scholarly journals rRNA Promoter Activity in the Fast-Growing Bacterium Vibrio natriegens

2002 ◽  
Vol 184 (5) ◽  
pp. 1349-1358 ◽  
Author(s):  
Sarah E. Aiyar ◽  
Tamas Gaal ◽  
Richard L. Gourse
Keyword(s):  
Growth Rate ◽  
Protein Synthesis ◽  
High Capacity ◽  
High Rate ◽  
Core Promoters ◽  
E Coli ◽  
Rrna Transcription ◽  
High Gene ◽  
Up Elements

ABSTRACT The bacterium Vibrio natriegens can double with a generation time of less than 10 min (R. G. Eagon, J. Bacteriol. 83:736-737, 1962), a growth rate that requires an extremely high rate of protein synthesis. We show here that V. natriegens' high potential for protein synthesis results from an increase in ribosome numbers with increasing growth rate, as has been found for other bacteria. We show that V. natriegens contains a large number of rRNA operons, and its rRNA promoters are extremely strong. The V. natriegens rRNA core promoters are at least as active in vitro as Escherichia coli rRNA core promoters with either E. coli RNA polymerase (RNAP) or V. natriegens RNAP, and they are activated by UP elements, as in E. coli. In addition, the E. coli transcription factor Fis activated V. natriegens rrn P1 promoters in vitro. We conclude that the high capacity for ribosome synthesis in V. natriegens results from a high capacity for rRNA transcription, and the high capacity for rRNA transcription results, at least in part, from the same factors that contribute most to high rates of rRNA transcription in E. coli, i.e., high gene dose and strong activation by UP elements and Fis.

scholarly journals Escherichia coli Promoters with UP Elements of Different Strengths: Modular Structure of Bacterial Promoters

1998 ◽  
Vol 180 (20) ◽  
pp. 5375-5383 ◽  
Author(s):  
Wilma Ross ◽  
Sarah E. Aiyar ◽  
Julia Salomon ◽  
Richard L. Gourse
Keyword(s):  
Escherichia Coli ◽  
Core Promoter ◽  
Consensus Sequence ◽  
Α Subunit ◽  
Core Promoters ◽  
E Coli ◽  
Promoter Recognition ◽  
Transcription In Vitro ◽  
Up Elements

ABSTRACT The α subunit of Escherichia coli RNA polymerase (RNAP) participates in promoter recognition through specific interactions with UP element DNA, a region upstream of the recognition hexamers for the ς subunit (the −10 and −35 hexamers). UP elements have been described in only a small number of promoters, including the rRNA promoter rrnB P1, where the sequence has a very large (30- to 70-fold) effect on promoter activity. Here, we analyzed the effects of upstream sequences from several additional E. coli promoters (rrnD P1, rrnB P2, λp R, lac, merT, and RNA II). The relative effects of different upstream sequences were compared in the context of their own core promoters or as hybrids to thelac core promoter. Different upstream sequences had different effects, increasing transcription from 1.5- to ∼90-fold, and several had the properties of UP elements: they increased transcription in vitro in the absence of accessory protein factors, and transcription stimulation required the C-terminal domain of the RNAP α subunit. The effects of the upstream sequences correlated generally with their degree of similarity to an UP element consensus sequence derived previously. Protection of upstream sequences by RNAP in footprinting experiments occurred in all cases and was thus not a reliable indicator of UP element strength. These data support a modular view of bacterial promoters in which activity reflects the composite effects of RNAP interactions with appropriately spaced recognition elements (−10, −35, and UP elements), each of which contributes to activity depending on its similarity to the consensus.

scholarly journals Development and comparison of cell-free protein synthesis systems derived from typical bacterial chassis

2021 ◽  
Vol 8 (1) ◽  
Author(s):  
Liyuan Zhang ◽  
Xiaomei Lin ◽  
Ting Wang ◽  
Wei Guo ◽  
Yuan Lu
Keyword(s):  
Protein Synthesis ◽  
Protein Production ◽  
Influential Factors ◽  
Competent Cell ◽  
Free Protein ◽  
Application Range ◽  
E Coli ◽  
Short Time ◽  
Cell Free Protein Synthesis

