scholarly journals Multiplexed characterization of rationally designed promoter architectures deconstructs combinatorial logic for IPTG-inducible systems

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Timothy C. Yu ◽  
Winnie L. Liu ◽  
Marcia S. Brinck ◽  
Jessica E. Davis ◽  
Jeremy Shek ◽  
...  
Keyword(s):  
Gene Expression ◽  
Inducible Promoter ◽  
Transcriptional Responses ◽  
Promoter Elements ◽  
Sequence Composition ◽  
Inducible Promoters ◽  
Mechanics Model ◽  
The Individual ◽  
Repressor Binding Site

AbstractA crucial step towards engineering biological systems is the ability to precisely tune the genetic response to environmental stimuli. In the case of Escherichia coli inducible promoters, our incomplete understanding of the relationship between sequence composition and gene expression hinders our ability to predictably control transcriptional responses. Here, we profile the expression dynamics of 8269 rationally designed, IPTG-inducible promoters that collectively explore the individual and combinatorial effects of RNA polymerase and LacI repressor binding site strengths. We then fit a statistical mechanics model to measured expression that accurately models gene expression and reveals properties of theoretically optimal inducible promoters. Furthermore, we characterize three alternative promoter architectures and show that repositioning binding sites within promoters influences the types of combinatorial effects observed between promoter elements. In total, this approach enables us to deconstruct relationships between inducible promoter elements and discover practical insights for engineering inducible promoters with desirable characteristics.

scholarly journals Multiplexed characterization of rationally designed promoter architectures deconstructs combinatorial logic for IPTG-inducible systems

2020 ◽  
Author(s):  
Timothy C. Yu ◽  
Winnie L. Liu ◽  
Marcia Brinck ◽  
Jessica E. Davis ◽  
Jeremy Shek ◽  
...  
Keyword(s):  
Gene Expression ◽  
Inducible Promoter ◽  
Transcriptional Responses ◽  
Promoter Elements ◽  
Sequence Composition ◽  
Inducible Promoters ◽  
Mechanics Model ◽  
The Individual ◽  
Repressor Binding Site

AbstractA crucial step towards engineering biological systems is the ability to precisely tune the genetic response to environmental stimuli. In the case of Escherichia coli inducible promoters, our incomplete understanding of the relationship between sequence composition and gene expression hinders our ability to predictably control transcriptional responses. Here, we profile the expression dynamics of 8,269 rationally designed IPTG-inducible promoters that collectively explore the individual and combinatorial effects of RNA polymerase and LacI repressor binding site strengths. Using these data, we fit a statistical mechanics model that accurately models gene expression and reveals properties of theoretically optimal inducible promoters. Furthermore, we characterize three novel promoter architectures and show that repositioning binding sites within promoters influences the types of combinatorial effects observed between promoter elements. In total, this approach enables us to deconstruct relationships between inducible promoter elements and discover practical insights for engineering inducible promoters with desirable characteristics.

scholarly journals An Estradiol-Inducible Promoter Enables Fast, Graduated Control of Gene Expression in Fission Yeast

2016 ◽  
Author(s):  
Makoto J. Ohira ◽  
David G. Hendrickson ◽  
R. Scott McIsaac ◽  
Nicholas Rhind
Keyword(s):  
Gene Expression ◽  
Fission Yeast ◽  
Dose Response ◽  
Dynamic Range ◽  
Inducible Promoter ◽  
Experimental Manipulation ◽  
Control Of Gene Expression ◽  
Inducible Promoters ◽  
Linear Dose ◽  
Promoter System

ABSTRACTThe fission yeast Schizosaccharomyces pombe lacks a diverse toolkit of inducible promoters for experimental manipulation. Available inducible promoters suffer from slow induction kinetics, limited control of expression levels and/or a requirement for defined growth medium. In particular, no S. pombe inducible promoter systems exhibit a linear dose response, which would allow expression to be tuned to specific levels. We have adapted a fast, orthogonal promoter system with a large dynamic range and a linear dose response, based on β-estradiol-regulated function of the human estrogen receptor, for use in S. pombe. We show that this promoter system, termed Z3EV, turns on quickly, can reach a maximal induction of 20 fold, and exhibits a linear dose response over its entire induction range, with few off target effects. We demonstrate the utility of this system by regulating the mitotic inhibitor Wee1 to create a strain in which cell size is regulated by β-estradiol concentration. This promoter system will be of great utility for experimentally regulating gene expression in fission yeast.

scholarly journals Arbitrarily primed PCR fingerprinting of RNA and DNA in Entamoeba histolytica

2000 ◽  
Vol 42 (5) ◽  
pp. 249-253 ◽  
Author(s):  
Paulo Renato VALLE ◽  
Maria Betânia G. SOUZA ◽  
Edna M. PIRES ◽  
Edward F. SILVA ◽  
Maria A. GOMES
Keyword(s):  
Gene Expression ◽  
Rapd Analysis ◽  
Polymorphic Markers ◽  
Biological Features ◽  
Pcr Products ◽  
The Individual ◽  
Arbitrarily Primed Pcr ◽  
High Degree ◽  
Rna And Dna

Differences were detected in the gene expression of strains of E. histolytica using RNA (RAP-PCR) and DNA fingerprinting (RAPD). Analysis of the electrophoretic profiles of the gels revealed some polymorphic markers that could be used in the individual characterization of the strains. The 260 bands generated by using five different primers for RAP-PCR, as well as RAPD, were employed in the construction of dendograms. The dendogram obtained based on the RAPD products permitted the distinction of symptomatic and asymptomatic isolates, as well the correlation between the polymorphism exhibited and the virulence of the strains. The dendogram obtained for the RAP-PCR products did not show a correlation with the virulence of the strains but revealed a high degree of intraspecific transcriptional variability that could be related to other biological features, whether or not these are involved in the pathogenesis of amebiasis.

