RESUMEN
Gene expression noise can reduce cellular fitness or facilitate processes such as alternative metabolism, antibiotic resistance, and differentiation. Unfortunately, efforts to study the impacts of noise have been hampered by a scaling relationship between noise and expression level from individual promoters. Here, we use theory to demonstrate that mean and noise can be controlled independently by expressing two copies of a gene from separate inducible promoters in the same cell. We engineer low and high noise inducible promoters to validate this result in Escherichia coli, and develop a model that predicts the experimental distributions. Finally, we use our method to reveal that the response of a promoter to a repressor is less sensitive with higher repressor noise and explain this result using a law from probability theory. Our approach can be applied to investigate the effects of noise on diverse biological pathways or program cellular heterogeneity for synthetic biology applications.
Asunto(s)
Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica , Genes Bacterianos , Regiones Promotoras Genéticas , Proteínas Represoras/genética , Escherichia coli/efectos de los fármacos , Escherichia coli/metabolismo , Citometría de Flujo , Genes Reporteros , Ingeniería Genética/métodos , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Plásmidos/química , Plásmidos/metabolismo , Proteínas Represoras/metabolismo , Proteínas Represoras/farmacología , Transformación Bacteriana , Proteína Fluorescente RojaRESUMEN
Optogenetic systems enable unmatched precision for controlling molecular biological processes but require the use of specialized electrical and optical hardware. In an effort to make working with optogenetic systems more accessible, we recently described an open-source hardware platform called the Light Plate Apparatus (LPA). The LPA is a device capable of delivering two independent light signals from standard LEDs to wells of a 24-well culture plate. Basic to advanced level light programs can be created for the LPA in Iris, an easy to use, open-source web application. In this chapter, we describe each step required to build, program, and use the LPA.
Asunto(s)
Optogenética/instrumentación , Diseño de Equipo , Rayos Láser , Luz , Impresión Tridimensional , Programas InformáticosRESUMEN
Two-component systems (TCSs) are the largest family of multi-step signal transduction pathways and valuable sensors for synthetic biology. However, most TCSs remain uncharacterized or difficult to harness for applications. Major challenges are that many TCS output promoters are unknown, subject to cross-regulation, or silent in heterologous hosts. Here, we demonstrate that the two largest families of response regulator DNA-binding domains can be interchanged with remarkable flexibility, enabling the corresponding TCSs to be rewired to synthetic output promoters. We exploit this plasticity to eliminate cross-regulation, un-silence a gram-negative TCS in a gram-positive host, and engineer a system with over 1,300-fold activation. Finally, we apply DNA-binding domain swapping to screen uncharacterized Shewanella oneidensis TCSs in Escherichia coli, leading to the discovery of a previously uncharacterized pH sensor. This work should accelerate fundamental TCS studies and enable the engineering of a large family of genetically encoded sensors with diverse applications.
Asunto(s)
ADN Bacteriano/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Ingeniería Genética , Shewanella/genética , Shewanella/metabolismo , ADN Bacteriano/genéticaRESUMEN
In optogenetics, researchers use light and genetically encoded photoreceptors to control biological processes with unmatched precision. However, outside of neuroscience, the impact of optogenetics has been limited by a lack of user-friendly, flexible, accessible hardware. Here, we engineer the Light Plate Apparatus (LPA), a device that can deliver two independent 310 to 1550 nm light signals to each well of a 24-well plate with intensity control over three orders of magnitude and millisecond resolution. Signals are programmed using an intuitive web tool named Iris. All components can be purchased for under $400 and the device can be assembled and calibrated by a non-expert in one day. We use the LPA to precisely control gene expression from blue, green, and red light responsive optogenetic tools in bacteria, yeast, and mammalian cells and simplify the entrainment of cyanobacterial circadian rhythm. The LPA dramatically reduces the entry barrier to optogenetics and photobiology experiments.