RESUMO
Morphogenetic engineering is an emerging field that explores the design and implementation of self-organized patterns, morphologies, and architectures in systems composed of multiple agents such as cells and swarm robots. Synthetic biology, on the other hand, aims to develop tools and formalisms that increase reproducibility, tractability, and efficiency in the engineering of biological systems. We seek to apply synthetic biology approaches to the engineering of morphologies in multicellular systems. Here, we describe the engineering of two mechanisms, symmetry-breaking and domain-specific cell regulation, as elementary functions for the prototyping of morphogenetic instructions in bacterial colonies. The former represents an artificial patterning mechanism based on plasmid segregation while the latter plays the role of artificial cell differentiation by spatial colocalization of ubiquitous and segregated components. This separation of patterning from actuation facilitates the design-build-test-improve engineering cycle. We created computational modules for CellModeller representing these basic functions and used it to guide the design process and explore the design space in silico. We applied these tools to encode spatially structured functions such as metabolic complementation, RNAPT7 gene expression, and CRISPRi/Cas9 regulation. Finally, as a proof of concept, we used CRISPRi/Cas technology to regulate cell growth by controlling methionine synthesis. These mechanisms start from single cells enabling the study of morphogenetic principles and the engineering of novel population scale structures from the bottom up.
Assuntos
Bactérias/genética , Sistemas CRISPR-Cas/genética , Simulação por Computador , Expressão Gênica/genética , Engenharia Genética/métodos , Metionina/genética , RNA/genética , Reprodutibilidade dos Testes , Biologia Sintética/métodosRESUMO
Yeast integrating plasmids (YIPs) are a versatile tool for stable integration in Saccharomyces cerevisiae. However, current YIP systems necessitate time- and labor-intensive methods for cloning and selection marker rescue. Here, we describe the design, construction, and validation of a new YIP system capable of accelerating the stable integration of multiple expression constructs into different loci in the yeast S. cerevisiae. These 'directed pop-out' plasmids enable a simple, two-step integration protocol that results in a scarless integration alongside a complete rescue of the selection marker. These plasmids combine three key features: a dedicated 'YIPout' fragment directs a recombination event that rescues the selection marker while avoiding undesired excision of the target DNA sequence, a multifragment modular DNA assembly system simplifies cloning, and a new set of counterselectable markers enables serial integration followed by a transformation-free marker rescue event. We constructed and tested directed pop-out YIPs for integration of fluorescent reporter genes into four yeast loci. We validated our new YIP design by integrating three reporter genes into three different loci with transformation-free rescue of selection markers. These new YIP designs will facilitate the construction of yeast strains that express complex heterologous metabolic pathways.
Assuntos
Expressão Gênica , Genética Microbiana/métodos , Biologia Molecular/métodos , Mutagênese Insercional , Saccharomyces cerevisiae/genética , Genes Reporter , Plasmídeos , Recombinação Genética , Seleção GenéticaRESUMO
We report an optogenetic method based on Arabidopsis thaliana cryptochrome 2 for rapid and reversible protein oligomerization in response to blue light. We demonstrated its utility by photoactivating the ß-catenin pathway, achieving a transcriptional response higher than that obtained with the natural ligand Wnt3a. We also demonstrated the modularity of this approach by photoactivating RhoA with high spatiotemporal resolution, thereby suggesting a previously unknown mode of activation for this Rho GTPase.