scholarly journals A rhamnose-inducible system for precise and temporal control of gene expression in cyanobacteria

2017 ◽  
Author(s):  
Ciarán L. Kelly ◽  
Andrew Hitchcock ◽  
Antonio Torres-Méndez ◽  
John T. Heap
Keyword(s):  
Gene Expression ◽  
Escherichia Coli ◽  
Linear Response ◽  
Inducible Promoter ◽  
External Control ◽  
Basal Transcription ◽  
Control Of Gene Expression ◽  
Biotechnological Potential ◽  
Genetic Tools ◽  
Inducible Promoters

ABSTRACTCyanobacteria are important for fundamental studies of photosynthesis and have great biotechnological potential. In order to better study and fully exploit these organisms, the limited repertoire of genetic tools and parts must be expanded. A small number of inducible promoters have been used in cyanobacteria, allowing dynamic external control of gene expression through the addition of specific inducer molecules. However, the inducible promoters used to date suffer from various drawbacks including toxicity of inducers, leaky expression in the absence of inducer and inducer photolability, the latter being particularly relevant to cyanobacteria which, as photoautotrophs, are grown under light. Here we introduce the rhamnose-induciblerhaBADpromoter ofEscherichia coliinto the model freshwater cyanobacteriumSynechocystissp. PCC 6803 and demonstrate it has superior properties to previously reported cyanobacterial inducible promoter systems, such as a non-toxic, photostable, non-metabolizable inducer, a linear response to inducer concentration and crucially no basal transcription in the absence of inducer.

scholarly journals Symbioflor2® Escherichia coli Genotypes Enhance Ileal and Colonic Gene Expression Associated with Mucosal Defense in Gnotobiotic Mice

2020 ◽  
Vol 8 (4) ◽  
pp. 512
Author(s):  
Unai Escribano-Vazquez ◽  
Claudia Beimfohr ◽  
Deborah Bellet ◽  
Muriel Thomas ◽  
Kurt Zimmermann ◽  
...  
Keyword(s):  
Gene Expression ◽  
Escherichia Coli ◽  
Transcriptional Responses ◽  
E Coli ◽  
Intestinal Physiology ◽  
Beneficial Action ◽  
Probiotic Product ◽  
Gnotobiotic Mice ◽  
Irritable Bowel ◽  
The Individual

Symbioflor2® is a probiotic product composed of six Escherichia coli genotypes, which has a beneficial effect on irritable bowel syndrome. Our objective was to understand the individual impact of each of the six genotypes on the host, together with the combined impact of the six in the compound Symbioflor2®. Gnotobiotic mice were mono-associated with one of the six genotypes or associated with the compound product. Ileal and colonic gene expression profiling was carried out, and data were compared between the different groups of gnotobiotic mice, along with that obtained from conventional (CV) mice and mice colonized with the probiotic E. coli Nissle 1917. We show that Symbioflor2® genotypes induce intestinal transcriptional responses involved in defense and immune mechanisms. Using mice associated with Symbioflor2®, we reveal that the product elicits a balanced response from the host without any predominance of a single genotype. The Nissle strain and the six bacterial genotypes have different effects on the intestinal gene expression, suggesting that the impacts of these probiotics are not redundant. Our data show the effect of the Symbioflor2® genotypes at the molecular level in the digestive tract, which further highlights their beneficial action on several aspects of intestinal physiology.

scholarly journals Regulation of Sesquiterpene Cyclase Gene Expression (Characterization of an Elicitor- and Pathogen-Inducible Promoter)

1997 ◽  
Vol 115 (2) ◽  
pp. 437-451 ◽  
Author(s):  
S. Yin ◽  
L. Mei ◽  
J. Newman ◽  
K. Back ◽  
J. Chappell
Keyword(s):  
Gene Expression ◽  
Inducible Promoter ◽  
Sesquiterpene Cyclase ◽  
Expression Characterization

Separation and Characterization of Standard Propoxyphene Diastereomers and Their Determination in Pharmaceutical and Illicit Preparations

1981 ◽  
Vol 64 (4) ◽  
pp. 875-883
Author(s):  
Shiv K Soni ◽  
Daniel Van Gelder
Keyword(s):  
High Pressure ◽  
Optical Isomers ◽  
Gold Chloride ◽  
Melting Points ◽  
Eutectic Melting ◽  
Racemic Mixtures ◽  
The Individual ◽  
Isomeric Mixtures ◽  
Asymmetric Carbon

Abstract Due to the existence of 2 asymmetric carbon atoms in: the propoxyphene molecule, there are 4 diastereomers: alpha dextro, alpha levo, beta dextro, and beta levo. Only α-d-propoxyphene is included under the federal Controlled Substances Act. Baseline separations of propoxyphene from various incipients (aspirin, caffeine, phenacetin, and acetaminophen) present in pharmaceutical and illicit preparations, and between the alpha and beta diastereomers, were achieved by high pressure liquid chromatography. The column eluant was collected and propoxyphene was extracted. The optical isomers were differentiated and characterized by melting points and by chemical microcrystalline tests. Using hot stage thermomicroscopy, the eutectic melting points of binary isomeric mixtures of propoxyphene bases and salts were found to be depressed about 10° and 15-30°C, respectively, below the individual isomer melting points. The characteristic microcrystals formed with the alpha racemic mixtures by using a glycerin-aqueous gold chloride reagent were not produced by the beta racemic mixtures.

Sign in / Sign up

Export Citation Format

Share Document