scholarly journals Beta-Glucuronidase (GusA) reporter enzyme assay for Escherichia coli

2019 ◽  
Author(s):  
Madeleine Huber ◽  
Jörg Soppa
Keyword(s):  
Escherichia Coli ◽  
Translational Regulation ◽  
Enzyme Assay ◽  
Microtiter Plate ◽  
E Coli ◽  
Reporter Enzyme ◽  
Keio Collection ◽  
Beta Galactosidase ◽  
Transcriptional Fusions ◽  
Microtiter Plate Format

Abstract The beta-Glucuronidase (GusA) is a long-known reporter enzyme for many different species [1]. The E. coli gusA gene is often used in plant research because plants lack an endogenous gusA gene. In E. coli, the transcript of the gusA gene is more stable than that of the highly used reporter gene beta-galactosidase (lacZ) [2]. The GusA activity can be determined using the artificial substrate p-nitrophenyl-β-D-glucopyranosid (pNPG). pNPG is converted to glucoronic acid and para-nitrophenol (pNP), which can be quantified spectrometrically at 405 nm. To avoid background, it is best to use an E. coli strain with a deletion of the chromosomal gusA gene, which is available e.g. at the Keio collection [3]. The gusA gene can be used for transcriptional fusions, e.g. to characterize promoters, and also for translational fusions, e.g. to study translational regulation. The assay was adapted to the microtiter plate format to enable the parallel handling of a large number of samples. The “procedure” (see below) describes an application with the gusA gene in a translational fusion with the gene of interest cloned under the control of the inducible arabinose promoter PBAD.

scholarly journals Glycerol-3-phosphate dehydrogenase (GlpD) reporter enzyme assay for Escherichia coli

2019 ◽  
Author(s):  
Madeleine Huber ◽  
Jörg Soppa
Keyword(s):  
Translational Regulation ◽  
Enzyme Assay ◽  
Microtiter Plate ◽  
Dihydroxyacetone Phosphate ◽  
Electron Carrier ◽  
E Coli ◽  
Reporter Enzyme ◽  
Glycerol 3 Phosphate Dehydrogenase ◽  
Transcriptional Fusions ◽  
Microtiter Plate Format

Abstract Glycerol-3-phosphate dehydrogenase (GlpD) is a recently introduced reporter enzyme for E. coli [1]. GlpD calalyzes the oxidation of Glycerin-3-phosphate (G3P) to dihydroxyacetone-phosphate (DHAP). The oxidation is coupled to the reduction of the artificial yellow substrate tetrazol-3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromid (MTT) to a blue-violet formazan, which is mediated by the electron carrier phenazin-methanosulfate (PMS). This leads to an increase in absorption at 570 nm, which is measured to quantify the GlpD activity. To avoid background, it is best to use an E. coli strain with a deletion of the chromosomal glpD gene, which is available e.g. at the Keio collection [2]. The glpD gene can be used for transcriptional fusions, e.g. to characterize promoters, and also for translational fusions, e.g. to study translational regulation. The assay was adapted to the microtiter plate format to enable the parallel handling of a large number of samples. The “procedure” (see below) describes an application with the glpD gene in a translational fusion with the gene of interest cloned under the control of the inducible arabinose promoter PBAD.

scholarly journals Dihydrofolate reductase (DHFR) reporter enzyme assay for Haloferax volcanii

2019 ◽  
Author(s):  
Madeleine Huber ◽  
Jörg Soppa
Keyword(s):  
Dihydrofolate Reductase ◽  
Enzyme Assay ◽  
Background Level ◽  
Microtiter Plate ◽  
Haloferax Volcanii ◽  
Expression Level ◽  
Wildtype Strain ◽  
Translational Fusion ◽  
Reporter Enzyme ◽  
Microtiter Plate Format

Abstract The dihydrofolate reductase (DHFR) is routinely used a reporter enzyme for H. volcanii. The DHFR catalyzes the reduction of dihydrofolate to tetrahydrofolate and the concomitant oxidation of NADPH to NADP+. This leads to a reduction of extinction at 340 nm, which is measured to quantify the DHFR activity. To avoid background, it is best to use an H. volcanii strain with a deletion of the chromosomal dhfr gene, which is available upon request ([email protected]). However, the expression level of the chromosomal dhfr gene is very low, so that it is also possible to use the wildtype strain and subtract the DHFR background level. The assay was adapted to the microtiter plate format to enable the parallel handling of a large number of samples. The “procedure” (see below) describes an application with the dhfr gene in a translational fusion with the gene of interest.

