Improving Reproducibility in Synthetic Biology

Synthetic biology holds great promise to deliver transformative technologies to the world in the coming years. However, several challenges still remain to be addressed before it can deliver on its promises. One of the most important issues to address is the lack of reproducibility within research of the life sciences. This problem is beginning to be recognised by the community and solutions are being developed to tackle the problem. The recent emergence of automated facilities that are open for use by researchers (such as biofoundries and cloud labs) may be one of the ways that synthetic biologists can improve the quality and reproducibility of their work. In this perspective article, we outline these and some of the other technologies that are currently being developed which we believe may help to transform how synthetic biologists approach their research activities

Frontiers in microfluidics, a teaching resource review

This is a literature teaching resource review for biologically inspired microfluidics courses or exploring the diverse applications of microfluidics. The structure is around key papers and model organisms. While courses gradually change over time, a focus remains on understanding how microfluidics has developed as well as what it can and cannot do for researchers. As a primary starting point, we cover micro-fluid mechanics principles and microfabrication of devices. A variety of applications are discussed using model prokaryotic and eukaryotic organisms from the set of bacteria (Escherichia coli), trypanosomes (Trypanosoma brucei), yeast (Saccharomyces cerevisiae), slime molds (Physarum polycephalum), worms (Caenorhabditis elegans), flies (Drosophila melangoster), plants (Arabidopsis thaliana), and mouse immune cells (Mus musculus). Other engineering and biochemical methods discussed include biomimetics, organ on a chip, inkjet, droplet microfluidics, biotic games, and diagnostics. While we have not yet reached the end-all lab on a chip, microfluidics can still be used effectively for specific applications

Engineering Micro/Nanorobots Towards Biomedical Applications: Targeted Delivery, Surgery and Detoxification

Over the past decade, micro/nanomotors have emerged as novel and versatile nanotools demonstrating considerable promise for many environmental and biological applications. This dissertation aims to demonstrate unique advantages of micro/nanorobot platforms.The first theme focuses on developing the use of ultrasound propelled nanostructures for diverse applications, including “on-the-move” capture and the transport of multiple cargoes and internalization and movement inside live MCF-7 cancer cells.The second theme explores the use of acoustically triggered microcannons. This principle was tested towards mechanochemical blood clot degradation and enhancing drug permeability through the epidermis.The third theme describes the use of biohybrid robotics systems for detoxification and decontamination of environmental pollutants, including bacteria (E. coli), nerve agent (methyl paraoxon) and heavy metal ions (Cd and Pb) from aqueous solutions.In the not-so-distant future, Micro/nanorobots could serve as robust and versatile platform for potentially improving medical diagnosis and treatment

The Experiment Data Depot: A Web-Based Software Tool for Biological Experimental Data Storage, Sharing, and Visualization

Although recent advances in synthetic biology allow us to produce biological designs more efficiently than ever, our ability to predict the end result of these designs is still nascent. Predictive models require large amounts of high-quality data to be parametrized and tested, which are not generally available. Here, we present the Experiment Data Depot (EDD), an online tool designed as a repository of experimental data and metadata. EDD provides a convenient way to upload a variety of data types, visualize these data, and export them in a standardized fashion for use with predictive algorithms. In this paper, we describe EDD and showcase its utility for three different use cases: storage of characterized synthetic biology parts, leveraging proteomics data to improve biofuel yield, and the use of extracellular metabolite concentrations to predict intracellular metabolic fluxes

Engineering 4D regulation toolbox to control spatiotemporal cell-free reconstitution

