A single-input binary counting module based on serine integrase site-specific recombination

A device that counts and records the number of events experienced by an individual cell could have many uses in experimental biology and biotechnology. Here, we report a DNA-based ‘latch’ that switches between two states upon each exposure to a repeated stimulus. The key component of the latch is a DNA segment whose orientation is inverted by the actions of ϕC31 integrase and its recombination directionality factor (RDF). Integrase expression is regulated by an external input, while RDF expression is controlled by the state of the latch, such that the orientation of the invertible segment switches efficiently each time the device receives an input pulse. Recombination occurs over a time scale of minutes after initiation of integrase expression. The latch requires a delay circuit, implemented with a transcriptional repressor expressed in only one state, to ensure that each input pulse results in only one inversion of the DNA segment. Development and optimization of the latch in living cells was driven by mathematical modelling of the recombination reactions and gene expression regulated by the switch. We discuss how N latches built with orthogonal site-specific recombination systems could be chained together to form a binary ripple counter that could count to 2N − 1

Developmental, Physiological, and Transcriptomic Analyses of Neurons involved in the Generation of Mammalian Breathing

Breathing is a rhythmic motor behavior with obvious physiological importance: breathing movements are essential for respiration, which sustains homeostasis and life itself in a wide array of animals including humans and all mammals. The breathing rhythm is produced by interneurons of the brainstem preBötzinger complex (preBötC) whose progenitors express the transcription factor Dbx1. However, the cellular and synaptic neural mechanisms underlying respiratory rhythmogenesis remain unclear. The first chapter of this dissertation examines a Dbx1 transgenic mouse line often exploited to study the neural control of breathing. It emphasizes the cellular fate of progenitors that express Dbx1 at different times during development. I couple tamoxifen-inducible Dbx1 Cre-driver mice with Cre-dependent reporters, then show that Dbx1-expressing progenitors give rise to preBötC neurons and glia. Further, I quantify the temporal assemblage of Dbx1 neurons and glia in the preBötC and provide practical guidance on breeding and tamoxifen administration strategies to bias reporter protein expression toward neurons (or glia), which can aid researchers in targeting studies to unravel their functions in respiratory neurobiology. The second chapter of this dissertation exploits the mouse model characterized in the first chapter and then focuses on mechanisms of respiratory rhythmogenesis. The breathing cycle consists of inspiratory and expiratory phases. Inspiratory burst-initiation and burst-sustaining mechanisms have been investigated by many groups. Here, I specifically investigate the role of short-term synaptic depression in burst termination and the inspiratory-expiratory phase transition using rhythmically active medullary slice preparations from Dbx1 Cre-driver mice coupled with channelrhodopsin reporters. I demonstrate the existence of a post- inspiratory refractory period that precludes light-evoked bursts in channelrhodopsin-expressing Dbx1-derived preBötC neurons. I show that postsynaptic factors cannot account for the refractory period, and that presynaptic vesicle depletion most likely underlies the refractory period. The third chapter of this dissertation focuses on transcriptomic analysis of Dbx1 preBötC neurons, and differences in gene expression between Dbx1-derived and non- Dbx1-derived preBötC neurons. I analyze and quantify the expression of over 20,000 genes, and make the raw data publicly available for further analysis. I argue that this full transcriptome approach will enable our research group (and others) to devise physiological studies that target specific subunits and isoforms of ion channels and integral membrane proteins to examine the role(s) of Dxb1- derived neurons and glia at the molecular level of breathing behavior. In addition to predictable gene candidates (such as ion channels, etc) this transcriptome analysis delivers unanticipated novel gene candidates that can be investigated in future respiratory physiology experiments. Knowing the site (preBötC) and cell class (Dbx1) at the point of origin of respiration, this dissertation provides tools and specific investigations that advance understanding of the neural mechanisms of breathing

