Journal: Microbiology Spectrum

Article Title: Control of Helicobacter pylori with engineered probiotics secreting selective guided antimicrobial peptides

doi: 10.1128/spectrum.02014-23

Figure Lengend Snippet: Precision targeting of H. pylori using probiotic delivery of guided antimicrobial peptides (gAMPs). The probiotic Lactococcus lactis carries the Escherichia coli / L. lactis shuttle vector, pTKR, for the expression of gAMPs in the mouse stomach, allowing rapid engineering in E. coli and transfer to L. lactis . The gAMPs were placed downstream of the acid-inducible P1 promoter and the usp secretion signal peptide to allow secretion in the acidic stomach environment. The guide attached to the N-terminus of the AMP was that portion of the human thrombin protein, Multimerin-1, that binds to H. pylori .

Article Snippet: E. coli 10β (DH10B derivative) was purchased from New England Biolabs (NEB #C3019I) and was grown in Luira-Betani (LB) broth/agar (Thermo Scientific) at 37°C with aeration.

Techniques: Plasmid Preparation, Expressing

Journal: Microbiology Spectrum

Article Title: Control of Helicobacter pylori with engineered probiotics secreting selective guided antimicrobial peptides

doi: 10.1128/spectrum.02014-23

Figure Lengend Snippet: MM1-guided GFP (MM1-GFP) protein binds specifically to H. pylori cells. ( A ) Protein preparations of MM1-GFP, but not GFP, bound to H. pylori cells. Fluorescence intensity of cells of target bacterium H. pylori 60,190 WT untreated (red) or treated with GFP (green) or MM1-GFP (blue) with averaged median fluorescence ( n = 3) in relative fluorescence units (RFUs) obtained from BD FACSverse flow cytometer using blue 488 nm laser and a 488/10 bandpass filter; standard deviation shown; statistical significance (one-way analysis of variance, one-way ANOVA; ns, not significant; *** P ≤ 0.001). (B and C) Neither MM1-GFP nor GFP protein significantly bound to off-target bacterial cells. Flow cytometry as in ( A ) for the cells of off-target bacteria Lactobacillus plantarum ( B ) or Escherichia coli K12 ( C ). ( D ) Confocal microscopy demonstrated that MM1-GFP, but not GFP, bound strongly to Helicobacter pylori cells. Imaging of H. pylori 60,190 WT cells untreated (first column) or treated with GFP (second column) or MM1-GFP (third column). Top row: visualization of GFP or MM1-GFP fluorescence at 488 nm. Middle row: visualization of bacterial cells (CellBrite stain, 640 nm). Bottom row: merged images.

Article Snippet: E. coli 10β (DH10B derivative) was purchased from New England Biolabs (NEB #C3019I) and was grown in Luira-Betani (LB) broth/agar (Thermo Scientific) at 37°C with aeration.

Techniques: Fluorescence, Flow Cytometry, Standard Deviation, Bacteria, Confocal Microscopy, Imaging, Staining

Journal: Microbiology Spectrum

Article Title: Control of Helicobacter pylori with engineered probiotics secreting selective guided antimicrobial peptides

doi: 10.1128/spectrum.02014-23

Figure Lengend Snippet: gAMP probiotics selectively kill H. pylori when co-cultured in vitro . Guided (red) or unmodified (blue) versions of three AMPs (alyteserin, CRAMP, and laterosporulin) were expressed by engineered L. lactis, which was co-cultured with the target ( H. pylori ) or the non-target bacteria, E. coli and Lactobacillus . Eight different initial probiotic concentrations were tested for each AMP or gAMP (x-axis), and the titer of the H. pylori or off-target bacterium was measured after 24 hours of co-culture (y-axis). Titers were determined starting with qPCR using vacA primers for H. pylori , DE3-T7 polymerase primers for E. coli, recA primers for Lactobacillus , and acma primers for L. lactis . The corresponding CFU values were calculated from standard curves of C T versus CFU using CFU values obtained by bacterial dilution and plating . The limits of qPCR detection differed between bacterial species resulting in flat-lining at different low-end levels.

