Utilizing in vitro DNA assembly to engineer a synthetic T7 Nanoluc reporter phage for Escherichia coli detection

2019 ◽  
Vol 11 (3) ◽  
pp. 63-68 ◽  
Author(s):  
Elsi M Pulkkinen ◽  
Troy C Hinkley ◽  
Sam R Nugen
Keyword(s):  
Escherichia Coli ◽  
Liquid Culture ◽  
Detection Methods ◽  
Luciferase Expression ◽  
Dna Assembly ◽  
Bacterial Populations ◽  
Low Concentrations ◽  
Health And Disease ◽  
T7 Phage

Abstract Bacteria have major role in regulating human health and disease, therefore, there is a continuing need to develop new detection methods and therapeutics to combat them. Bacteriophages can be used to infect specific bacteria, which make them good candidates for detecting and editing bacterial populations. However, creating phage-based detection assays is somewhat limited by the difficulties in the engineering of phages. We present here a synthetic biology strategy to engineer phages using a simple in vitro method. We used this method to insert a NanoLuc luciferase expression cassette into the T7 phage, in order to construct the NRGp6 reporter phage. The synthetic NRGp6 phage was used to efficiently detect low concentrations of Escherichia coli from liquid culture. We envision that our approach will benefit synthetic biologists for constructing different kinds of engineered phages, and enable new approaches for phage-based therapeutics and diagnostics.

scholarly journals In Vivo Bioluminescence Imaging for the Study of Intestinal Colonization by Escherichia coli in Mice

2009 ◽  
Vol 76 (1) ◽  
pp. 264-274 ◽  
Author(s):  
M.-L. Foucault ◽  
L. Thomas ◽  
S. Goussard ◽  
B. R. Branchini ◽  
C. Grillot-Courvalin
Keyword(s):  
Escherichia Coli ◽  
Bioluminescence Imaging ◽  
Light Emission ◽  
Direct Method ◽  
Firefly Luciferase ◽  
Intestinal Colonization ◽  
Bacterial Populations ◽  
Bioluminescence Signal

ABSTRACT Bioluminescence imaging (BLI) is emerging as a powerful tool for real-time monitoring of infections in living animals. However, since luciferases are oxygenases, it has been suggested that the requirement for oxygen may limit the use of BLI in anaerobic environments, such as the lumen of the gut. Strains of Escherichia coli harboring the genes for either the bacterial luciferase from Photorhabdus luminescens or the PpyRE-TS and PpyGR-TS firefly luciferase mutants of Photinus pyralis (red and green thermostable P. pyralis luciferase mutants, respectively) have been engineered and used to monitor intestinal colonization in the streptomycin-treated mouse model. There was excellent correlation between the bioluminescence signal measured in the feces (R 2 = 0.98) or transcutaneously in the abdominal region of whole animals (R 2 = 0.99) and the CFU counts in the feces of bacteria harboring the luxABCDE operon. Stability in vivo of the bioluminescence signal was achieved by constructing plasmid pAT881(pGB2ΩPamiluxABCDE), which allowed long-term monitoring of intestinal colonization without the need for antibiotic selection for plasmid maintenance. Levels of intestinal colonization by various strains of E. coli could be compared directly by simple recording of the bioluminescence signal in living animals. The difference in spectra of light emission of the PpyRE-TS and PpyGR-TS firefly luciferase mutants and dual bioluminescence detection allowed direct in vitro and in vivo quantification of two bacterial populations by measurement of red and green emitted signals and thus monitoring of the two populations simultaneously. This system offers a simple and direct method to study in vitro and in vivo competition between mutants and the parental strain. BLI is a useful tool to study intestinal colonization.

scholarly journals A rationally designed c-di-AMP FRET biosensor to monitor nucleotide dynamics

2021 ◽  
Author(s):  
Alex J. Pollock ◽  
Philip H. Choi ◽  
Shivam A. Zaver ◽  
Liang Tong ◽  
Joshua J. Woodward
Keyword(s):  
Single Cells ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Second Messenger ◽  
Adenosine Monophosphate ◽  
Luria Broth ◽  
Effector Proteins ◽  
Detection Methods ◽  
Bacterial Populations

