Cooperativity to increase Turing pattern space for synthetic biology

It is hard to bridge the gap between mathematical formulations and biological implementations of Turing patterns, yet this is necessary for both understanding and engineering these networks with synthetic biology approaches. Here, we model a reaction-diffusion system with two morphogens in a monostable regime, inspired by components that we recently described in a synthetic biology study in mammalian cells. The model employs a single promoter to express both the activator and inhibitor genes and produces Turing patterns over large regions of parameter space, using biologically interpretable Hill function reactions. We applied a stability analysis and identified rules for choosing biologically tunable parameter relationships to increase the likelihood of successful patterning. We show how to control Turing pattern sizes and time evolution by manipulating the values for production and degradation relationships. More importantly, our analysis predicts that steep dose-response functions arising from cooperativity are mandatory for Turing patterns. Greater steepness increases parameter space and even reduces the requirement for differential diffusion between activator and inhibitor. These results demonstrate some of the limitations of linear scenarios for reaction-diffusion systems and will help to guide projects to engineer synthetic Turing patterns.Centro Regional de Estudios Genómico

Genetically encoded sender-receiver system in 3D mammalian cell culture

Engineering spatial patterning in mammalian cells, employing entirely genetically encoded components, requires solving several problems. These include how to code secreted activator or inhibitor molecules and how to send concentration-dependent signals to neighboring cells, to control gene expression. The Madin-Darby Canine Kidney (MDCK) cell line is a potential engineering scaffold as it forms hollow spheres (cysts) in 3D culture and tubulates in response to extracellular hepatocyte growth factor (HGF). We first aimed to graft a synthetic patterning system onto single developing MDCK cysts. We therefore developed a new localized transfection method to engineer distinct sender and receiver regions. A stable reporter line enabled reversible EGFP activation by HGF and modulation by a secreted repressor (a truncated HGF variant, NK4). By expanding the scale to wide fields of cysts, we generated morphogen diffusion gradients, controlling reporter gene expression. Together, these components provide a toolkit for engineering cell-cell communication networks in 3D cell culture.Centro Regional de Estudios Genómico

A study on autocatalysis through synthetic biology. Exploration of spatiotemporal dynamics in the presence or absence of synthetic autocatalytic Hepatocyte Growth Factor signaling in mammalian cells

Una de las preguntas sin resolver en la biologia celular, es como las células mamíferas logran generar patrones estables como organos o seres vivos, en un entorno variable. El concepto matemático del bucle de retreoalimentación, es una herramienta que puede generar orden. En esta tesis, presento dos proyectos que forman parte de una idea iterativa para recrear patrones biológicos sintéticamente en células mamíferas. En la primera parte, presento la creación de una linea celular que funge como detector de niveles de la hormona HGF a través de una reportero transcripcional. En la segunda parte, demuestro la reprogramación de esta células con fin de producir HGF en respuesta a HGF, en efecto creando un bucle de retroalimentación positivo. En ambos proyectos, utilizo microscopia cuantitativa espaciotemporal para analyzar y medir la evolución dinámica de las células en respuesta a un estímulo de HGF.One unanswered riddle in biology is how can mammalian cells organize to generate ordered patterns such as organs and living beings, in an ever changing environment. An underlying mathematical principle for the generation of order is given by feedback motifs. Here, I present two projects which are part of an effort to recreate stable ordered patterns in a cellular system through information encoded in DNA. In the first part, I present a receiver mammalian cell line which can accurately sense the diffusible Hepatocyte Growth Factor (HGF) through a transcriptional reporter. In the second part I reprogrammed this cell line so that it produces more HGF in response to HGF, in effect creating an autocatalytic positive feedback. In both cases, I have used spatiotemporal quantitative microscopy analysis to monitor the dynamic evolution of the cell lines in response to a HGF stimulus

A study on autocatalysis through synthetic biology. Exploration of spatiotemporal dynamics in the presence or absence of synthetic autocatalytic Hepatocyte Growth Factor signaling in mammalian cells

Una de las preguntas sin resolver en la biologia celular, es como las células mamíferas logran generar patrones estables como organos o seres vivos, en un entorno variable. El concepto matemático del bucle de retreoalimentación, es una herramienta que puede generar orden. En esta tesis, presento dos proyectos que forman parte de una idea iterativa para recrear patrones biológicos sintéticamente en células mamíferas. En la primera parte, presento la creación de una linea celular que funge como detector de niveles de la hormona HGF a través de una reportero transcripcional. En la segunda parte, demuestro la reprogramación de esta células con fin de producir HGF en respuesta a HGF, en efecto creando un bucle de retroalimentación positivo. En ambos proyectos, utilizo microscopia cuantitativa espaciotemporal para analyzar y medir la evolución dinámica de las células en respuesta a un estímulo de HGF.One unanswered riddle in biology is how can mammalian cells organize to generate ordered patterns such as organs and living beings, in an ever changing environment. An underlying mathematical principle for the generation of order is given by feedback motifs. Here, I present two projects which are part of an effort to recreate stable ordered patterns in a cellular system through information encoded in DNA. In the first part, I present a receiver mammalian cell line which can accurately sense the diffusible Hepatocyte Growth Factor (HGF) through a transcriptional reporter. In the second part I reprogrammed this cell line so that it produces more HGF in response to HGF, in effect creating an autocatalytic positive feedback. In both cases, I have used spatiotemporal quantitative microscopy analysis to monitor the dynamic evolution of the cell lines in response to a HGF stimulus