RESUMEN
By assembly and evolutionary engineering of T7-phage-based transcriptional switches made from endogenous components of the bet operon on the Escherichia coli chromosome, genetic switches inducible by choline, a safe and inexpensive compound, were constructed. The functional plasticity of the BetI repressor was revealed by rapid and high-frequency identification of functional variants with various properties, including those with high stringency, high maximum expression level, and reversed phenotypes, from a pool of BetI mutants. The plasmid expression of BetI mutants resulted in the choline-inducible (Bet-ON) or choline-repressible (Bet-OFF) switching of genes under the pT7/betO sequence at unprecedentedly high levels, while keeping the minimal leaky expression in uninduced conditions.
Asunto(s)
Bacteriófago T7 , Colina/farmacología , Evolución Molecular Dirigida , Proteínas de Escherichia coli , Escherichia coli , Proteínas Represoras , Bacteriófago T7/genética , Bacteriófago T7/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Proteínas Represoras/genética , Proteínas Represoras/metabolismoRESUMEN
The evolutionary design of genetic switches and circuits requires iterative rounds of positive (ON-) and negative (OFF-) selection. We previously reported a rapid OFF selection system based on the kinase activity of herpes simplex virus thymidine kinase (hsvTK) on the artificial mutator nucleoside dP. By fusing hsvTK with the kanamycin resistance marker aminoglycoside-(3')-phosphotransferase (APH), we established a novel selector system for genetic switches. Due to the bactericidal nature of kanamycin and nucleoside-based lethal mutagenesis, both positive and negative selection could be completed within several hours. Using this new selector system, we isolated a series of homoserine lactone-inducible genetic switches with different expression efficiencies from libraries of the Vibrio fischeri lux promoter in two days, using only liquid handling.
Asunto(s)
Proteínas Bacterianas/genética , Genes de Cambio , Kanamicina Quinasa/genética , Timidina Quinasa/genética , Proteínas Virales/genética , 4-Butirolactona/análogos & derivados , 4-Butirolactona/química , 4-Butirolactona/metabolismo , Aliivibrio fischeri/genética , Escherichia coli/metabolismo , Biblioteca de Genes , Vectores Genéticos/genética , Vectores Genéticos/metabolismo , Regiones Promotoras Genéticas , Proteínas Represoras/genética , Simplexvirus/genética , Espectrometría de Fluorescencia , Transactivadores/genéticaRESUMEN
Ever-increasing repertories of RNA-based switching devices are enabling synthetic biologists to construct compact, self-standing, and easy-to-integrate regulatory circuits. However, it is rather rare that the existing RNA-based expression controllers happen to have the exact specification needed for particular applications from the beginning. Evolutionary design of is powerful strategy for quickly tuning functions/specification of genetic switches. Presented here are the steps required for rapid and efficient enrichment of genetic switches with desired specification using recently developed nucleoside kinase-based dual selection system. Here, the library of genetic switches, created by randomizing either the part or the entire sequence coding switching components, is subjected to OFF (negative) selection and ON (positive) selection in various conditions. The entire selection process is completed only by liquid handling, facilitating the parallel and continuous operations of multiple selection projects. This automation-liable platform for genetic selection of functional switches has potential applications for development of RNA-based biosensors, expression controllers, and their integrated forms (genetic circuits).
Asunto(s)
Evolución Molecular Dirigida/métodos , Riboswitch , Aliivibrio fischeri/genética , Secuencia de Bases , Escherichia coli/genética , Escherichia coli/metabolismo , Biblioteca de Genes , Mutagénesis , Proteínas Represoras/genética , Simplexvirus/enzimología , Biología Sintética/métodos , Timidina Quinasa/metabolismo , Transactivadores/genéticaRESUMEN
A simple microfluidic system has been presented to perform continuous two-parameter cell sorting based on size and surface markers. Immunomagnetic bead-conjugated cells are initially sorted based on size by utilizing the hydrodynamic filtration (HDF) scheme, introduced into individual separation lanes, and simultaneously focused onto one sidewall by the hydrodynamic effect. Cells are then subjected to magnetophoretic separation in the lateral direction, and finally they are individually recovered through multiple outlet branches. We successfully demonstrated the continuous sorting of JM (human lymphocyte cell line) cells using anti-CD4 immunomagnetic beads and confirmed that accurate size- and surface marker-based sorting was achieved. In addition, the sorting of cell mixtures was performed at purification ratios higher than 90%. The proposed system enables two-dimensional cell sorting without necessitating complicated setups and operations, and thus, it can be a useful tool for general biological experiments including cell-based disease diagnosis, stem cell engineering, and cellular physiological studies.