THE ROLE OF GENE EXPRESSION NOISE IN MAMMALIAN CELL SURVIVAL

Drug resistance and metastasis remain obstacles to effective cancer treatment. A major challenge contributing to this problem is cellular heterogeneity. Even in the same environment, cells with identical genomes can display cell-to-cell differences in gene expression, also known as gene expression noise. Gene expression noise can vary in magnitude in a population or in fluctuation time scales, which is influenced by gene regulatory networks. Currently, it is unclear how gene expression noise from gene regulatory networks contributes to drug survival outcomes in mammalian cells. An isogenic cell line with a noise-modulating genetic system tuned to the same mean is required. Additionally, how modulating endogenous mean gene expression and noise in living cells influences pro-survival metastatic state transitions remains unanswered. To address these knowledge gaps, I implemented an exogenous synthetic biology approach to control noise for the drug resistance gene PuroR in drug survival while complementing with endogenous expression measurements of the pro-metastatic gene BACH1 as a correlate for metastatic survival. For exogenous control, I developed synthetic gene circuits in Chinese Hamster Ovary (CHO) cells based on positive and negative feedback that tune noise for PuroR at identical mean expression. At a decoupled noise point, isogenic cells were treated with various Puromycin concentrations. Evolution experiments revealed that noise hurts drug resistance during low drug dosage while facilitating resistance at a high Puromycin concentration. Drug adaptation for the low-noise gene circuit relied on intra-circuit mutations while the high-noise circuit did not and became re-sensitized to drug after removing circuit induction. To implement the endogenous approach, I tagged the endogenous BACH1 gene with the mCherry fluorescent protein in six HEK293 clones. Molecular perturbations such as serum starvation and long-term hemin treatment altered mean fluorescence in at least one clone. Additionally, monitoring migration after cell wounding revealed increased non-uniform fluorescence at the wound edge. The increased mean fluorescence for the potentially bistable HEK293 clone 2C10 during hemin treatment may reflect altered BACH1 state transitions. Overall, noise enhanced the probability of cells to reach an expression level that confers survival during drug treatment while hemin perturbations may induce a pro-survival metastatic transition via BACH1 expression

On the role of parvalbumin interneurons in neuronal network activity in the prefrontal cortex

The prefrontal cortex (PFC) is an area important for executive functions, the initiation and temporal organization of goal-directed behavior, as well as social behaviors. Inhibitory interneurons expressing parvalbumin (PV) have a vital role in modulating PFC circuit plasticity and output, as inhibition by PV interneurons on excitatory pyramidal neurons regulates the excitability of the network. Thus, dysfunctions of prefrontal PV interneurons are implicated in the pathophysiology of a range of PFC-dependent neuropsychiatric disorders characterized by excitation and inhibition (E/I) imbalance and impaired gamma oscillations. In particular, the hypofunction of receptors important for neurotransmission and regulating cellular functions, such as the N-methyl-D-aspartate receptors (NMDARs) and the tyrosine receptor kinase B (trkB), has been implicated in PV dysfunction. Notably, this hypofunction is known to impair the normal development of PV interneurons. However, it can also affect adult brain activity. The effects of altered receptors on PV interneurons are multiple, from impaired morphological connectivity to disruption of intrinsic activity, but have not yet been fully characterized. Moreover, the effects of deficits of PV neuron-mediated inhibition on neuronal network activity are complex, involved with compensatory mechanisms, and not fully understood either. For instance, the E/I imbalance due to PV inhibition has been suggested to functionally disrupt the cortex, which can be observed through an abnormal increase in broadband gamma activity. But as the synchronous activity of cortical PV interneurons is necessary for the generation of cortical gamma oscillations, it is paradoxical that deficient PV inhibition is associated with increased broadband gamma power. This thesis aims to examine the role of PV interneurons in shaping neuronal network activity in the mouse PFC by investigating the microscopic to macroscopic functional effects of disrupting receptors necessary for the proper activity of PV interneurons. In paper I, we observed that the increase of broadband gamma power due to NMDAR hypofunction in PV neurons is associated with asynchronies of network activity, confirming that dysfunction of neuronal inhibition can cause desynchronization at multiple time scales (affecting entrainment of spikes by the LFP, as well as cross-frequency coupling and brain states fragmentation). In Paper II, we prompted and analyzed the rippling effect of PV dysfunction in the adult PFC by expressing a dominant-negative trkB receptor specifically in PV interneurons. Despite avoiding interfering with the development of the brain, we found pronounced morphological and functional alterations in the targeted PV interneurons. These changes were associated with unusual aggressive behavior coupled with gamma-band alterations and a decreased modulation of prefrontal excitatory neuronal populations by PV interneurons. Thus, the work presented in this thesis furthers our understanding of the role of PV function in PFC circuitry, particularly of two receptors that are central to the role of PV interneurons in coordinating local circuit activity. A better understanding of the potential mechanisms that could explain the neuronal changes seen in individuals with neuropsychiatric dysfunctions could lead to using gamma oscillations or BDNF-trkB levels as biomarkers in psychiatric disorders. It also presents possibilities for potential treatments designed around reestablishing E/I balance by modifying receptor levels in particular cell types

