Search

The implementation of Boolean logic circuits in cells have become a very active field within synthetic biology. Although these are mostly focussed on the genetic components alone, the context in which the circuit performs is crucial for its outcome. We characterise 20 genetic NOT logic gates in up to 7 bacterial-based contexts each, to generate 135 different functions. The contexts we focus on are combinations of four plasmid backbones and three hosts, two Escherichia coli and one Pseudomonas putida strains. Each gate shows seven different dynamic behaviours, depending on the context. That is, gates can be fine-tuned by changing only contextual parameters, thus improving the compatibility between gates. Finally, we analyse portability by measuring, scoring, and comparing gate performance across contexts. Rather than being a limitation, we argue that the effect of the genetic background on synthetic constructs expands functionality, and advocate for considering context as a fundamental design parameter.

Pathogen-associated infections represent one of the major threats to human health and require reliable methods for immediate and robust identification of pathogenic microorganisms. Here, an inexpensive cellulase-linked immunomagnetic methodology was developed for the specific and ultrasensitive analysis of bacteria at their single-cell levels within a 3 h procedure. Detection of a model bacterium, Escherichia coli , was performed in a sandwich reaction with E. coli -specific either aptamer or antibody (Ab)-modified magnetic beads (MBs) and Ab/aptamer reporter molecules linked to cellulase. The cellulase-labeled immuno-aptamer sandwich applied onto nitrocellulose-film-modified electrodes digested the film and changed its electrical conductivity. Electrode's chronocoulometric responses at 0.3 V, in the absence of any redox indicators, allowed a single E. coli cell detection and from 1 to 4 × 10 4 CFU mL -1 E. coli quantification. No interference/cross-reactivity from Salmonella enteritidis , Enterobacter agglomerans , Pseudomonas putida , Staphylococcus aureus , and Bacillus subtilis was observed when the assay was performed on Ab-modified MBs, and E. coli could be quantified in tap water and milk. This electrochemically label-free methodology is sufficiently fast, highly specific, and sensitive to be used in direct in-field applications. The assay can be adapted for specific detection of other bacterial strains of either the same or different species and offers new analytical tools for fast, specific, and reliable analysis of bacteria in the clinic, food, and environment.

In vitro synthesis of ATP catalyzed by the ATP-synthase requires membrane vesicles, in which the ATP-synthase is present within the bilayer membrane. Inverted vesicle prepared from Gram negative cells (e.g., Escherichia coli or Pseudomonas putida) can be readily obtained and used for in vitro ATP-synthesis. Up to now, quantification of ATP synthesized by membrane vesicles has been mostly analyzed via bioluminescence-based assays. Alternatively, vesicle respiration and the associated ATP level can be determined using biosensors, which not only provide high selectivity, but allow ATP measurements without the sample being illuminated. Here, we present a microbiosensor for ATP in combination with scanning electrochemical microscopy (SECM) using an innovative two-compartment electrochemical cell for the determination of ATP levels at E.coli or P. putida inverted vesicles. For a protein concentration of 22 mg/ml, a total amount of 0.29 ± 0.03 μM/μl ATP per vesicle was determined in case of E.coli; in turn, P. putida derived vesicles yielded 0.48 ± 0.02 μM/μl ATP per vesicle at a total protein concentration of 25.2 mg/ml. Inhibition experiments with Venturicidin A clearly revealed that the respiratory chain enzyme complex responsible for ATP generation is effectively involved., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Many bacteria perform a run-and-tumble random walk to explore their surrounding and to perform chemotaxis. In this article we present a novel method to infer the relevant parameters of bacterial motion from experimental trajectories including the tumbling events. We introduce a stochastic model for the orientation angle, where a shot-noise process initiates tumbles, and analytically calculate conditional moments, reminiscent of Kramers-Moyal coefficients. Matching them with the moments calculated from experimental trajectories of the bacteria E. coli and Pseudomonas putida, we are able to infer their respective tumble rates, the rotational diffusion constants, and the distributions of tumble angles in good agreement with results from conventional tumble recognizers. We also define a novel tumble recognizer, which explicitly quantifies the error in recognizing tumbles. In the presence of a chemical gradient we condition the moments on the bacterial direction of motion and thereby explore the chemotaxis strategy. For both bacteria we recover and quantify the classical chemotactic strategy, where the tumble rate is smallest along the chemical gradient. In addition, for E. coli we detect some cells, which bias their mean tumble angle towards smaller values. Our findings are supported by a scaling analysis of appropriate ratios of conditional moments, which are directly calculated from experimental data., Competing Interests: The authors have declared that no competing interests exist.