Article Snippet: E. coli 10β (DH10B derivative) was purchased from New England Biolabs (NEB #C3019I) and was grown in Luira-Betani (LB) broth/agar (Thermo Scientific) at 37°C with aeration.

Techniques: Probiotics, Cell Culture, In Vitro, Bacteria, Co-Culture Assay

Journal: Microbiology Spectrum

Article Title: Control of Helicobacter pylori with engineered probiotics secreting selective guided antimicrobial peptides

doi: 10.1128/spectrum.02014-23

Figure Lengend Snippet: gAMP and AMP probiotics control H. pylori in the mouse model both as a therapeutic and a prophylactic. ( A ) For both therapeutic and prophylactic experiments, the H. pylori infection was established by oral gavage for four consecutive days with 250 µL of resuspended H. pylori (~5 × 10 7 CFU/mL). For therapeutic experiments, the infection was followed by a dose of 250 µL of resuspended L. lactis (~5 × 10 7 CFU/mL) on day 5 of the regimen. For prophylactic experiments, the probiotic was provided on day 0, followed by a H. pylori challenge on days 3–6. Immediately before administration of both H. pylori and L. lactis , mice stomach samples were extracted by reverse oral gavage method. Further samples were extracted on day 8 and 10 by the same method. For each treatment listed in ( B ) and ( C ), at least six mice were used. ( B ) H. pylori titer measured over the time course of the therapeutic experiment. On each day, probiotics expressing gAMP or AMP were compared to antibiotic treatment or negative controls. H. pylori titers in the reverse oral gavage stomach samples were determined by qPCR using the CFU vs C T standard curve for H. pylori . The strength of infection is color-coded. Complete data with significance values are presented in . ( C ) H. pylori titer measured over the time course of the prophylactic experiment. The same probiotic and negative control treatments and H. pylori titer determinations were used as in ( B ).

Article Snippet: E. coli 10β (DH10B derivative) was purchased from New England Biolabs (NEB #C3019I) and was grown in Luira-Betani (LB) broth/agar (Thermo Scientific) at 37°C with aeration.

Techniques: Probiotics, Control, Infection, Expressing, Negative Control

Journal: Microbiology Spectrum

Article Title: Control of Helicobacter pylori with engineered probiotics secreting selective guided antimicrobial peptides

doi: 10.1128/spectrum.02014-23

Figure Lengend Snippet: Probiotic gAMP/AMP treatment in vivo reverses the degradation of taxonomic richness caused by H. pylori infection. ( A and C ) The left two panels cover the therapeutic experiment, displaying the taxonomic analyses of the samples from . ( B and D ) The right two panels cover the prophylactic experiment, with taxonomic analyses of the samples of . ( A and B ) The top panels display the major genera found on each sampling day for each treatment, with H. pylori infection leading to domination by Acinetobacter and Staphylococcus and a relief from this domination being provided by the probiotic treatments. ( C and D ) The bottom panels chart the taxonomic richness by a simple ASV log 10 count over time for each treatment group, with a rebound or retention of taxonomic richness in the probiotic treatment group, especially the gAMP probiotic group.

Article Snippet: E. coli 10β (DH10B derivative) was purchased from New England Biolabs (NEB #C3019I) and was grown in Luira-Betani (LB) broth/agar (Thermo Scientific) at 37°C with aeration.

Techniques: In Vivo, Infection, Sampling

Journal: Microbiology Spectrum

Article Title: Control of Helicobacter pylori with engineered probiotics secreting selective guided antimicrobial peptides