AbstractCell-free protein synthesis (CFPS) systems have become an ideal choice for pathway prototyping, protein production, and biosensing, due to their high controllability, tolerance, stability, and ability to produce proteins in a short time. At present, the widely used CFPS systems are mainly based on Escherichia coli strain. Bacillus subtilis, Corynebacterium glutamate, and Vibrio natriegens are potential chassis cells for many biotechnological applications with their respective characteristics. Therefore, to expand the platform of the CFPS systems and options for protein production, four prokaryotes, E. coli, B. subtilis, C. glutamate, and V. natriegens were selected as host organisms to construct the CFPS systems and be compared. Moreover, the process parameters of the CFPS system were optimized, including the codon usage, plasmid synthesis competent cell selection, plasmid concentration, ribosomal binding site (RBS), and CFPS system reagent components. By optimizing and comparing the main influencing factors of different CFPS systems, the systems can be optimized directly for the most influential factors to further improve the protein yield of the systems. In addition, to demonstrate the applicability of the CFPS systems, it was proved that the four CFPS systems all had the potential to produce therapeutic proteins, and they could produce the receptor-binding domain (RBD) protein of SARS-CoV-2 with functional activity. They not only could expand the potential options for in vitro protein production, but also could increase the application range of the system by expanding the cell-free protein synthesis platform.

An in vitro bioassay for a mulluscan gonadotropin

1975 ◽  
Vol 62 (2) ◽  
pp. 433-446
Author(s):  
M. J. Wells ◽  
R. K. O'Dor ◽  
S. K. Buckley
Keyword(s):  
Protein Synthesis ◽  
High Rate ◽  
Follicle Cells ◽  
Octopus Vulgaris ◽  
Amino Acid Incorporation ◽  
Gland Extract ◽  
Acid Incorporation ◽  
Kinetics Of

1. Protein synthesis occurs at a high rate in the ovaries of maturing Octopus vulgaris and can be measured from the incorporation of [14C]leucine in vivo and in isolated groups of eggs in vitro. 2. Removal of the optic glands in vivo 1--3 days prior to testing markedly reduces amino acid incorporation in vivo or in vitro. After 5 days in vivo incorporation stops. 3. The rate of incorporation in vitro is increased by the addition of optic gland extract. 4. Analysis of the kinetics of leucine uptake and incorporation in vitro indicates that the hormone has an effect on the inward transport of leucine which is independent of its action on protein synthesis. 5. Electron-microscope studies of the follicle cells and ova show that the former are the site of protein synthesis. 6. Changes in either uptake or incorporation into protein by the follicle cells can be used as a qualitative biolobical assay for the optic gland hormone. Uptake is very easy to measure but incorporation is the more sensitive parameter. Either is potentially suitable as a quantitative assay for this and perhaps also for other molluscan gonadotropins.

Effect of Tannins on the In Vitro Growth of Escherichia coli O157:H7 and In Vivo Growth of Generic Escherichia coli Excreted from Steers

2007 ◽  
Vol 70 (3) ◽  
pp. 543-550 ◽  
Author(s):  
BYENG R. MIN ◽  
WILLIAM E. PINCHAK ◽  
ROBIN C. ANDERSON ◽  
TODD R. CALLAWAY
Keyword(s):  
Escherichia Coli ◽  
Growth Rate ◽  
Bacterial Growth ◽  
Escherichia Coli O157 ◽  
Tannin Extract ◽  
Bacterial Growth Rate ◽  
Linear Effect ◽  
E Coli

The effect of commercially available chestnut and mimosa tannins in vitro (experiment 1) or in vivo (experiment 2) on the growth or recovery of Escherichia coli O157:H7 or generic fecal E. coli was evaluated. In experiment 1, the mean growth rate of E. coli O157:H7, determined via the measurement of optical density at 600 nm during anaerobic culture in tryptic soy broth at 37°C, was reduced (P < 0.05) with as little as 400 μg of either tannin extract per ml of culture fluid. The addition of 200, 400, 600, 800, and 1,200 μg of tannins per ml significantly (P < 0.01) reduced the specific bacterial growth rate when compared with the nontannin control. The specific growth rate decreased with increasing dose levels up to 800 μg of tannins per ml. Bacterial growth inhibition effects in chestnut tannins were less pronounced than in mimosa tannins. Chestnut tannin extract addition ranged from 0 to 1,200 μg/ml, and a linear effect (P < 0.05) was observed in cultures incubated for 6 h against the recovery of viable cells, determined via the plating of each strain onto MacConkey agar, of E. coli O157:H7 strains 933 and 86-24, but not against strain 6058. Similar tests with mimosa tannin extract showed a linear effect (P < 0.05) against the recovery of E. coli O157:H7 strain 933 only. The bactericidal effect observed in cultures incubated for 24 h with the tannin preparations was similar, although it was less than that observed from cultures incubated for 6 h. When chestnut tannins (15 g of tannins per day) were infused intraruminally to steers fed a Bermuda grass hay diet in experiment 2, fecal E. coli shedding was lower on days 3 (P < 0.03), 12 (P = 0.08), and 15 (P < 0.001) when compared with animals that were fed a similar diet without tannin supplementation. It was concluded that dietary levels and sources of tannins potentially reduce the shedding of E. coli from the gastrointestinal tract.