Modular construction of mammalian gene circuits using TALE transcriptional repressors

An important goal of synthetic biology is the rational design and predictable implementation of synthetic gene circuits using standardized and interchangeable parts. However, engineering of complex circuits in mammalian cells is currently limited by the availability of well-characterized and orthogonal transcriptional repressors. Here, we introduce a library of 26 reversible transcription activator–like effector repressors (TALERs) that bind newly designed hybrid promoters and exert transcriptional repression through steric hindrance of key transcriptional initiation elements. We demonstrate that using the input-output transfer curves of our TALERs enables accurate prediction of the behavior of modularly assembled TALER cascade and switch circuits. We also show that TALER switches using feedback regulation exhibit improved accuracy for microRNA-based HeLa cancer cell classification versus HEK293 cells. Our TALER library is a valuable toolkit for modular engineering of synthetic circuits, enabling programmable manipulation of mammalian cells and helping elucidate design principles of coupled transcriptional and microRNA-mediated post-transcriptional regulation.National Institutes of Health (U.S.) (Grant 5R01CA155320-04)National Institutes of Health (U.S.) (Grant P50GM098792)National Institutes of Health (U.S.) (Grant 1R01CA173712-01

Investigating the Role of Phox2B-expressing Glutamatergic Parafacial Zone Neurons in Sleep Wake Control

Inhibitory GABAergic neurons in the parafacial zone (PZGABA) are essential for slow wave sleep (SWS). Since existing literature about the heterogenous population of PZ neurons is lacking, questions remain regarding the non-GABAergic sleep active PZ neurons. This study seeks to determine if glutamatergic PZ neurons expressing the transcription factor Phox2B (PZPhox2B) participate in sleep-wake control. Phox2B-IRES-Cre mice received injections of adeno-associated virus containing Cre-dependent diphtheria toxin subunit A (DTA) DNA into the PZ (PZPhox2B-DTA). Analysis of injection sites revealed transfection covering the PZ and the locus coeruleus, also known to express Phox2B. We recorded the sleep-wake cycle of PZPhox2B-DTA mice and compared them with control mice, analyzing their sleep-wake quantity, fragmentation, and power spectral distribution. We found total amounts and cortical power for wakefulness, SWS, and REM sleep of PZPhox2B-DTA mice were unaffected. There was fragmentation in wakefulness during the active period for PZPhox2B-DTA mice, seen as a significant reduction in the amount of time and number of episodes spent in the longest bout; however, wakefulness during the rest period was not significantly altered. No significant change was found in the bout numbers and amounts for SWS and REM sleep of PZPhox2B-DTA mice. I was unable to confirm targeted ablation of PZPhox2B-DTA neurons due to a lack of reliable antibody staining. Therefore, it remains possible that ablation of PZPhox2B neurons was incomplete and the wakeful fragmentation is due to neuronal ablation outside of the PZ, such as in the neighboring LC