Bottom-up reconstituting well-characterized functional molecular entities, parts and modules towards a synthetic cell will give new insights into the general mechanisms and molecular origins of life. However, a remaining central challenge is how to organize cellular processes spatiotemporally from their component parts in vitro. To this end, we developed a 4D regulation toolbox to facilitate a bottom-up reconstitution in both time and space. The spatiotemporal regulation of the 4D toolbox covers the aspects from dynamic gene transcription & translation, reversible protein interaction, spatially protein positioning, sequential protein assembly, extends to defining geometrical membrane boundaries and mimicking cellular anisotropic microenvironment. Firstly, we developed a thermo-genetic regulation toolbox based on synthetic RNA thermometers, for temporally controlling protein expression in vitro. We validated RNA thermometers from in vivo to in vitro and tuned RNA thermometers through utilizing cell free protein synthesis system. Then we generated the thermo-sensitive protocell by encapsulating thermo-regulated transcription and translation machine in water-in-oil droplets. With the temperature sensing devices, the protocells can be operated with logic AND gates, differentially processing temperature stimuli into biological signals. Secondly, we engineered the PhyB-PIF6 system to spatiotemporally target proteins by light onto model membranes and thus sequentially guide protein pattern formation and structural assembly in vitro from the bottom up. We show that complex micrometer-sized protein patterns can be printed on timescales of seconds. Moreover, when printing self-assembling proteins such as the bacterial cytoskeleton protein FtsZ, the targeted assembly into filaments and large-scale structures such as artificial rings can be accomplished. To develop an artificial anisotropic membrane environment, we introduced a 3D printed protein hydrogel device to induce pH-stimulated reversible shape changes in trapped vesicles. Deformations towards unusual quadratic or triangular shapes can be accomplished. Mechanical force induced by the cages to phase-separated membrane vesicles can lead to spontaneous shape deformations. Moreover, the shape-tunable vesicle provides a spatially well-defined microenvironment for reconstituting shape-dependent protein systems, such as reaction-diffusion system that request explicitly non-spherical geometries. By taking advantages of the 3D printed hydrogel, we programmably engineered contractible scaffolds for actin-myosin motor reconstitution in 3D space. Nanoscale actomyosin motor as a bio-actuator could generate, transmit active contraction and then drive large-scale shape-morphing of complex 3D hydrogel scaffolds. In summary, by developing the spatiotemporal toolbox, this thesis introduces a promising step towards establishing bottom-up reconstitution in space and time, which could also guide future efforts in hierarchically building up the next level of complexity towards a minimal cell

Part I: Polyrotaxanes as MRI Contrast Agents and NPC Therapeutics. Part II: Development of an Analytic-Directed Synthesis System

The work described in this dissertation is separated into two parts. Part I describes the development of cyclodextrin-based polyrotaxanes (PRs) as both MRI contrast agents and as potential therapeutics for Niemann-Pick Type C (NPC) disease. Polyrotaxanes are a class of supramolecular materials which are constructed through the non-covalent threading of macrocyclic molecules onto a polymer core that are retained by the covalent attachment of bulky molecules to the ends of the polymer as end-caps. This unique architecture provides a rigid and rod-like morphology that imparts attractive biological and mechanical properties compared to other nanomaterials used in biological applications. In our case, we have developed PRs constructed from cyclodextrin derivatives threaded onto Pluronic triblock copolymer cores. Their utility as MRI contrast agents as well as NPC therapeutics is reported in Chapter 2. The development of a greener and more scalable synthesis of PRs using a solid-state approach is described in Chapter 3. Part II of this thesis describes the development of a multi-scale automated synthesis system guided by mass spectrometry. Continuous-flow and high throughput experimentation are two technologies which are rapidly changing the way modern synthesis is conducted. Continuous-flow reactors allow for a level of control over reaction parameters that is unparalleled relative to batch systems, thus providing the capability to execute reactions faster, greener, and safer than ever before. Chapter 4 describes the development of a continuous-flow platform for the synthesis of diphenhydramine which includes a continuous-flow reactor, on-line mass spectrometric monitoring, and continuous-flow crystallization. Also integrated in this effort is the use of accelerated reactions in microdroplets to guide continuous-flow synthesis. High throughput experimentation technologies have already transformed the way in which biological assays are conducted and are rapidly making their way into the organic synthetic process. The development of a high throughput reaction screening approach based on desorption electrospray ionization mass spectrometry (DESI-MS) is described in Chapters 5 and 6. This system, capable of executing and analyzing thousands of reactions per hour, has the potential to dramatically accelerate the process of reaction optimization and discovery

An aptamer-based sensing platform for luteinising hormone pulsatility measurement