scholarly journals Peptide-Based Formulation from Lactic Acid Bacteria Impairs the Pathogen Growth in Ananas Comosus (Pineapple)

Coatings ◽  
2020 ◽  
Vol 10 (5) ◽  
pp. 457 ◽  
Author(s):  
Gabriela N. Tenea ◽  
Daniela Olmedo ◽  
Clara Ortega
Keyword(s):  
Significant Proportion ◽  
High Capacity ◽  
Total Cell ◽  
Ananas Comosus ◽  
Street Vending ◽  
E Coli ◽  
Cell Counts ◽  
Total Cell Density

Worldwide, street vending commerce has grown exponentially, representing in some countries, including Ecuador, a significant proportion of food consumed by the urban population. Pineapple is one of the common fruits sold as ready-to-eat slices by ambulant vendors in the street or on public transport at risk of contamination by various microorganisms. Previously, we selected Lactobacillus plantarum UTNCys5-4 and Lactococcus lactis subsp. lactis Gt28 strains producing peptides with high capacity to inhibit pathogen growth in vitro. In this study, the effect of different edited formulations containing a mixture of Cys5-4/Gt28 peptides was evaluated in vitro and ex vitro against a pathogenic cocktail containing E. coli (2), Salmonella (2) and Shigella (1). The growth of bacterial cocktail co-inoculated with cell-free supernatant containing peptides (formulation T1) and precipitated peptides (formulation T6), in a ratio of Cys5-4/Gt28:1:1 (v/v), results in a decrease of total cell viability with 1.85 and 1.2 log CFU/mL orders of magnitude at 6 h of incubation. About the same decrease (1.9 log CFU/g) was observed when pineapple slices artificially inoculated with the pathogenic cocktail were coated with T1 formulation, indicating the capacity to diminish simultaneous pathogens in situ, thus demonstrating its great biological control and protection. However, the E. coli cell counts reduced by 2.08 log CFU/g while Salmonella and Shigella cell counts reduced by 1.43 and 1.91 log CFU/g, respectively, at 5 days of refrigeration. In the untreated pineapple slices, the total cell density was maintained during storage, suggesting the adaptation of the pathogens to the fruit matrix. The peptide-based formulation exerted a bacteriolytic mode of action inducing pathogenic cell death. The results indicate that coating pineapple slices with peptide-based formulation is a promising approach to protect them from further contamination by microbial spoilage as well as an alternative to increase the food safety.

In vitro protein synthesis directed by R17 viral ribonucleic acid. II. On the fate of mRNA in the cell-free system

1969 ◽  
Vol 47 (12) ◽  
pp. 1179-1186 ◽  
Author(s):  
Satomi J. Igarashi
Keyword(s):  
Amino Acids ◽  
Protein Synthesis ◽  
Amino Acid Incorporation ◽  
Free System ◽  
Cell Free System ◽  
E Coli ◽  
Acid Incorporation ◽  
Binding Factor ◽  
Vitro Protein

In the crude E. coli B cell-free system, mRNA was hydrolyzed by contaminating nuclease activities before significant polymerization of amino acids took place. Ribosomes appeared to be one of the sources of nuclease. A modified high-salt washing procedure was developed to remove nuclease from ribosomes. RNase-free ribosomes thus obtained appeared to be inactive in poly-U-directed phenylalanine incorporation, unless poly-U binding factor was added to the system. R17 RNA could not direct amino acid incorporation in the presence of RNase-free ribosomes because binding of intact R17 RNA to ribosomes did not take place even in the presence of poly-U binding factor.

scholarly journals Expression of novel fusion antiviral proteins Ricin A Chain-Pokeweed Antiviral Proteins (RTA-PAPs) in Escherichia coli and their inhibition of protein synthesis and of hepatitis B virus in vitro

2018 ◽  
Author(s):  
Yasser Hassan ◽  
Sherry Ogg ◽  
Hui Ge
Keyword(s):  
Protein Synthesis ◽  
Expression System ◽  
Therapeutic Index ◽  
Antiviral Protein ◽  
E Coli ◽  
Protein Isoform ◽  
Antiviral Proteins ◽  
A Chain