scholarly journals Enhancement of the Efficiency of Secretion of Heterologous Lipase in Escherichia coli by Directed Evolution of the ABC Transporter System

2005 ◽  
Vol 71 (7) ◽  
pp. 3468-3474 ◽  
Author(s):  
Gyeong Tae Eom ◽  
Jae Kwang Song ◽  
Jung Hoon Ahn ◽  
Yeon Soo Seo ◽  
Joon Shick Rhee
Keyword(s):  
Escherichia Coli ◽  
Directed Evolution ◽  
Abc Transporter ◽  
Microtiter Plate ◽  
Single Amino Acid ◽  
Wild Type ◽  
E Coli ◽  
Single Amino Acid Change ◽  
Secretion Efficiency

ABSTRACT The ABC transporter (TliDEF) from Pseudomonas fluorescens SIK W1, which mediated the secretion of a thermostable lipase (TliA) into the extracellular space in Escherichia coli, was engineered using directed evolution (error-prone PCR) to improve its secretion efficiency. TliD mutants with increased secretion efficiency were identified by coexpressing the mutated tliD library with the wild-type tliA lipase in E. coli and by screening the library with a tributyrin-emulsified indicator plate assay and a microtiter plate-based assay. Four selected mutants from one round of error-prone PCR mutagenesis, T6, T8, T24, and T35, showed 3.2-, 2.6-, 2.9-, and 3.0-fold increases in the level of secretion of TliA lipase, respectively, but had almost the same level of expression of TliD in the membrane as the strain with the wild-type TliDEF transporter. These results indicated that the improved secretion of TliA lipase was mediated by the transporter mutations. Each mutant had a single amino acid change in the predicted cytoplasmic regions in the membrane domain of TliD, implying that the corresponding region of TliD was important for the improved and successful secretion of the target protein. We therefore concluded that the efficiency of secretion of a heterologous protein in E. coli can be enhanced by in vitro engineering of the ABC transporter.

scholarly journals Stochastic transcriptional pulses orchestrate flagellar biosynthesis in Escherichia coli

2020 ◽  
Vol 6 (6) ◽  
pp. eaax0947 ◽  
Author(s):  
J. Mark Kim ◽  
Mayra Garcia-Alcala ◽  
Enrique Balleza ◽  
Philippe Cluzel
Keyword(s):  
Escherichia Coli ◽  
Translational Regulation ◽  
Temporal Dynamics ◽  
Transcriptional Networks ◽  
Cell Level ◽  
E Coli ◽  
Flagellar Biosynthesis ◽  
Flagellar Assembly ◽  
Regulatory Dynamics ◽  
Flagellum Biosynthesis

The classic picture of flagellum biosynthesis in Escherichia coli, inferred from population measurements, depicts a deterministic program where promoters are sequentially up-regulated and are maintained steadily active throughout exponential growth. However, complex regulatory dynamics at the single-cell level can be masked by bulk measurements. Here, we discover that in individual E. coli cells, flagellar promoters are stochastically activated in pulses. These pulses are coordinated within specific classes of promoters and comprise “on” and “off” states, each of which can span multiple generations. We demonstrate that in this pulsing program, the regulatory logic of flagellar assembly dictates which promoters skip pulses. Surprisingly, pulses do not require specific transcriptional or translational regulation of the flagellar master regulator, FlhDC, but instead appears to be essentially governed by an autonomous posttranslational circuit. Our results suggest that even topologically simple transcriptional networks can generate unexpectedly rich temporal dynamics and phenotypic heterogeneities.

scholarly journals Novel Translation Initiation Regulation Mechanism in Escherichia coli ptrB Mediated by a 5′-Terminal AUG