Normal fertility in human involves highly orchestrated communication across the hypothalamic-pituitary-gonadal (HPG) axis. The pulsatile release of Luteinising Hormone (LH) is a critical element for downstream regulation of sex steroid hormone synthesis and the production of mature eggs. Changes in LH pulsatile pattern have been linked to hypothalamic dysfunction, resulting in multiple reproductive and growth disorders including Polycystic Ovary Syndrome (PCOS), Hypothalamic Amenorrhea (HA), and delayed/precocious puberty. Therefore, assessing the pulsatility of LH is important not only for academic investigation of infertility, but also for clinical decisions and monitoring of treatment. However, there is currently no clinically available tool for measuring human LH pulsatility. The immunoassay system is expensive and requires large volumes of patient blood, limiting its application for LH pulsatility monitoring. In this thesis, I propose a novel method using aptamer-enabled sensing technology to develop a device platform to measure LH pulsatility. I first generated a novel aptamer binding molecule against LH by a nitrocellulose membrane-based in vitro selection then characterised its high affinity and specific binding properties by multiple biophysical/chemical methods. I then developed a sensitive electrochemical-based detection method using this aptamer. The principal mechanism is that structure switching upon binding is associated with the electron transfer rate changes of the MB redox label. I then customised this assay to numerous device platforms under our rapid prototyping strategy including 96 well automated platform, continuous sensing platform and chip-based multiple electrode platform. The best-performing device was found to be the AELECAP (Automated ELEctroChemical Aptamer Platform) – a 96-well plate based automatic micro-wire sensing platform capable of measuring a series of low volume luteinising hormone within a short time. Clinical samples were evaluated using AELECAP. A series of clinical samples were measured including LH pulsatility profile of menopause female (high LH amplitude), normal female/male (normal LH amplitude) and female with hypothalamic amenorrhea (no LH pulsatility). Total patient numbers were 12 of each type, with 50 blood samples collected every 10 mins in 8 hours. Results showed that the system can distinguish LH pulsatile pattern among the cohorts and pulsatility profiles were consistent with the result measured by clinical assays. AELECAP shows high potential as a novel approach for clinical aptamer-based sensing. AELECAP competes with current automated immunometric assays system with lower costs, lower reagent use, and a simpler setup. There is potential for this approach to be further developed as a tool for infertility research and to assist clinicians in personalised treatment with hormonal therapy.Open Acces

Engineering 4D regulation toolbox to control spatiotemporal cell-free reconstitution

Bottom-up reconstituting well-characterized functional molecular entities, parts and modules towards a synthetic cell will give new insights into the general mechanisms and molecular origins of life. However, a remaining central challenge is how to organize cellular processes spatiotemporally from their component parts in vitro. To this end, we developed a 4D regulation toolbox to facilitate a bottom-up reconstitution in both time and space. The spatiotemporal regulation of the 4D toolbox covers the aspects from dynamic gene transcription & translation, reversible protein interaction, spatially protein positioning, sequential protein assembly, extends to defining geometrical membrane boundaries and mimicking cellular anisotropic microenvironment. Firstly, we developed a thermo-genetic regulation toolbox based on synthetic RNA thermometers, for temporally controlling protein expression in vitro. We validated RNA thermometers from in vivo to in vitro and tuned RNA thermometers through utilizing cell free protein synthesis system. Then we generated the thermo-sensitive protocell by encapsulating thermo-regulated transcription and translation machine in water-in-oil droplets. With the temperature sensing devices, the protocells can be operated with logic AND gates, differentially processing temperature stimuli into biological signals. Secondly, we engineered the PhyB-PIF6 system to spatiotemporally target proteins by light onto model membranes and thus sequentially guide protein pattern formation and structural assembly in vitro from the bottom up. We show that complex micrometer-sized protein patterns can be printed on timescales of seconds. Moreover, when printing self-assembling proteins such as the bacterial cytoskeleton protein FtsZ, the targeted assembly into filaments and large-scale structures such as artificial rings can be accomplished. To develop an artificial anisotropic membrane environment, we introduced a 3D printed protein hydrogel device to induce pH-stimulated reversible shape changes in trapped vesicles. Deformations towards unusual quadratic or triangular shapes can be accomplished. Mechanical force induced by the cages to phase-separated membrane vesicles can lead to spontaneous shape deformations. Moreover, the shape-tunable vesicle provides a spatially well-defined microenvironment for reconstituting shape-dependent protein systems, such as reaction-diffusion system that request explicitly non-spherical geometries. By taking advantages of the 3D printed hydrogel, we programmably engineered contractible scaffolds for actin-myosin motor reconstitution in 3D space. Nanoscale actomyosin motor as a bio-actuator could generate, transmit active contraction and then drive large-scale shape-morphing of complex 3D hydrogel scaffolds. In summary, by developing the spatiotemporal toolbox, this thesis introduces a promising step towards establishing bottom-up reconstitution in space and time, which could also guide future efforts in hierarchically building up the next level of complexity towards a minimal cell