AbstractRicin A chain (RTA) and Pokeweed antiviral proteins (PAPs) are plant-derived N-glycosidase ribosomal-inactivating proteins (RIPs) isolated from Ricinus communis and Phytolacca Americana respectively. This study was to investigate the potential antiviral value of novel fusion proteins between RTA and PAPs (RTA-PAPs). In brief, RTA-Pokeweed antiviral protein isoform 1 from seeds (RTA-PAPS1) was produced in E. coli in vivo expression system, purified from inclusion bodies using gel filtration chromatography and protein synthesis inhibitory activity assayed by comparison to the production of a control protein Luciferase. The antiviral activity of the RTA-PAPS1 against Hepatitis B virus (HBV) in HepAD38 cells was then determined using a dose response assay by quantifying supernatant HBV DNA compared to control virus infected HepAD38 cells. The cytotoxicity in HepAD38 cells was determined by measuring cell viability using a tetrazolium dye uptake assay. Results showed that RTA-PAPS1 could effectively be recovered and purified from inclusion bodies. The refolded protein was bioactive with 50% protein synthesis inhibitory concentration (IC50) of 0.06nM (3.63ng/ml). The results also showed that RTA-PAPS1 had a synergetic activity against HBV with a half-maximal response concentration value (EC50) of 0.03nM (1.82ng/ml) and a therapeutic index of >21818. The fusion protein was further optimized using in silico tools, produced in E. coli in vivo expression system, purified by three-step process from soluble lysate and protein synthesis inhibition activity assayed. Results showed that the optimized protein RTA mutant-Pokeweed antiviral protein isoform 1 from leaves (RTAM-PAP1) could be recovered and purified from soluble lysates with gain of function activity on protein synthesis inhibition with an IC50 of 0.03nM (1.82ng/ml). Collectively, our results demonstrate that RTA-PAPs are amenable to effective production and purification in native form, possess significant antiviral activity against HBV in vitro with a high therapeutic index and, thus, meriting further development as potential antiviral agents against chronic HBV infection.

Compartmentalization of an all-E. coli Cell-Free Expression System for the Construction of a Minimal Cell

2016 ◽  
Vol 22 (2) ◽  
pp. 185-195 ◽  
Author(s):  
Filippo Caschera ◽  
Vincent Noireaux
Keyword(s):  
Protein Synthesis ◽  
Expression System ◽  
Coli Cell ◽  
Free Expression ◽  
Genetic Circuit ◽  
Translation System ◽  
Minimal Cell ◽  
E Coli ◽  
Synthetic Cell

Cell-free expression is a technology used to synthesize minimal biological cells from natural molecular components. We have developed a versatile and powerful all-E. coli cell-free transcription–translation system energized by a robust metabolism, with the far objective of constructing a synthetic cell capable of self-reproduction. Inorganic phosphate (iP), a byproduct of protein synthesis, is recycled through polysugar catabolism to regenerate ATP (adenosine triphosphate) and thus supports long-lived and highly efficient protein synthesis in vitro. This cell-free TX-TL system is encapsulated into cell-sized unilamellar liposomes to express synthetic DNA programs. In this work, we study the compartmentalization of cell-free TX-TL reactions, one of the aspects of minimal cell module integration. We analyze the signals of various liposome populations by fluorescence microscopy for one and for two reporter genes, and for an inducible genetic circuit. We show that small nutrient molecules and proteins are encapsulated uniformly in the liposomes with small fluctuations. However, cell-free expression displays large fluctuations in signals among the same population, which are due to heterogeneous encapsulation of the DNA template. Consequently, the correlations of gene expression with the compartment dimension are difficult to predict accurately. Larger vesicles can have either low or high protein yields.

scholarly journals Biosynthesis of Escherichia coli adenosine deaminase using cell-free protein synthesis

2021 ◽  
Vol 66 (3) ◽  
pp. 271-276
Author(s):  
I. S. Kazlouski ◽  
A. I. Zinchenko
Keyword(s):  
Escherichia Coli ◽  
Protein Synthesis ◽  
Adenosine Deaminase ◽  
Submerged Cultivation ◽  
Process Conditions ◽  
Experimental Sample ◽  
Free Protein ◽  
E Coli ◽  
Cell Free Protein Synthesis

One of the recent perspective trends of molecular biotechnology is cell-free protein synthesis (CFPS). The procedure of CFPS is based on in vitro reconstruction of all stages of a biosynthesis of protein in a whole cell, including a transcription, an aminoacylation of tRNA and translation of mRNA by ribosomes.Previously, we constructed a strain Escherichia coli that produces homologous adenosine deaminase (ADase). In the present study, as an alternative to canonical submerged cultivation in a fermenter, the possibility of the ADase synthesis in the system of CFPS was studied. For synthesis of this enzyme we used the E. coli-30 cell extract, T7 bacteriophage RNA polymerase, and high-copy plasmid vector pET42mut with gene ADase inserted into it.As a result of the work we have demonstrated for the first time the possibility of synthesis of ADase E. coli in the CFPS system. In a partially optimized process conditions, an experimental sample of recombinant AD with an activity of 530 U/ml of enzyme preparation was obtained.

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