2017 ◽  
Vol 199 (14) ◽  
Author(s):  
Heather J. Beck ◽  
Gary R. Janssen
Keyword(s):  
Escherichia Coli ◽  
Translation Initiation ◽  
Translational Regulation ◽  
Current Knowledge ◽  
Upstream Open Reading Frame ◽  
Model Organisms ◽  
Regulation Mechanism ◽  
Rna Regulation ◽  
Content Type ◽  
E Coli

ABSTRACT Alternative translation initiation mechanisms, distinct from the Shine-Dalgarno (SD) sequence-dependent mechanism, are more prevalent in bacteria than once anticipated. Translation of Escherichia coli ptrB instead requires an AUG triplet at the 5′ terminus of its mRNA. The 5′-terminal AUG (5′-uAUG) acts as a ribosomal recognition signal to attract ribosomes to the ptrB mRNA rather than functioning as an initiation codon to support translation of an upstream open reading frame. ptrB expression exhibits a stronger dependence on the 5′-uAUG than the predicted SD sequence; however, strengthening the predicted ptrB SD sequence relieves the necessity for the 5′-uAUG. Additional sequences within the ptrB 5′ untranslated region (5′-UTR) work cumulatively with the 5′-uAUG to control expression of the downstream ptrB coding sequence (CDS), thereby compensating for the weak SD sequence. Replacement of 5′-UTRs from other mRNAs with the ptrB 5′-UTR sequence showed a similar dependence on the 5′-uAUG for CDS expression, suggesting that the regulatory features contained within the ptrB 5′-UTR are sufficient to control the expression of other E. coli CDSs. Demonstration that the 5′-uAUG present on the ptrB leader mRNA is involved in ribosome binding and expression of the downstream ptrB CDS revealed a novel form of translational regulation. Due to the abundance of AUG triplets at the 5′ termini of E. coli mRNAs and the ability of ptrB 5′-UTR regulation to function independently of gene context, the regulatory effects of 5′-uAUGs on downstream CDSs may be widespread throughout the E. coli genome. IMPORTANCE As the field of synthetic biology continues to grow, a complete understanding of basic biological principles will be necessary. The increasing complexity of the synthetic systems highlights the gaps in our current knowledge of RNA regulation. This study demonstrates that there are novel ways to regulate canonical Shine-Dalgarno-led mRNAs in Escherichia coli, illustrating that our understanding of the fundamental processes of translation and RNA regulation is still incomplete. Even for E. coli, one of the most-studied model organisms, genes with translation initiation mechanisms that do not fit the canonical Shine-Dalgarno sequence paradigm are being revealed. Uncovering diverse mechanisms that control translational expression will allow synthetic biologists to finely tune protein production of desired gene products.

scholarly journals Evaluating the Five Escherichia coli Derivative Strains as Platform for Arginine Deiminase Overproduction

2020 ◽  
Author(s):  
Sara Abdollahi ◽  
Mohammad Hossein Morowvat ◽  
Amir Savardashtaki ◽  
Cambyz Irajie ◽  
Sohrab Najafipour ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Recombinant Protein ◽  
Microtiter Plate ◽  
Arginine Deiminase ◽  
Expression Vectors ◽  
Efficacy Evaluation ◽  
Microtiter Plate Assay ◽  
E Coli ◽  
Bacterial Enzyme ◽  
Host Strains

Abstract Escherichia coli is one of the most preferred host microorganisms for the production of recombinant proteins due to its well-characterized genome, availability of various expression vectors and host strains. Choosing a proper host strain for the overproduction of a desired recombinant protein is very important because of the diversity of genetically modified expression strains. This study attempted to evaluate the five host strains including BL21 (DE3), Rosetta (DE3), DH5α, XL1-BLUE and SHuffle in terms of arginine deiminase (ADI) production and enzyme activity. Arginine deiminase (ADI) was chosen a bacterial enzyme which degrades L-arginine. It is effective in treatment of some types of human cancers like melanoma and hepatocellular carcinoma (HCC) which are arginine-auxotrophic. Five mentioned E. coli strains were cultivated. The pET-3a was used as the expression vector. The competent E. coli cells were obtained through CaCl 2 method. It was then transformed with the construct of pET3a-ADI using heat shock strategy. The ADI production levels were examined by 10% SDS-PAGE analysis. The ability of host strains for expression of the requested recombinant protein was compared. The enzymatic activity of the obtained recombinant ADI from each studied strain was assessed by a colorimetric 96-well microtiter plate assay. All the five strains exhibited a significant band at 46 kDa. BL21 (DE3) produced the highest amount of ADI protein followed by Rosetta (DE3). The following activity assay showed that ADI from BL21 (DE3) and Rosetta (DE3) had the most activity. There are some genetic and metabolic differences among the various E. coli strains, leading to differences in the amount of recombinant protein production. The results of this study can be used for the efficacy evaluation of the five studied strains for the production of similar pharmaceutical enzymes. The strains also could be analyzed in terms of proteomics.

