RESUMEN
Synthetic minimal cells are a class of bioreactors that have some, but not all, functions of live cells. Here, we report a critical step toward the development of a bottom-up minimal cell: cellular export of functional protein and RNA products. We used cell-penetrating peptide tags to translocate payloads across a synthetic cell vesicle membrane. We demonstrated efficient transport of active enzymes and transport of nucleic acid payloads by RNA-binding proteins. We investigated influence of a concentration gradient alongside other factors on the efficiency of the translocation, and we show a method to increase product accumulation in one location. We demonstrate the use of this technology to engineer molecular communication between different populations of synthetic cells, to exchange protein and nucleic acid signals. The synthetic minimal cell production and export of proteins or nucleic acids allows experimental designs that approach the complexity and relevancy of natural biological systems. A record of this paper's transparent peer review process is included in the supplemental information.
Asunto(s)
Células Artificiales , Péptidos de Penetración Celular , Ácidos Nucleicos , Ácidos Nucleicos/metabolismo , Células Artificiales/metabolismo , Proteínas , Péptidos de Penetración Celular/química , Péptidos de Penetración Celular/metabolismoRESUMEN
Liposomal encapsulation serves as the basis for the engineering of biomimetic and novel synthetic cells. Liposomes are normally formed using such methods as thin film rehydration (TFH), density-mediated reverse emulsion encapsulation (REE), or one of many microfluidics-based approaches-with the latter of these two methods being used mainly for the encapsulation of various lumen constituents such as cell-free protein expression reactions. Here, we describe the simultaneous formation and encapsulation of liposomes and various cell-mimetic lumen chemistries, respectively, using a 3D-printable microcapillary-based microfluidics device based off of the droplet-shooting and size-filtration (DSSF) liposome preparation method.
Asunto(s)
Liposomas , Microfluídica , Biomimética , Emulsiones , Microfluídica/métodos , Impresión TridimensionalRESUMEN
Synthetic cells can mimic the intricate complexities of live cells, while mitigating the level of noise that is present natural systems; however, many crucial processes still need to be demonstrated in synthetic cells to use them to comprehensively study and engineer biology. Here we demonstrate key functionalities of synthetic cells previously available only to natural life: differentiation and mating. This work presents a toolset for engineering combinatorial genetic circuits in synthetic cells. We demonstrate how progenitor populations can differentiate into new lineages in response to small molecule stimuli or as a result of fusion, and we provide practical demonstration of utility for metabolic engineering. This work provides a tool for bioengineering and for natural pathway studies, as well as paving the way toward the construction of live artificial cells.
Asunto(s)
Células Artificiales , Células Artificiales/metabolismo , Bioingeniería , Comunicación Celular , Redes Reguladoras de Genes , Ingeniería Metabólica , Biología SintéticaRESUMEN
Thermal denaturation is a common technique in the biophysical study of nucleic acids. These experiments are typically performed by monitoring the increase in absorbance (hyperchromism) of a sample at 260nm with temperature (Mergny & Lacroix, 2003; Puglisi & Tinoco, 1989). This wavelength is chosen as nucleic acids of mixed sequence typically exhibit their maximum absorbance here. Exceptions exist, however, some noncanonical nucleic acid structures exhibit differing spectral changes with temperature, resulting in other wavelengths being convenient reporters of secondary structure. In the case of nucleic acids that bind visible light-absorbing ligands, such as fluorogenic aptamers, another wavelength can be a convenient reporter of secondary structure stability and RNA-ligand recognition. As it can be difficult, if not impossible, to know which wavelength to employ a priori, we have developed a system for obtaining the full UV-visible spectrum of a sample at each wavelength, allowing for the subsequent extraction of the absorbance-temperature profile at the desired wavelength. Here, we describe the apparatus and software used to do so. We also describe another technique for the use of a qPCR instrument for measuring secondary structure stability of fluorescent nucleic acid-ligand complexes.