doi: 10.1128/spectrum.02014-23

Figure Lengend Snippet: The Microbial Dysbiosis Index (MDI) comprised 10 co-varying taxa that also correlated with H. pylori infection. ( A ) Compositional Correlation network of 10 genera revealed by the CCREPE analysis, whose composition co-varied among the mice stomach samples on days 0 and 5, which were pre- and post -H. pylori infection, respectively. Eight of these were positively correlated with each other (blue lines), while two others ( Staphylococcus and Acinetobacter ) were positively correlated with each other but inversely correlated with the other 8 (red lines). All the correlations were significant ( P < 0.05, q < 0.10). ( B ) The 10 genera that co-varied significantly also had a significant change in their mean relative abundance among the samples of day 0 vs day 5, with 8 of them decreasing following H. pylori infection while Staphylococcus and Acinetobacter saw a significant increase. This further validates the importance of these genera as markers for mice stomach microbial health in our experiment. ( C ) Principal Components Analysis (PCA) of the taxonomic relationship between the microbiota of all 350 samples in the study. Dysbiotic samples (red) clustered separately from non-dysbiotic samples (blue), demonstrating that MDI correlated with the taxonomic relatedness of the samples generally. Jitter was used to allow all samples to be in view. ( D ) The same PCA as in ( C ) but with ordinates overlayed corresponding to the 10 genera of the MDI, with longer vectors indicating more correspondence to the abundance of that genus. Staphylococcus and Acinetobacter are again seen as strong predictors for H. pylori /no treatment samples (red). No jitter was used in order to report the unaltered PCA output.

Article Snippet: E. coli 10β (DH10B derivative) was purchased from New England Biolabs (NEB #C3019I) and was grown in Luira-Betani (LB) broth/agar (Thermo Scientific) at 37°C with aeration.

Techniques: Infection

Journal: Microbiology Spectrum

Article Title: Control of Helicobacter pylori with engineered probiotics secreting selective guided antimicrobial peptides

doi: 10.1128/spectrum.02014-23

Figure Lengend Snippet: Probiotic gAMP treatment protects against microbial dysbiosis. ( A ) At the start of the therapeutic experiment (day 0), the MDIs of all samples (dark blue) were negative (healthy) and had predominantly high taxonomic richness (circle size). After H. pylori infection and probiotic treatment (days 8 and 10 combined), untreated (“only H. pylori ”) and empty probiotic-treated samples were dysbiotic with low taxonomic richness, while AMP and gAMP probiotic-treated samples were healthy and had high taxonomic richness. Antibiotic treatment relieved dysbiosis but had low taxonomic richness. ( B ) In the prophylactic experiment, all probiotic treatments protected against H. pylori -induced dysbiosis, with gAMP probiotics promoting the most robust taxonomic richness. Infected mice without the prophylactic yielded only dysbiotic samples. ( C ) Dysbiosis in the therapeutic experiment, tracked by day. All treatments showed a negative (healthy) MDI for day 0 and a positive (dysbiotic) MDI for day 5 since these samples were collected just before oral inoculation with H. pylori or probiotic, respectively. The bar colors correspond to the colors and treatments in ( A ). Within 3 days, dysbiosis had been alleviated by gAMP and AMP probiotics and antibiotics but not by the empty vector probiotic. ( D ) Dysbiosis in the prophylactic experiment, tracked by day. All samples had healthy MDI values before (day 0) and 3 days after probiotic treatment, but H. pylori induced dysbiosis by days 8 and 10 in samples lacking prophylaxis (null treatment, magenta). All probiotic treatments protected against H. pylori -induced dysbiosis.

Article Snippet: E. coli 10β (DH10B derivative) was purchased from New England Biolabs (NEB #C3019I) and was grown in Luira-Betani (LB) broth/agar (Thermo Scientific) at 37°C with aeration.

Techniques: Infection, Probiotics, Plasmid Preparation

Journal: bioRxiv

Article Title: High-throughput protein characterization by complementation using DNA barcoded fragment libraries

doi: 10.1101/2024.05.08.593210

Figure Lengend Snippet: Complementation of CysA activity by Bacillus subtilis CysP and by novel sulfate transporter TK06_RS10770 from Pseudomonas fluorescens FW300-N2E2. First panel shows the native E. coli system. Second panel shows the function of the sulfate permease (CysP) from B. subtilis , along with its AlphaFold predicted structure. The final panel shows the AlphaFold predicted structure for TK06_RS10770 of P. fluorescens FW300-N2E2.

Article Snippet: Genome fragment library generation cloning was carried out in New England Biolabs 10-β high-efficiency electrocompetent E. coli cells.

