e coli i top10 i  (ATCC)

Journal: Antibiotics

Article Title: Development of New Antimicrobial Peptides by Directional Selection

doi: 10.3390/antibiotics14111120

Figure Lengend Snippet: Schematic overview of the experimental approach. A library of expression plasmids encoding random mutants of well-characterized antimicrobial peptides (Hm-AMP2, melittin and cecropin) was generated by random mutagenesis with partially degenerate oligonucleotides. The plasmid library was transformed into E. coli , followed by induction of recombinant AMP expression. Clones with the strongest growth inhibition were selected for subsequent rounds of mutagenesis and screening. The most promising variants were synthesized and tested for their properties.

Article Snippet: The strain used for recombinant plasmid construction was E. coli TOP10 (F − mcrA Δ(mrr-hsdRMS-mcrBC) φ80lacZΔM15 ΔlacX74 nupG recA1 araD139 Δ(ara-leu)7697 galE15 galK16 rpsL(Str^R) endA1 λ − ) (Invitrogen, Carlsbad, CA, USA).

Techniques: Expressing, Generated, Mutagenesis, Plasmid Preparation, Transformation Assay, Recombinant, Clone Assay, Inhibition, Synthesized

Journal: Antibiotics

Article Title: Development of New Antimicrobial Peptides by Directional Selection

doi: 10.3390/antibiotics14111120

Figure Lengend Snippet: Activity of synthetic peptides melittin ( A ), cecropin ( B ), and Hm-AMP2 ( C ) in relation to introduced mutations. In the alignment on the left, shades of blue indicate amino acid sequence similarities. On the right, antimicrobial activity against E. coli and B. subtilis , cytotoxicity toward Expi293F cells, and the calculated therapeutic index based on peptide activities are shown. MEL—melittin, CECR—cecropin, AMP2—Hm-AMP2 peptide. AMP’s cytotoxicity was measured as the IC 90 , the concentration that inhibits 90% of cell growth. NA (not applicable) indicates that peptide showed no activity within tested concentration range. Minimal inhibitory concentration (MIC) and IC 90 are given in µM. The therapeutic index (TI) was defined as the lowest IC 90 divided by the highest MIC.

Article Snippet: The strain used for recombinant plasmid construction was E. coli TOP10 (F − mcrA Δ(mrr-hsdRMS-mcrBC) φ80lacZΔM15 ΔlacX74 nupG recA1 araD139 Δ(ara-leu)7697 galE15 galK16 rpsL(Str^R) endA1 λ − ) (Invitrogen, Carlsbad, CA, USA).

Techniques: Activity Assay, Sequencing, Concentration Assay

Journal: Cell Reports Medicine

Article Title: Designer probiotic-based living drugs for uric acid homeostasis control in hyperuricemic mice and rats

doi: 10.1016/j.xcrm.2025.102379

Figure Lengend Snippet: Design and characterization of a probiotic UA sensor (A) Schematic design of the UA sensor for modulating gene expression. Without UA, HucR binds to the synthetic promoter P HucR and represses the expression of a gene of interest (GOI). In the presence of UA, UA is transported from the culture medium into bacterial cells via a urate transporter YgfU, resulting in the dissociation of HucR from P HucR and initiation of gene expression. (B) Red fluorescent protein (RFP) expression of the UA sensor with and without the urate transporter YgfU. EcN cells were transformed with either pGN123 or pGN245 and cultivated with 0- and 250-μM UA. The fluorescence intensity was evaluated after 12 h using a microplate reader. (C) Screening various ribosome binding sites (RBSs) for YgfU expression. (D) Optimization of the UA-responsive promoter. The number of tandem operator repeats within P HucR increased from one (P HucR1 ) to four (P HucR4 ). (E) Screening constitutive promoters of different strengths (P const ) to drive HucR and YgfU expression. (F) Dose-dependent RFP expression profile. PULSE cells expressing RFP (PULSE RFP ) were grown with varying concentrations of UA for 12 h. (G) Time-dependent RFP expression kinetics of the UA sensor. The fluorescence intensity of PULSE RFP cells was determined after induction with 250 μM UA for the indicated durations. (H) UA sensor-mediated gene expression in different bacteria strains. Five bacteria strains, including E. coli and Salmonella , transformed with the engineered UA sensor were incubated with 0 and 250 μM UA for 12 h. Data in (B) to (H) are expressed as means ± SD; n = 3 independent experiments. Each data point represents the mean of three technical replicates. p values were calculated by one-way ANOVA. (NS, not significant, ∗∗∗ p < 0.001). See also and and .

Article Snippet: E. coli TOP10 , Sangon Biotech , Cat#B528412.

Techniques: Gene Expression, Expressing, Transformation Assay, Fluorescence, Binding Assay, Bacteria, Incubation

Journal: Cell Reports Medicine

Article Title: Designer probiotic-based living drugs for uric acid homeostasis control in hyperuricemic mice and rats

doi: 10.1016/j.xcrm.2025.102379

Figure Lengend Snippet: PULSE-mediated UA control in acute hyperuricemic mice (A) Schematic for UA-triggered LuxCDABE expression in PULSE cells. (B) Time schedule for PULSE-mediated gene expression following oral delivery in mice. Wild-type mice were orally administered either wild-type E. coli Nissle (EcN; G1) or PULSE cells expressing luxCDABE as a reporter (PULSE Lux ; G2–G5). Thirty minutes later, the mice were gavaged with 1 mL of PBS (G2), 0.25 mM UA (G3), 0.5 mM UA (G4), or 1 mM UA (G1 and G5). Bioluminescence signal intensity was measured using an in vivo imaging system at 1 and 2 h post-gavage. (C and D) PULSE-mediated reporter gene expression in engineered probiotics following oral delivery in wild-type mice. (E) Time schedule for development of an acute hyperuricemia mouse model. Kunming mice (5 weeks old, male) were fed a high-purine diet (150 g/kg yeast extract and 1 g/kg ethambutol hydrochloride) for four weeks instead of a normal chow diet, and then administered hypoxanthine (HX, 1.5 g/kg) and potassium oxonate (PO, 125 mg/kg). (F) Serum UA levels were quantified using a UA assay kit every 30 min following administration. (G) Time schedule for oral administration of PULSE cells to control serum UA levels in acute hyperuricemic mice. Mice fed a high-purine diet for four weeks were fasted for 4 h, then gavaged with PBS, EcN, PULSE smUOX , PULSE LamB-smUOX , PULSE InakN-smUOX , or PULSE mix (a 1:1:1 mixture of PULSE smUOX , PULSE LamB-smUOX , and PULSE InakN-smUOX ) at a dose of 10 9 CFU. Six hours after bacterial gavage, mice were orally administered HX (1.5 g/kg) and PO (125 mg/kg). (H) Serum UA levels were quantified using a UA assay kit 7.5 h after bacterial gavage. Data in (D), (F), and (H) are presented as means ± SEM. In (D) and (F), n = 5 mice; in (H), n = 6 mice . In (F) and (H), each data value represents the mean of three technical replicates. p values were calculated by one-way ANOVA/two-way ANOVA. (NS, not significant, ∗ p < 0.05 and ∗∗∗ p < 0.001). See also .

Article Snippet: E. coli TOP10 , Sangon Biotech , Cat#B528412.

Techniques: Control, Expressing, Gene Expression, In Vivo Imaging, Probiotics