ABSTRACT3’3’-cyclic di-adenosine monophosphate (c-di-AMP) is an important nucleotide second messenger found throughout the bacterial domain of life. C-di-AMP is essential in many bacteria and regulates a diverse array of effector proteins controlling pathogenesis, cell wall homeostasis, osmoregulation, and central metabolism. Despite the ubiquity and importance of c-di-AMP, methods to detect this signaling molecule are limited, particularly at single cell resolution. In this work, crystallization of the Listeria monocytogenes c-di-AMP effector protein Lmo0553 enabled structure guided design of a Förster resonance energy transfer (FRET) based biosensor, which we have named CDA5. CDA5 is a fully genetically encodable, specific, and reversible biosensor which allows for the detection of c-di-AMP dynamics both in vitro and within live single cells in a nondestructive manner. Our initial studies identify a unimodal distribution of c-di-AMP in Bacillus subtilis which decreases rapidly when cells are grown in diluted Luria Broth. Furthermore, we find that B. subtilis mutants lacking either a c-di-AMP phosphodiesterase or cyclase have respectively higher and lower FRET responses, again in a unimodal manner. These findings provide novel insight into c-di-AMP distribution within bacterial populations and establish CDA5 as a powerful platform for characterizing new aspects of c-di-AMP regulation.ImportanceC-di-AMP is an important nucleotide second messenger for which detection methods are severely limited. In this work we engineer and implement a c-di-AMP specific FRET biosensor to remedy this dearth. We present this biosensor, CDA5, as a versatile tool to investigate previously intractable facets of c-di-AMP biology.

scholarly journals Blood Volume Required for Detection of Low Levels and Ultralow Levels of Organisms Responsible for Neonatal Bacteremia by Use of Bactec Peds Plus/F, Plus Aerobic/F Medium, and the BD Bactec FX System: anIn VitroStudy

2015 ◽  
Vol 53 (11) ◽  
pp. 3609-3613 ◽  
Author(s):  
Diana P. Lancaster ◽  
David F. Friedman ◽  
Kathleen Chiotos ◽  
Kaede V. Sullivan
Keyword(s):  
Escherichia Coli ◽  
Staphylococcus Epidermidis ◽  
Candida Parapsilosis ◽  
Content Type ◽  
Low Concentrations ◽  
Low Levels ◽  
Blood Volumes ◽  
Bactec Fx ◽  
Ml Detection

We used anin vitrotechnique to investigate blood volumes required to detect bacteremia and fungemia with low concentrations of an organism. At 1 to 10 CFU/ml,Escherichia coli,Staphylococcus epidermidis,Staphylococcus aureus,Listeria monocytogenes,Candida albicans, andCandida parapsilosisisolates were detected in volumes as low as 0.5 ml. Detection ofStreptococcus agalactiaeand detection of bacteremia at <1 CFU/ml were unreliable.

scholarly journals Compensatory mutations modulate the competitiveness and dynamics of plasmid-mediated colistin resistance in Escherichia coli clones

2020 ◽  
Vol 14 (3) ◽  
pp. 861-865 ◽  
Author(s):  
Qiu E. Yang ◽  
Craig MacLean ◽  
Andrei Papkou ◽  
Manon Pritchard ◽  
Lydia Powell ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Fitness Landscape ◽  
Fitness Cost ◽  
Colistin Resistance ◽  
Bacterial Populations ◽  
Compensatory Evolution ◽  
Neutral Mutations ◽  
The Cost ◽  
Negative Epistasis

AbstractThe emergence of mobile colistin resistance (mcr) threatens to undermine the clinical efficacy of the last antibiotic that can be used to treat serious infections caused by Gram-negative pathogens. Here we measure the fitness cost of a newly discovered MCR-3 using in vitro growth and competition assays. mcr-3 expression confers a lower fitness cost than mcr-1, as determined by competitive ability and cell viability. Consistent with these findings, plasmids carrying mcr-3 have higher stability than mcr-1 plasmids across a range of Escherichia coli strains. Crucially, mcr-3 plasmids can stably persist, even in the absence of colistin. Recent compensatory evolution has helped to offset the cost of mcr-3 expression, as demonstrated by the high fitness of mcr-3.5 as opposed to mcr-3.1. Reconstructing all of the possible evolutionary trajectories from mcr-3.1 to mcr-3.5 reveals a complex fitness landscape shaped by negative epistasis between compensatory and neutral mutations. Our findings highlight the importance of fitness costs and compensatory evolution in driving the dynamics and stability of mobile colistin resistance in bacterial populations, and they highlight the need to understand how processes (other than colistin use) impact mcr dynamics.

Catalase: an old enzyme with a new role?