Développement d'un système de différenciation modulable par la lumière pour la création et le contrôle de consortiums microbiens dans S. cerevisiae, sa caractérisation en cellule unique pour le développement de modèles prédictifs, et son utilisation pour l'expression hétérologue

Les consortiums microbiens artificiels cherchent à exploiter la division du travail pour optimiser des fonctions et possèdent un immense potentiel pour la bioproduction. Les approches de co-culture, le mode préférentiel pour générer des consortiums, restent limitées dans leur capacité à donner naissance à des consortiums stables ayant des compositions précisément ajustées. J'ai développé ici un système de différenciation artificielle dans la levure boulanger capable de générer à partir d'une seule souche des consortiums microbiens stables avec des fonctionnalités choisies et ayant une composition définie par l'utilisateur dans l'espace et dans le temps, grâce à une modification génétique pilotée par optogénétique. Grâce à une dynamique rapide, reproductible et ajustable par la lumière, mon système permet un contrôle dynamique de la composition des consortiums dans des cultures continues pendant de longues périodes. Je démontre également que notre système peut être étendu de manière simple pour donner naissance à des consortiums avec de multiples sous-populations. Cette stratégie de différenciation artificielle établit un nouveau paradigme pour la création de consortiums microbiens complexes qui sont simples à mettre en oeuvre, contrôlables avec précision et polyvalents à utiliser.En plus de cela, j'ai caractérisé le système au niveau de la cellule unique dans différents contextes en changeant la structure du bruit du facteur de transcription optogénétique qui induit la différenciation. J'ai découvert que le changement de la structure du bruit introduisait un couplage complexe entre les niveaux de la population de cellule et des cellules individuelles, qui ne peut être prédit par un simple modèle d'équations différentielles ordinaires. L'utilisation d'un modèle stochastique bien caractérisé a permis de rétablir la prévisibilité.Enfin, j'ai couplé le système de différenciation avec un system d'arrêt de croissance et de bioproduction de sorte que les cellules différenciées arrêtent de croître et commencent à produire une protéine d'intérêt. J'ai comparé l'efficacité de l'approche basée sur la différenciation avec des équivalents constitutifs et inductibles. J'ai constaté que la production n'était pas monotone par rapport à la fraction de différenciation mais qu'elle pouvait surpasser l'expression induite par un promoteur constitutif fort.Artificial microbial consortia seek to leverage division-of-labour to optimize function and possess immense potential for bioproduction. Co-culturing approaches, the preferred mode of generating a consortium, remain limited in their ability to give rise to stable consortia having finely tuned compositions. Here, I developed an artificial differentiation system in budding yeast capable of generating stable microbial consortia with custom functionalities from a single strain at user-defined composition in space and in time based on optogenetically-driven genetic rewiring. Owing to fast, reproducible, and light-tunable dynamics, my system enables dynamic control of consortia composition in continuous cultures for extended periods independently of the cell density. I further demonstrate that our system can be extended in a straightforward manner to give rise to consortia with multiple subpopulations. This artificial differentiation strategy establishes a novel paradigm for the creation of complex microbial consortia that are simple to implement, precisely controllable, and versatile to use.In addition to this, I characterized the system at the single cell level in different genetic contexts by changing the noise structure of the optogenetic transcription factor that drives differentiation. I found that changing the noise structure introduced complex coupling between the population and the single cell level, which cannot be predicted by a simple population model. A stochastic model of differentiation composed in a stochastic model of plasmid fluctuations not only restored predictability, but revealed mechanistic insights into the functioning of the system. The latter was exploited to demonstrate control of expression of a constitutively expressed gene (proxy for plasmid copy number).Lastly, I coupled the differentiation system with a growth arrest and production module such that differentiated cells stop growing and start producing a protein of interest. Growth arrest was effected via hijacking of the mating pheromone pathway and production was carried out by an orthogonal transcription factor. I developed a light inducible reference to assess the increase in production upon growth arrest. Comparing the efficiency of the differentiation-based approach with constitutive and inducible counterparts, I found that production was non-monotonic with respect to differentiation fraction and could outcompete constitutive expression. However, production did not increase upon growth arrest

