A Standardized Inverter Package Borne by Broad Host Range Plasmids for Genetic Circuit Design in Gram-Negative Bacteria. 2021

Huseyin Tas, and Ángel Goñi-Moreno, and Víctor de Lorenzo
Systems Biology Department, Centro Nacional de Biotecnología-CSIC, Campus de Cantoblanco, Madrid, 28049, Spain.

Genetically encoded logic gates, especially inverters-NOT gates-are the building blocks for designing circuits, engineering biosensors, or decision-making devices in synthetic biology. However, the repertoire of inverters readily available for different species is rather limited. In this work, a large whole of NOT gates that was shown to function previously in a specific strain of Escherichia coli, was recreated as broad host range (BHR) collection of constructs assembled in low, medium, and high copy number plasmid backbones of the SEVA (Standard European Vector Architecture) collection. The input/output function of each of the gates was characterized and parametrized in the environmental bacterium and metabolic engineering chassis Pseudomonas putida. Comparisons of the resulting fluorescence cytometry data with those published for the same gates in Escherichia coli provided useful hints on the portability of the corresponding gates. The hereby described inverter package (20 different versions of 12 distinct gates) borne by BHR plasmids thus becomes a toolbox of choice for designing genetic circuitries in a variety of Gram-negative species other than E. coli.

UI MeSH Term Description Entries
D010957 Plasmids Extrachromosomal, usually CIRCULAR DNA molecules that are self-replicating and transferable from one organism to another. They are found in a variety of bacterial, archaeal, fungal, algal, and plant species. They are used in GENETIC ENGINEERING as CLONING VECTORS. Episomes,Episome,Plasmid
D004926 Escherichia coli A species of gram-negative, facultatively anaerobic, rod-shaped bacteria (GRAM-NEGATIVE FACULTATIVELY ANAEROBIC RODS) commonly found in the lower part of the intestine of warm-blooded animals. It is usually nonpathogenic, but some strains are known to produce DIARRHEA and pyogenic infections. Pathogenic strains (virotypes) are classified by their specific pathogenic mechanisms such as toxins (ENTEROTOXIGENIC ESCHERICHIA COLI), etc. Alkalescens-Dispar Group,Bacillus coli,Bacterium coli,Bacterium coli commune,Diffusely Adherent Escherichia coli,E coli,EAggEC,Enteroaggregative Escherichia coli,Enterococcus coli,Diffusely Adherent E. coli,Enteroaggregative E. coli,Enteroinvasive E. coli,Enteroinvasive Escherichia coli
D005434 Flow Cytometry Technique using an instrument system for making, processing, and displaying one or more measurements on individual cells obtained from a cell suspension. Cells are usually stained with one or more fluorescent dyes specific to cell components of interest, e.g., DNA, and fluorescence of each cell is measured as it rapidly transverses the excitation beam (laser or mercury arc lamp). Fluorescence provides a quantitative measure of various biochemical and biophysical properties of the cell, as well as a basis for cell sorting. Other measurable optical parameters include light absorption and light scattering, the latter being applicable to the measurement of cell size, shape, density, granularity, and stain uptake. Cytofluorometry, Flow,Cytometry, Flow,Flow Microfluorimetry,Fluorescence-Activated Cell Sorting,Microfluorometry, Flow,Cell Sorting, Fluorescence-Activated,Cell Sortings, Fluorescence-Activated,Cytofluorometries, Flow,Cytometries, Flow,Flow Cytofluorometries,Flow Cytofluorometry,Flow Cytometries,Flow Microfluorometries,Flow Microfluorometry,Fluorescence Activated Cell Sorting,Fluorescence-Activated Cell Sortings,Microfluorimetry, Flow,Microfluorometries, Flow,Sorting, Fluorescence-Activated Cell,Sortings, Fluorescence-Activated Cell
D015723 Gene Library A large collection of DNA fragments cloned (CLONING, MOLECULAR) from a given organism, tissue, organ, or cell type. It may contain complete genomic sequences (GENOMIC LIBRARY) or complementary DNA sequences, the latter being formed from messenger RNA and lacking intron sequences. DNA Library,cDNA Library,DNA Libraries,Gene Libraries,Libraries, DNA,Libraries, Gene,Libraries, cDNA,Library, DNA,Library, Gene,Library, cDNA,cDNA Libraries
D016958 Pseudomonas putida A species of gram-negative, aerobic bacteria isolated from soil and water as well as clinical specimens. Occasionally it is an opportunistic pathogen.
D053263 Gene Regulatory Networks Interacting DNA-encoded regulatory subsystems in the GENOME that coordinate input from activator and repressor TRANSCRIPTION FACTORS during development, cell differentiation, or in response to environmental cues. The networks function to ultimately specify expression of particular sets of GENES for specific conditions, times, or locations. Gene Circuits,Gene Modules,Gene Networks,Transcriptional Networks,Gene Module,Circuit, Gene,Circuits, Gene,Gene Circuit,Gene Network,Gene Regulatory Network,Module, Gene,Modules, Gene,Network, Gene,Network, Gene Regulatory,Network, Transcriptional,Networks, Gene,Networks, Gene Regulatory,Networks, Transcriptional,Regulatory Network, Gene,Regulatory Networks, Gene,Transcriptional Network
D058507 Host Specificity The properties of a pathogen that makes it capable of infecting one or more specific hosts. The pathogen can include PARASITES as well as VIRUSES; BACTERIA; FUNGI; or PLANTS. Host Specialization,Host Range,Host Species Specificity,Host Ranges,Host Specializations,Host Species Specificities,Host Specificities,Range, Host,Ranges, Host,Specialization, Host,Specializations, Host,Specificities, Host,Specificities, Host Species,Specificity, Host,Specificity, Host Species
D060847 Metabolic Engineering Methods and techniques used to genetically modify cells' biosynthetic product output and develop conditions for growing the cells as BIOREACTORS. Engineering, Metabolic

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