scholarly journals Kinetics and mechanism of the bactericidal action of human neutrophils against Escherichia coli

Blood ◽  
1984 ◽  
Vol 64 (3) ◽  
pp. 635-641
Author(s):  
MN Hamers ◽  
AA Bot ◽  
RS Weening ◽  
HJ Sips ◽  
D Roos
Keyword(s):  
Escherichia Coli ◽  
Computer Program ◽  
Rate Constants ◽  
Bacterial Cell ◽  
Cell Envelope ◽  
Human Neutrophils ◽  
Galactosidase Activity ◽  
E Coli ◽  
Bacterial Proteins ◽  
Beta Galactosidase

A mutant strain of Escherichia coli (E. coli ML-35) was used to follow the kinetics of phagocytosis, perforation of the bacterial cell envelope, and inactivation of bacterial proteins by human neutrophils. This particular E. coli mutant strain has no lactose permease, but constitutively forms the cytoplasmic enzyme beta-galactosidase. This implies that the artificial substrate ortho-nitrophenyl-beta-D- galactopyranoside cannot reach the beta-galactosidase unless the bacterial cell envelope has been perforated. Thus, the integrity of the E. coli envelope can be measured simply by the activity of beta- galactosidase with this substrate. Indeed, ingestion of E. coli ML-35 by human neutrophils was followed by perforation of the bacteria (increase in beta-galactosidase activity). Subsequently, the beta- galactosidase activity decreased due to inactivation of the enzyme. With a simple mathematical model and a curve-fitting computer program, we have determined the first-order rate constants for phagocytosis, perforation, and beta-galactosidase inactivation. With 32 normal donors, we found an interdonor variation in these rate constants of 20% to 30% (SD) and an assay variance of 5%. The perforation process closely correlated with the loss of colony-forming capacity of the bacteria. This new assay measures phagocytosis and killing in a fast, simple, and accurate way; it is not hindered by extracellular bacteria. Moreover, this method also measures the postkilling event of inactivation of a bacterial protein, which permits a better detection of neutrophils deficient in this function. The assay can also be used for screening neutrophil functions without the use of a computer program. A simple calculation suffices to detect neutrophil abnormalities. Neutrophils from patients with chronic granulomatous disease (CGD) showed an impaired rate of perforation and thus also of inactivation. Neutrophils from myeloperoxidase-deficient patients or from a patient with the Chediak-Higashi syndrome only showed a retarded inactivation of beta-galactosidase, but normal ingestion and perforation. The role of myeloperoxidase in the killing process is discussed. Although myeloperoxidase does not seem to be a prerequisite for perforation, it probably plays a role in bacterial destruction by normal cells, because the inactivation of bacterial proteins seems strictly myeloperoxidase dependent.

scholarly journals Rapid Accumulation of Motility-Activating Mutations in Resting Liquid Culture ofEscherichia coli

2019 ◽  
Vol 201 (19) ◽  
Author(s):  
Darren J. Parker ◽  
Pınar Demetci ◽  
Gene-Wei Li
Keyword(s):  
Gene Expression ◽  
Escherichia Coli ◽  
Liquid Medium ◽  
Regulatory Networks ◽  
Gene Knockout ◽  
Single Gene ◽  
Point Mutations ◽  
Content Type ◽  
E Coli ◽  
Keio Collection

