Dataset Information

Improved Escherichia coli Bactofection and Cytotoxicity by Heterologous Expression of Bacteriophage ΦX174 Lysis Gene E.

ABSTRACT: Bactofection offers a gene delivery option particularly useful in the context of immune modulation. The bacterial host naturally attracts recognition and cellular uptake by antigen presenting cells (APCs) as the initial step in triggering an immune response. Moreover, depending on the bacterial vector, molecular biology tools are available to influence and/or overcome additional steps and barriers to effective antigen presentation. In this work, molecular engineering was applied using Escherichia coli as a bactofection vector. In particular, the bacteriophage ΦX174 lysis E (LyE) gene was designed for variable expression across strains containing different levels of lysteriolysin O (LLO). The objective was to generate a bacterial vector with improved attenuation and delivery characteristics. The resulting strains exhibited enhanced gene and protein release and inducible cellular death. In addition, the new vectors demonstrated improved gene delivery and cytotoxicity profiles to RAW264.7 macrophage APCs.

SUBMITTER: Chung TC

PROVIDER: S-EPMC9896019 | biostudies-literature | 2015 May

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Improved Escherichia coli Bactofection and Cytotoxicity by Heterologous Expression of Bacteriophage ΦX174 Lysis Gene E.

Chung Tai-Chun TC Jones Charles H CH Gollakota Akhila A Kamal Ahmadi Mahmoud M Rane Snehal S Zhang Guojian G Pfeifer Blaine A BA

Molecular pharmaceutics 20150416 5

Bactofection offers a gene delivery option particularly useful in the context of immune modulation. The bacterial host naturally attracts recognition and cellular uptake by antigen presenting cells (APCs) as the initial step in triggering an immune response. Moreover, depending on the bacterial vector, molecular biology tools are available to influence and/or overcome additional steps and barriers to effective antigen presentation. In this work, molecular engineering was applied using Escherichi ...[more]

PMID: 25849744

Similar Datasets

Project description:Rapid detection of bacterial pathogens is a critical unmet need for both food and environmental samples such as irrigation water. As a part of the Food safety Modernization Act (FSMA), The Produce Safety rule has established several requirements for testing for the presence of generic Escherichia coli in water, but the current method available for testing (EPA M1603) demands specified multiple colony verification and highly trained personnel to perform these tests. The purpose of the study was to assess a phage induced bacterial lysis using quantitative image analysis to achieve rapid detection of E. coli at low concentrations within 8 hours. This study aimed to develop a simple yet highly sensitive and specific approach to detect target bacteria in complex matrices. In the study, E. coli cells were first enriched in tryptic soy broth (TSB), followed by T7 phage induced lysis, concentration, staining and fluorescent imaging. Image analysis was conducted including image pre-processing, image segmentation and quantitatively analysis of cellular morphological features (area, eccentricity and full width at half maximum). Challenge experiments using realistic matrices, including simulated fresh produce wash water, coconut water and spinach wash water, demonstrated the method can be applied for use in situations that occur in food processing facilities. The results indicated E. coli cells that are lysed by T7 phages demonstrated significantly (P < 0.05) higher extracellular DNA release, altered cellular shape (from rod to circular) and diffused fluorescent signal intensity. Using this biosensing strategy, a sensitivity to detect Escherichia coli at 10 CFU/ml within 8 hours was achieved, both in laboratory medium and in complex matrices. The proposed phage based biosensing strategy enables rapid detection of bacteria and is applicable to analysis of food systems. Furthermore, the steps involved in this assay can be automated to enable detection of target bacteria in food facilities without extensive resources.

Project description:BACKGROUND: Solar energy is the ultimate energy source on the Earth. The conversion of solar energy into fuels and energy sources can be an ideal solution to address energy problems. The recent discovery of proteorhodopsin in uncultured marine ?-proteobacteria has made it possible to construct recombinant Escherichia coli with the function of light-driven proton pumps. Protons that translocate across membranes by proteorhodopsin generate a proton motive force for ATP synthesis by ATPase. Excess protons can also be substrates for hydrogen (H(2)) production by hydrogenase in the periplasmic space. In the present work, we investigated the effect of the co-expression of proteorhodopsin and hydrogenase on H(2) production yield under light conditions. RESULTS: Recombinant E. coli BL21(DE3) co-expressing proteorhodopsin and [NiFe]-hydrogenase from Hydrogenovibrio marinus produced ~1.3-fold more H(2) in the presence of exogenous retinal than in the absence of retinal under light conditions (70 ?mole photon/(m2·s)). We also observed the synergistic effect of proteorhodopsin with endogenous retinal on H(2) production (~1.3-fold more) with a dual plasmid system compared to the strain with a single plasmid for the sole expression of hydrogenase. The increase of light intensity from 70 to 130 ?mole photon/(m(2)·s) led to an increase (~1.8-fold) in H(2) production from 287.3 to 525.7 mL H(2)/L-culture in the culture of recombinant E. coli co-expressing hydrogenase and proteorhodopsin in conjunction with endogenous retinal. The conversion efficiency of light energy to H(2) achieved in this study was ~3.4%. CONCLUSION: Here, we report for the first time the potential application of proteorhodopsin for the production of biohydrogen, a promising alternative fuel. We showed that H(2) production was enhanced by the co-expression of proteorhodopsin and [NiFe]-hydrogenase in recombinant E. coli BL21(DE3) in a light intensity-dependent manner. These results demonstrate that E. coli can be applied as light-powered cell factories for biohydrogen production by introducing proteorhodopsin.

Dataset Information

Improved Escherichia coli Bactofection and Cytotoxicity by Heterologous Expression of Bacteriophage ΦX174 Lysis Gene E.

Publications

Improved Escherichia coli Bactofection and Cytotoxicity by Heterologous Expression of Bacteriophage ΦX174 Lysis Gene E.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets