and unforeseen interactions with the host cell can cause deviations from a desired function, resulting in the need for time-consuming reassembly to fix these issues. Here, we use a regulatory motif that controls transcription and translation to create genetic devices whose response functions can be dynamically tuned. This allows us, after construction, to shift the on and off states of a sensor by 4.5- and 28-fold, respectively, and modify genetic NOT and NOR logic gates to allow their transitions between states to be varied over a >6-fold range. In all cases, tuning leads to trade-offs in the fold-change and the ability to distinguish cellular states. This work lays the foundation for adaptive genetic circuits that can be tuned after their physical assembly to maintain functionality across diverse environments and design contexts

Massively parallel characterization of engineered transcript isoforms using direct RNA sequencing

Transcriptional terminators signal where transcribing RNA polymerases (RNAPs) should halt and disassociate from DNA. However, because termination is stochastic, two different forms of transcript could be produced: one ending at the terminator and the other reading through. An ability to control the abundance of these transcript isoforms would offer bioengineers a mechanism to regulate multi-gene constructs at the level of transcription. Here, we explore this possibility by repurposing terminators as ‘transcriptional valves’ that can tune the proportion of RNAP read-through. Using one-pot combinatorial DNA assembly, we iteratively construct 1780 transcriptional valves for T7 RNAP and show how nanopore-based direct RNA sequencing (dRNA-seq) can be used to characterize entire libraries of valves simultaneously at a nucleotide resolution in vitro and unravel genetic design principles to tune and insulate termination. Finally, we engineer valves for multiplexed regulation of CRISPR guide RNAs. This work provides new avenues for controlling transcription and demonstrates the benefits of long-read sequencing for exploring complex sequence-function landscapes

From Materials to Devices: (I) Ultrathin Flexible Implantable Bio-probes with Biodegradable Sacrificial Layers (II) Carrier Spin Injection and Transport in Diamond

abstract: My research has been focusing on the innovations of material and structure designs, and the development of fabrication processes of novel nanoelectronics devices. My first project addresses the long-existing challenge of implantable neural probes, where high rigidity and high flexibility for the probe need to be satisfied at the same time. Two types of probes that can be used out of the box have been demonstrated, including (1) a compact probe that spontaneously forms three-dimensional bend-up devices only after implantation, and (2) an ultra-flexible probe as thin as 2 µm attached to a small silicon shaft that can be accurately delivered into the tissue and then get fully released in situ without altering its shape and position as the support is fully retracted. This work provides a general strategy to prepare ultra-small and flexible implantable probes that allow high insertion accuracy and minimal surgical damages with best biocompatibility. My second project focuses on the injection and characterization of carrier spins in single crystal diamond based nanoscale devices. The conventional diamond-based quantum information process that exploits nitrogen vacancy centers faces a major barrier of large scale communication. Electron/hole spin in diamond devices, on the other hand, could also be a good candidate for quantum computing due to the very small spin-orbit coupling and great coherent transport length of spin. To date, there has been no demonstration of carrier spin transport in diamond. In this work, I try to answer this fundamental question of how to inject and characterize electron spins in Boron doped diamond. Nanoscale diamond devices have been fabricated to investigate this question, including Hall bar device for material characterization, and lateral spin valve for injecting spin-polarized current into a mesoscopic diamond bar and detecting induced pure spin current. The preliminary results show signatures of spin transport in heavily doped diamond films. Looking into the future, the knowledge we obtained in these two projects, including the strategy to integrate thin-film nanoelectronics devices on a flexible bio-probe configuration, and how to build spintronic devices with diamond structures, could be unified in the exploration of spin-based sensors in biological systems.Dissertation/ThesisDoctoral Dissertation Materials Science and Engineering 201