ABSTRACTExpression of motility genes is a potentially beneficial but costly process in bacteria. Interestingly, many isolate strains ofEscherichia colipossess motility genes but have lost the ability to activate them under conditions in which motility is advantageous, raising the question of how they respond to these situations. Through transcriptome profiling of strains in theE. colisingle-gene knockout Keio collection, we noticed drastic upregulation of motility genes in many of the deletion strains compared to levels in their weakly motile parent strain (BW25113). We show that this switch to a motile phenotype is not a direct consequence of the genes deleted but is instead due to a variety of secondary mutations that increase the expression of the major motility regulator, FlhDC. Importantly, we find that this switch can be reproduced by growing poorly motileE. colistrains in nonshaking liquid medium overnight but not in shaking liquid medium. Individual isolates after the nonshaking overnight incubations acquired distinct mutations upstream of theflhDCoperon, including different insertion sequence (IS) elements and, to a lesser extent, point mutations. The rapidity with which genetic changes sweep through the populations grown without shaking shows that poorly motile strains can quickly adapt to a motile lifestyle by genetic rewiring.IMPORTANCEThe ability to tune gene expression in times of need outside preordained regulatory networks is an essential evolutionary process that allows organisms to survive and compete. Here, we show that upon overnight incubation in liquid medium without shaking, populations of largely nonmotileEscherichia colibacteria can rapidly accumulate mutants that have constitutive motility. This effect contributes to widespread secondary mutations in the single-gene knockout library, the Keio collection. As a result, 49/71 (69%) of the Keio strains tested exhibited various degrees of motility, whereas their parental strain is poorly motile. These observations highlight the plasticity of gene expression even in the absence of preexisting regulatory programs and should raise awareness of procedures for handling laboratory strains ofE. coli.

scholarly journals Kinetics and mechanism of the bactericidal action of human neutrophils against Escherichia coli

Blood ◽  
1984 ◽  
Vol 64 (3) ◽  
pp. 635-641 ◽  
Author(s):  
MN Hamers ◽  
AA Bot ◽  
RS Weening ◽  
HJ Sips ◽  
D Roos
Keyword(s):  
Escherichia Coli ◽  
Computer Program ◽  
Rate Constants ◽  
Bacterial Cell ◽  
Cell Envelope ◽  
Human Neutrophils ◽  
Galactosidase Activity ◽  
E Coli ◽  
Bacterial Proteins ◽  
Beta Galactosidase

Abstract A mutant strain of Escherichia coli (E. coli ML-35) was used to follow the kinetics of phagocytosis, perforation of the bacterial cell envelope, and inactivation of bacterial proteins by human neutrophils. This particular E. coli mutant strain has no lactose permease, but constitutively forms the cytoplasmic enzyme beta-galactosidase. This implies that the artificial substrate ortho-nitrophenyl-beta-D- galactopyranoside cannot reach the beta-galactosidase unless the bacterial cell envelope has been perforated. Thus, the integrity of the E. coli envelope can be measured simply by the activity of beta- galactosidase with this substrate. Indeed, ingestion of E. coli ML-35 by human neutrophils was followed by perforation of the bacteria (increase in beta-galactosidase activity). Subsequently, the beta- galactosidase activity decreased due to inactivation of the enzyme. With a simple mathematical model and a curve-fitting computer program, we have determined the first-order rate constants for phagocytosis, perforation, and beta-galactosidase inactivation. With 32 normal donors, we found an interdonor variation in these rate constants of 20% to 30% (SD) and an assay variance of 5%. The perforation process closely correlated with the loss of colony-forming capacity of the bacteria. This new assay measures phagocytosis and killing in a fast, simple, and accurate way; it is not hindered by extracellular bacteria. Moreover, this method also measures the postkilling event of inactivation of a bacterial protein, which permits a better detection of neutrophils deficient in this function. The assay can also be used for screening neutrophil functions without the use of a computer program. A simple calculation suffices to detect neutrophil abnormalities. Neutrophils from patients with chronic granulomatous disease (CGD) showed an impaired rate of perforation and thus also of inactivation. Neutrophils from myeloperoxidase-deficient patients or from a patient with the Chediak-Higashi syndrome only showed a retarded inactivation of beta-galactosidase, but normal ingestion and perforation. The role of myeloperoxidase in the killing process is discussed. Although myeloperoxidase does not seem to be a prerequisite for perforation, it probably plays a role in bacterial destruction by normal cells, because the inactivation of bacterial proteins seems strictly myeloperoxidase dependent.

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