Top-down and bottom-up control of drug-induced sleep and anaesthesia

In recent decades, research has unravelled fascinating detail about the molecular mechanisms underpinning pharmacologic loss of consciousness (LOC). However, the systems-level mechanisms are far less clear. Recent genetic approaches, however, enable unprecedented dissection on neural pathways, and they are paving a way for this line of research. The focus of this thesis is to investigate the neuroanatomical substrates of commonly used drugs which reversibly render us unconscious. Zolpidem is a positive allosteric modulator (PAM) of the GABAA receptor which binds to the benzodiazepine (BZ) site. Because zolpidem binds 1-3,,2 containing GABAA receptors, which are widespread, it acts virtually everywhere. We do not know if zolpidem causes sleep by enhancing GABAergic inhibition throughout the entire brain, or if the therapeutic sleep-inducing property depends upon specific brain circuitry. 2I77 mice are devoid of zolpidem-sensitivity. But, zolpidem-sensitivity can be restored selectively in brain regions, enabling dissection of the circuitry involved in zolpidem’s effect. To isolate the therapeutic effect of zolpidem we deleted GABAA-2I77-subunits and replaced them with GABAA-2F77-subunits in HDC neurons or frontal-cortex in isolation. We were able to selectively restore zolpidem-sensitivity in target neurons. This conferred zolpidem-enhanced IPSCs locally. Compared with wild-type mice and zolpidem-insensitive 2I77lox mice, we found that GABAA-2F77 receptors in either HDC-neurons or frontal cortex alone were enough to rescue the majority of zolpidem-mediated sleep. The response in HDC-2F77 mice was similar to that of an H1-receptor antagonist. By producing a null effect in a negative-control area – the superior colliculus – we show that HDC neurons and the frontal cortex are both substrates involved in zolpidem-mediated sleep. We also investigated the role of synaptic-inhibition onto corticothalamic-neurons in anaesthetic-induced LOC and sleep-wake. To do this, we genetically ablated 2-subunits from layer-6 corticothalamic-cells by crossing Ntsr1-Cre mice with GABAA-2I77lox mice. We found this reduced isoflurane sensitivity, but left sleep-wake behaviours virtually unaffected.Open Acces

In vivo and in vitro characterization and application of tyrosine recombinases for metabolic engineering

Site-specific recombinases are a family of DNA-modifying enzymes that can recognize short specific DNA sequences and drive recombination between them to rearrange DNA fragments which results in excision, integration or inversion. Here I describe in vivo application of the recombinases for chassis optimization for heterologous pathway expression in synthetic yeast and in vitro application of recombinases for gene expression optimization. For in vivo application, two recombination systems, Cre/loxP and Dre/rox, were developed for driving orthogonal co-SCRaMbLE of normal synthetic chromosomes and the tRNA Neochromosome. The functions of two recombinases were engineered to be activated and controlled by two hormone molecules, b-estradiol and RU486. In addition, a SCRaMbLE-in device was designed to integrate a pathway of interest into a synthetic chromosome in yeast while driving the normal SCRaMbLE process at the same time for chassis level optimization by the Cre/loxP recombination system. For in vitro application, three recombinases, Cre, Dre and VCre, were explored to integrate promoters into a pathway of interest for gene expression level diversification in metabolic engineering. To attempt broader application of the recombinases for metabolic engineering, a side project in metabolic engineering was also involved in my PhD study. As part of a collaborative Synthetic Natural Product (SynNP) project applying synthetic biology to discover and design new antibiotics against tuberculosis and other infectious diseases, a YeastFab compatible assembly method was designed for large pathway construction and tested for heterologous expression of the RiPPs exemplar pathway of nocathiacin I in S. cerevisiae