Initial bacterial attachment, likely affected by local shear, could influence biofilm formation. However, there are contradictory reports for the shear effects on attachment of different bacteria onto different surfaces. In this study, four bacteria, Staphylococcus epidermidis, Pseudomonas aeruginosa, Pseudomonas putida, and Escherichia coli, were examined for their attachment to glass and octadecyltrichlorosilane (OTS) modified glass under different shears. Polystyrene particles were used to verify that their shear dependent attachment on glass and OTS could be interpreted using an analysis based on the extended Derjaguin-Landau-Verwey-Overbeek (XDLVO) theory. In particular, the critical shear force (F(c-shear)) could correlate with the maximum attractive force (F(MAX)(XDLVO)) toward the secondary energy minimum as F(c-shear)=cF(MAX)(XDLVO). For these particles, c of ~1 was obtained, the value was within the coefficient range (0.1-1) of substances sliding over glass. For S. epidermidis, E. coli and P. aeruginosa on glass, c was 0.3, <0.6 and 0.2, respectively. When considering potential protein adsorption on OTS during bacterial attachment, c of these species on OTS was slightly above 1. A greatly enhanced attachment of P. aeruginosa on OTS was also observed, probably due to the presence of flagella. For P. putida, the attachment first decreased slightly or maintained with shear and then increased. Such behaviors were probably caused by the increased secretion of extracellular polymeric substances (EPS) at higher shears by P. putida. The results from this study suggested that, without complications from surface features/EPS, the analysis based on the XDLVO theory could provide a basis for understanding shear effect on initial bacterial attachment., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Chemotaxis toward chemical pollutants provides a mechanism for bacteria to migrate to locations of high contamination, which may improve the effectiveness of bioremediation. A microfluidic device was designed to mimic the dissolution of an organic-phase contaminant from a single pore into a larger macropore representing a preferred pathway for microorganisms that are carried along by groundwater flow. The glass windows of the microfluidic device allowed direct image analysis of bacterial distributions within the vicinity of the organic contaminant. Concentrations of chemotactic bacteria P. putida F1 near the organic/aqueous interface were 25% greater than those of a nonchemotactic mutant in the vicinity of toluene for a fluid velocity of 0.5 m/d. For E. coli responding to phenol, the bacterial concentrations were 60% greater than the controls, also at a velocity of 0.5 m/d. Velocities in the macropore were varied over a range from 0.5 to 10 m/d, the lower end of which is typical of groundwater velocities. The accumulation of chemotactic bacteria near the NAPL chemoattractant source decreased as the fluid velocity increased. Good agreement between computer-based simulations, generated using reasonable values of the model parameters, and the experimental data for P. putida strains confirmed the contribution due to chemotaxis. The experimental data for E. coli required a larger chemotactic sensitivity coefficient than that for P. putida, which was consistent with parameter values reported in the literature., (Copyright © 2012 Wiley Periodicals, Inc.)

A good part of the contemporary synthetic biology agenda aims at reprogramming microorganisms to enhance existing functions and/or perform new tasks. Moreover, the functioning of complex regulatory networks, or even a single gene, is revealed only when perturbations are entered in the corresponding dynamic systems and the outcome monitored. These endeavors rely on the availability of genetic tools to successfully modify á la carte the chromosome of target bacteria. Key aspects to this end include the removal of undesired genomic segments, systems for the production of directed mutants and allelic replacements, random mutant libraries to discover new functions, and means to stably implant larger genetic networks into the genome of specific hosts. The list of gram-negative species that are appealing for such genetic refactoring operations is growingly expanding. However, the repertoire of available molecular techniques to do so is very limited beyond Escherichia coli. In this chapter, utilization of novel tools is described (exemplified in two plasmids systems: pBAM1 and pEMG) tailored for facilitating chromosomal engineering procedures in a wide variety of gram-negative microorganisms.

The purpose of this study is to develop a novel bacterial analysis method by coupling the flow field-flow fractionation (flow FFF) separation technique with detection by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. The composition of carrier liquid used for flow FFF was selected based on retention of bacterial cells and compatibility with the MALDI process. The coupling of flow FFF and MALDI-TOF MS was demonstrated for P. putida and E. coli. Fractions of the whole cells were collected after separation by FFF and further analyzed by MALDI-MS. Each fraction, collected over different time intervals, corresponded to different sizes and possibly different growth stages of bacteria. The bacterial analysis by flow FFF/MALDI-TOF MS was completed within 1 h with only preliminary optimization of the process.

The bionconversion of indene to cis-(1S,2R)-indandiol, a potential key intermediate in the synthesis of Merck's HIV protease inhibitor, CRIXIVAN trade mark, can be achieved using Rhodococcus, Pseudomonas putida, and Escherichia coli strains. This study reports on the application of multiparameter flow cytometry for the measurement of cytoplasmic membrane integrity and membrane depolarization as indicators of toxic effects of the substrate, product, and by-products using each of these strains. Measurements of oxygen uptake rate (OUR) and optical density (OD) as indicators of metabolic activity and biomass growth, respectively, were also made. Measurements of the cytoplasmic membrane potential, cell viability, and respiratory activity provided a sensitive set of parameters to assess toxicity in the indene bioconversion and provided the basis for process improvements and strain selection. The toxic concentrations of the substrate, product, and by-products for each strain have been determined. The results show that it is possible to accumulate cis-(1S,2R)-indandiol and cis-1-amino-2-indanol up to 20 g/L without significant negative effects on cell physiology using any of the strains tested. The Gram-negative P. putida (421-5 and GM 730) and E. coli strains were more resistant to indene and the isolated chemicals of the biotransformation than the Gram-positive Rhodoccoccus I24 strain, possibly due to the presence of the outer membrane and efflux pump mechanisms. P. putida GM 730 and the E. coli TDO 123 strains responded similarly to toxic effects, and the E. coli TDO 123 strain was more resistant than the P. putida 421-5 strain. In addition to the recommendations for strain selection, the identified targets for bioprocess improvement include a combination of genetic as well as process engineering approaches., (Copyright 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 81: 405-420, 2003.)