Techniques: Activity Assay

Journal: bioRxiv

Article Title: High-throughput protein characterization by complementation using DNA barcoded fragment libraries

doi: 10.1101/2024.05.08.593210

Figure Lengend Snippet: Identification of a protein, LRK54_RS05660 of R. denitrificans FW104-10B01 that complements Δ metB knockout. The top of the figure shows the two dominant pathways from homoserine to homocysteine used by bacteria, the two step (MetBC) and one step (MetZ) pathways. The lower portion of the figure shows crystal structure of E. coli MetB and the AlphaFold predicted structure for LRK54_RS05660, which bears structural similarity.

Article Snippet: Genome fragment library generation cloning was carried out in New England Biolabs 10-β high-efficiency electrocompetent E. coli cells.

Techniques: Knock-Out, Bacteria

Journal: bioRxiv

Article Title: High-throughput protein characterization by complementation using DNA barcoded fragment libraries

doi: 10.1101/2024.05.08.593210

Figure Lengend Snippet: HisC activity from TK06_RS12685 of Pseudomonas fluorescens FW300-N2E2. One the left is the AlphaFold predicted structure of TK06_RS12685. On the right is the activity of HisC from E. coli that it is being complemented.

Article Snippet: Genome fragment library generation cloning was carried out in New England Biolabs 10-β high-efficiency electrocompetent E. coli cells.

Techniques: Activity Assay

Journal: ACS Synthetic Biology

Article Title: Recoded Bacteriophage Genome for Bio-Orthogonal-Enabled Concentration and Detection of E. coli in Drinking Water

doi: 10.1021/acssynbio.5c00665

Figure Lengend Snippet: Overview of High Complexity Golden Gate Assembly (HC-GGA) design and assembly. A) The coliphage T7 genome was divided into 21 fragments for modular assembly. Genes containing native amber (TAG) stop codons are shown in orange, and the major capsid protein gene (gp10A) is highlighted in blue. B) These fragments were generated by PCR from either BsmBI-domesticated T7 genomic DNA or synthetic gBlocks, assembled using HC GGA into a complete genome, and transformed into E. coli NEB 10β cells by electroporation. Individual plaques were isolated and sequenced to confirm successful genome reconstruction. C) For the Amber Free (AF) and Amber Free/NanoLuc (AF/NL) variants, selected fragments (F2, F6, F9, F10, F14, F19, and F21) were replaced with synthetic versions. In the AF variant, all native amber codons were recoded to ocher (TAA) codons to prevent unintended incorporation of noncanonical amino acids. In the AF/NL variant, an amber codon was inserted at the end of gp10A to enable site-specific incorporation of L-homopropargylglycine, and a NanoLuc luciferase gene was inserted downstream of gp10B under a T7 promoter. Upon infection of E. coli by the engineered phage, the host expresses the NanoLuc protein. After lysis, NanoLuc interacts with its substrate to produce luminescence. This signal is only generated if the phage successfully binds to, concentrates, and infects its E. coli host, thereby enabling sensitive and specific detection of viable bacteria in water samples. Figure created in Biorender.

Article Snippet: A portion of the assembly reactions (1 μL) were transformed into E. coli NEB 10β (C3020, NEB) cells by electroporation utilizing a BioRad GenePulser Xcell Microbial System under standard conditions (1.8 kV, 25 μF, 200 Ω).

Techniques: Generated, Transformation Assay, Electroporation, Isolation, Variant Assay, Luciferase, Infection, Lysis, Bacteria

Journal: ACS Synthetic Biology

Article Title: Recoded Bacteriophage Genome for Bio-Orthogonal-Enabled Concentration and Detection of E. coli in Drinking Water

doi: 10.1021/acssynbio.5c00665

Figure Lengend Snippet: Schematic for phage conjugation to nanoparticles via click chemistry. Cobalt-containing azide functionalized magnetic nanoparticles were conjugated to phages containing an alkyne-modified noncanonical amino acid (L-HPG) incorporated into the major capsid protein using a copper-mediated cycloaddition resulting in magnetized phages which could be used to capture and detect their host E. coli . Figure created in Biorender.

Article Snippet: A portion of the assembly reactions (1 μL) were transformed into E. coli NEB 10β (C3020, NEB) cells by electroporation utilizing a BioRad GenePulser Xcell Microbial System under standard conditions (1.8 kV, 25 μF, 200 Ω).