1984 ◽  
Vol 62 (10) ◽  
pp. 1006-1014 ◽  
Author(s):  
Maire E. Percy
Keyword(s):  
Physiological Role ◽  
Electron Donors ◽  
Biologically Active ◽  
Intracellular Enzyme ◽  
Published Evidence ◽  
Erythrocyte Catalase ◽  
Low Concentrations ◽  
Adrenergic Antagonist ◽  
Health And Disease

Although animal catalase has been studied for decades, its physiological role has remained perplexing. It has two enzymatic functions, not only catalyzing the breakdown of H2O2 into O2 and H2O, but also in the presence of low concentrations of H2O2 catalyzing the oxidation of electron donors such as ethanol or phenols. In this article, I have summarized some well-known properties of the enzyme and have also described several recently discovered features. Of particular interest is the finding that, although catalase has been regarded as an intracellular enzyme, there is published evidence for its association with the plasma membrane of the erythrocyte. Moreover, recent work from my laboratory indicates that in vitro at alkaline pH in the presence of Mg2+, the biologically active diphenols (β-3,4-dihydroxyphenylalanine and the β-adrenergic agonists isoproterenol, norepinephrine, and epinephrine) appear to function as electron donor substrates for human erythrocyte catalase and inhibit the production of O2 from H2O2 at micromolar concentrations. The β-adrenergic antagonist propranolol inhibits O2 production much less effectively and appears to competitively inhibit the reaction of catalase with epinephrine. These observations suggest an analogy between catalase and the β-adrenergic hormone receptor and raise many questions of interest to basic science, health, and disease.

scholarly journals Purification and characterization of the imidazoleglycerol-phosphate dehydratase of Saccharomyces cerevisiae from recombinant Escherichia coli

1995 ◽  
Vol 306 (2) ◽  
pp. 385-397 ◽  
Author(s):  
T R Hawkes ◽  
P G Thomas ◽  
L S Edwards ◽  
S J Rayner ◽  
K W Wilkinson ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Saccharomyces Cerevisiae ◽  
Active Sites ◽  
Active Form ◽  
Recombinant Escherichia Coli ◽  
Assay Method ◽  
Progress Curve ◽  
Low Concentrations

The HIS3+ gene of Saccharomyces cerevisiae was overexpressed in Escherichia coli and the recombinant imidazoleglycerol-phosphate dehydratase (IGPD) purified to homogeneity. Laser-desorption and electrospray m.s. indicated a molecular ion within 2 units of that expected (23833.3) on the basis of the protein sequence, with about half of the polypeptide lacking the N-terminal formylmethionine residue. IGPD initially purified as an apoprotein was catalytically inactive and mainly a trimer of M(r) 70,000. Addition of Mn2+ (but not Mg2+) caused this to assemble to an active (40 units/mg) enzyme (Mn-IGPD) comprising of 24 subunits (M(r) 573,000) and containing 1.35 +/- 0.1 Mn atoms/polypeptide subunit. An enzyme with an identical activity and metal content was also obtained when the fermenter growth medium of recombinant Escherichia coli was supplemented with MnCl2, and IGPD was purified through as Mn-IGPD rather than as the apoenzyme and assembled in vitro. Inhibition by EDTA indicated that the intrinsic Mn2+ was essential for activity. The retention of activity over time after dilution to very low concentrations of enzyme (< 20 nM) indicated that the metal remained in tight association with the protein. A novel continuous assay method was developed to facilitate the kinetic characterization of Mn-IGPD. At pH 7.0, the Km for IGP was 0.10 +/- 0.02 mM and the Ki value for inhibition by 1,2,4-triazole, 0.12 +/- 0.02 mM. In contrast with other reports, thiols had no influence on catalytic activity. The activity of Mn-IGPD varied with enzyme concentration in such a way as to suggest that it dissociates to a less active form at very low concentrations. Significant inhibition by the product, imidazole acetol phosphate, was inferred from the shape of the progress curve. Titration with, the potent competitive inhibitor, 2-hydroxy-3-(1,2,4-triazol-1-yl)propyl phosphonate indicated that Mn-IGPD contained 0.9 +/- 0.1 catalytic sites/protomer. The activity nearly doubled in the presence of high concentrations of Mn2+; the apparent Ks for stimulation was 20 microM. The basis of this effect was obscure, since there was no corresponding increase in the titre of active sites. Neither was there a discernable shift in the values of Km or Ki (above), although exogenous Mn2+ did reduce the optimum pH for kcat, from 7.2 to 6.8. On the basis of a single site/subunit, the maximum rate of catalytic turnover at 30 degrees C was 32 s-1.

scholarly journals Methylase-assisted subcloning for high throughput BioBrick assembly