Techniques: Conjugation Assay, Modification

Journal: ACS Synthetic Biology

Article Title: Recoded Bacteriophage Genome for Bio-Orthogonal-Enabled Concentration and Detection of E. coli in Drinking Water

doi: 10.1021/acssynbio.5c00665

Figure Lengend Snippet: Schematic overview of the bacteriophage-based assay for detection of E. coli in drinking water. A) An overnight culture of E. coli strain ECOR 13 is grown and then inoculated into fresh media. B) The culture is monitored by measuring optical density at 600 nm (OD 600 ) to ensure an appropriate growth phase, followed by cooling on ice. C) Tap water is filtered through a 0.22 μm membrane and stored at 4 °C to simulate drinking water conditions. D) The E. coli culture is serially diluted using the filtered tap water to prepare test samples with defined bacterial concentrations. E) Phage-coated magnetic nanoparticles are added to each inoculated sample and incubated for 30 min to allow binding and infection. F) Magnetized phages and any bound E. coli are separated using a magnetic rack. G) A luminescent substrate specific to NanoLuc is added to the isolated complexes. H) Upon lysis of infected E. coli , NanoLuc is released and reacts with the substrate to produce a luminescent signal. I) Luminescence is measured using a plate reader, providing a quantitative readout of viable E. coli presence in the sample. Figure created in Biorender.

Article Snippet: A portion of the assembly reactions (1 μL) were transformed into E. coli NEB 10β (C3020, NEB) cells by electroporation utilizing a BioRad GenePulser Xcell Microbial System under standard conditions (1.8 kV, 25 μF, 200 Ω).

Techniques: Membrane, Incubation, Binding Assay, Infection, Isolation, Lysis

Journal: ACS Synthetic Biology

Article Title: Recoded Bacteriophage Genome for Bio-Orthogonal-Enabled Concentration and Detection of E. coli in Drinking Water

doi: 10.1021/acssynbio.5c00665

Figure Lengend Snippet: Magnetized phages were used to detect E. coli (ECOR 13) in 100 mL drinking water samples. Data points represent the average of triplicates and error bars represent standard deviations. The average signal of the negative controls, consisting of uninoculated samples (0 CFU) are represented by a dashed line with the standard deviations represented by dotted lines.

Article Snippet: A portion of the assembly reactions (1 μL) were transformed into E. coli NEB 10β (C3020, NEB) cells by electroporation utilizing a BioRad GenePulser Xcell Microbial System under standard conditions (1.8 kV, 25 μF, 200 Ω).

Techniques:

Journal: ACS Synthetic Biology

Article Title: Recoded Bacteriophage Genome for Bio-Orthogonal-Enabled Concentration and Detection of E. coli in Drinking Water

doi: 10.1021/acssynbio.5c00665

Figure Lengend Snippet: Schematic overview of the genetic engineering workflow for constructing modified T7 bacteriophage genomes. a) PCR amplification of 21 fragments from the BsmBI-domesticated T7 genome. b) SPRI-based size selection and nucleic acid purification, followed by validation of fragment size and homogeneity via gel electrophoresis and quantification using Qubit. c) Assembly of fragments using Golden Gate Assembly with BsmBI, cycled at 42 °C for 5 min and 16 °C for 5 min over 15 cycles. d) Electroporation of 1 μL of the circularized genome into competent E. coli 10-beta cells, followed by 1.5 h of recovery at 37 °C in stable outgrowth media. e) Dilution plating with E. coli host, isolation of plaques, and whole-genome sequencing to confirm successful assembly and modification.

Article Snippet: A portion of the assembly reactions (1 μL) were transformed into E. coli NEB 10β (C3020, NEB) cells by electroporation utilizing a BioRad GenePulser Xcell Microbial System under standard conditions (1.8 kV, 25 μF, 200 Ω).

Techniques: Modification, Amplification, Size Selection, Nucleic Acid Purification, Biomarker Discovery, Nucleic Acid Electrophoresis, Electroporation, Isolation, Sequencing