PeerJ ◽  
2020 ◽  
Vol 8 ◽  
pp. e9841
Author(s):  
Ichiro Matsumura
Keyword(s):  
Escherichia Coli ◽  
Dna Methyltransferase ◽  
Dna Assembly ◽  
Site Specific ◽  
Restriction Fragments ◽  
E Coli ◽  
Restriction Sites ◽  
Unique Restriction ◽  
Biobrick Parts

The BioBrick standard makes possible iterated pairwise assembly of cloned parts without any depletion of unique restriction sites. Every part that conforms to the standard is compatible with every other part, thereby fostering a worldwide user community. The assembly methods, however, are labor intensive or inefficient compared to some newer ones so the standard may be falling out of favor. An easier way to assemble BioBricks is described herein. Plasmids encoding BioBrick parts are purified from Escherichia coli cells that express a foreign site-specific DNA methyltransferase, so that each is subsequently protected in vitro from the activity of a particular restriction endonuclease. Each plasmid is double-digested and all resulting restriction fragments are ligated together without gel purification. The ligation products are subsequently double-digested with another pair of restriction endonucleases so only the desired insert-recipient vector construct retains the capacity to transform E. coli. This 4R/2M BioBrick assembly protocol is more efficient and accurate than established workflows including 3A assembly. It is also much easier than gel purification to miniaturize, automate and perform more assembly reactions in parallel. As such, it should streamline DNA assembly for the existing community of BioBrick users, and possibly encourage others to join.

The Effect of Low Concentrations of Ascorbic Acid in Microbial Adherence in vitro

2003 ◽  
Vol 73 (4) ◽  
pp. 245-250 ◽  
Author(s):  
Lianou ◽  
Fortis ◽  
Papavassilliou
Keyword(s):  
Escherichia Coli ◽  
Ascorbic Acid ◽  
Epithelial Cells ◽  
Klebsiella Pneumoniae ◽  
Low Concentrations ◽  
Buccal Epithelial Cells ◽  
Microbial Strains ◽  
Vitro Effect ◽  
Microbial Adherence

In the present study, we examined the in vitro effect of low concentrations of ascorbic acid (lower than normal plasma levels), on the adherence of five microbial strains (Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, and Pseudomonas aeruginosa) to human buccal epithelial cells (BEC) of healthy volunteers. The study reached the following conclusions: 1) the presence of ascorbic acid significantly reduces the ability of all tested strains (with the exception of Klebsiella pneumoniae) to adhere to BEC; 2) ascorbic acid interacts with the epithelial cells and decreases the adherence of all tested microorganisms. These findings may be of potential clinical significance.

scholarly journals A rationally designed c-di-AMP FRET biosensor to monitor nucleotide dynamics

2021 ◽  
Author(s):  
Alex J. Pollock ◽  
Philip H. Choi ◽  
Shivam A. Zaver ◽  
Liang Tong ◽  
Joshua J. Woodward
Keyword(s):  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Second Messenger ◽  
Adenosine Monophosphate ◽  
Luria Broth ◽  
Effector Proteins ◽  
Detection Methods ◽  
Live Cells ◽  
Bacterial Populations

3’3’-cyclic di-adenosine monophosphate (c-di-AMP) is an important nucleotide second messenger found throughout the bacterial domain of life. C-di-AMP is essential in many bacteria and regulates a diverse array of effector proteins controlling pathogenesis, cell wall homeostasis, osmoregulation, and central metabolism. Despite the ubiquity and importance of c-di-AMP, methods to detect this signaling molecule are limited, particularly at single cell resolution. In this work, crystallization of the Listeria monocytogenes c-di-AMP effector protein Lmo0553 enabled structure guided design of a Förster resonance energy transfer (FRET) based biosensor, which we have named CDA5. CDA5 is a fully genetically encodable, specific, and reversible biosensor which allows for the detection of c-di-AMP dynamics both in vitro and within live cells in a nondestructive manner. Our initial studies identify a distribution of c-di-AMP in Bacillus subtilis populations first grown in Luria Broth and then resuspended in diluted Luria Broth compatible with florescence analysis. Furthermore, we find that B. subtilis mutants lacking either a c-di-AMP phosphodiesterase or cyclase have respectively higher and lower FRET responses. These findings provide novel insight into the c-di-AMP distribution within bacterial populations and establish CDA5 as a powerful platform for characterizing new aspects of c-di-AMP regulation. Importance C-di-AMP is an important nucleotide second messenger for which detection methods are severely limited. In this work we engineer and implement a c-di-AMP specific FRET biosensor to remedy this dearth. We present this biosensor, CDA5, as a versatile tool to investigate previously intractable facets of c-di-AMP biology.

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