one-sample t-test or two-way anova graphpad prism 8.0  (GraphPad Software Inc)

Journal: ACS Applied Materials & Interfaces

Article Title: Motile and Chemotactic Minicells and Minicell-Driven Biohybrids Engineered for Active Cargo Delivery

doi: 10.1021/acsami.5c04638

Figure Lengend Snippet: Genetic engineering of flagellar regulation improves minicell motility. (A) A simplified schematic showing the regulation of flagellar gene expression and motor rotation in E. coli . See the text for details. (B) Swimming speed of minicells generated by respective genetically modified strains. Swimming speed was calculated for the motile minicells only. (C) A fraction of the motile minicells produced by indicated strains. (D) Examples of trajectories of individual minicells detected with single cell tracking for the minicells produced by Δ min (left) or Δ min Δ ycgR + flhDC (right) strains. 100 randomly chosen trajectories have been visualized for each sample. The scale bars are 20 μm. (E) Average duration of the runs computed with particle tracking analysis for minicells generated by indicated modified strains. In (B,C,E), each data point represents the average swimming speed of one population of purified minicells, while dark points show the mean among the averages of the replicated experiments (at least six for each strain). The error bars represent the standard error of the mean. A two-sample t -test was used to compare different strains and calculate significance values: P ≤ 0.05 (*), P ≤ 0.01 (**), P ≤ 0.001 (***), P > 0.5 (ns).

Article Snippet: Statistical significance values were calculated with a two-sample t -test (ttest2) in MATLAB.

Techniques: Gene Expression, Generated, Genetically Modified, Produced, Single Cell Tracking, Modification, Purification

Journal: ACS Applied Materials & Interfaces

Article Title: Motile and Chemotactic Minicells and Minicell-Driven Biohybrids Engineered for Active Cargo Delivery

doi: 10.1021/acsami.5c04638

Figure Lengend Snippet: Characterization of enhanced motility of engineered minicells. (A) Examples of wide-field fluorescence images of minicells, produced by Δ min and Δ min Δ ycgR + flhDC strains, labeled with Alexa Fluor 594 carboxylic acid succinimidyl ester dye and immobilized onto 2% agar pads. An example of a minicell, generated by the Δ min Δ ycgR + flhDC strain, with more than four flagella, is shown in the right panel. The scale bars are 5 μm. (B) Quantification of the fraction of flagellated minicells produced by Δ min and Δ min Δ ycgR + flhDC strains. The numbers of minicells with one flagellum, two flagella, and more than two flagella were normalized to the total number of flagellated minicells. Total number of flagellated minicells included in the quantification: 673 (Δ min ) and 613 (Δ min Δ ycgR + flhDC ). The error bars represent the standard deviation among three labeling experiments. (C) Violin plots showing the distributions of flagellar rotation frequency identified in the power spectral density (PSD) profiles for single minicells (examples of PSDs are shown in Figure S3 ). Total number of minicells included in the analysis: 61 (Δ min ), 107 (Δ min Δ ycgR ), and 107 (Δ min Δ ycgR + flhDC ). A two-sample t -test was used to compare different strains and calculate significance values: P ≤ 0.05 (*), P ≤ 0.01 (**), P ≤ 0.001 (***), P > 0.5 (ns). (D) Minicell swimming speed measured as a function of medium viscosity for Δ min Δ ycgR + flhDC minicells and normal-sized wild-type cells. The viscosity of the medium was modified with Ficoll 400 in the range between 0 and 10% (w/v) Ficoll (corresponding to 1–5.3 mPa·s). The calibration function between the percentage of Ficoll and medium viscosity, obtained by fitting the published data ( Figure S4a ). The data points in the graph were calculated as the average between three replicated experiments for wild-type and four replicates for minicells; the error bars represent the standard error of the mean. The range of viscosities, within which the swimming speed increase has been observed, is shown in the inset. The significance of the maximal increase was calculated using a paired one-side t -test ( p = 0.028). (E) Swimming speed of minicells produced by Δ min and Δ min Δ ycgR + flhDC strains measured in a time-course experiment over 4 days after the purification. During the time-course, minicells were kept at 18 °C in Tryptone broth (TB) daily supplemented with 1% glucose. Three experiments were performed for each strain. The horizontal dashed line indicates the lowest threshold for swimming velocity (measured for nonmotile minicells produced by the Δ min Δ fliC strain).

Article Snippet: Statistical significance values were calculated with a two-sample t -test (ttest2) in MATLAB.

Techniques: Fluorescence, Produced, Labeling, Generated, Standard Deviation, Viscosity, Modification, Purification

Journal: ACS Applied Materials & Interfaces

Article Title: Motile and Chemotactic Minicells and Minicell-Driven Biohybrids Engineered for Active Cargo Delivery

doi: 10.1021/acsami.5c04638

Figure Lengend Snippet: Engineered minicells exhibit enhanced chemotactic performance. (A) A schematic of the experimental setup of the microfluidic device for quantitative measurements of chemotactic response. Flagellated minicells are illustrated in green (shown not to the scale). Minicell movement is recorded in the middle of a microfluidic channel with an established linear gradient of a nonmetabolizable chemoattractant α-methyl- dl -aspartate (0–1 mM MeAsp). Single trajectories of minicells are tracked and the runs are separated based on their directionality: either up (|α| < π/4) or down (3π/4 < |α| < π) the attractant gradient. (B) Chemotactic drift measured for the minicells produced by Δ min , Δ min + flhDC , and Δ min Δ ycgR + flhDC strains in the presence of a 0–1 mM MeAsp gradient. Each data point represents the average chemotactic drift for a population of purified and tracked minicells, while the bars show the mean among the averages of the replicated experiments (at least five for each strain). The error bars are the standard errors of the mean. A two-sample t -test was used to calculate significance values: P ≤ 0.05 (*), P ≤ 0.01 (**), P ≤ 0.001 (***), P > 0.5 (ns). (C) Run duration distributions from a representative experiment in the presence of the MeAsp gradient (0–1 mM) for the minicells produced by Δ min , Δ min + flhDC , and Δ min Δ ycgR + flhDC strains. The runs shorter than 5 frames (0.1 s) were excluded from the analysis. All other runs were separated based on their directionality: either up or down the gradient (as shown in A). The average run durations in both directions are shown in the legends. The number of analyzed trajectories: 909 (Δ min ), 5121 (Δ min + flhDC ), and 2107 (Δ min Δ ycgR + flhDC ). (D) Average run duration in the presence of the 0–1 mM MeAsp gradient for the minicells produced by Δ min , Δ min + flhDC , and Δ min Δ ycgR + flhDC strains. The runs were conditioned on their directionalityeither up or down the gradient of MeAsp. The average values were calculated for at least four replicated experiments for each strain. The error bars are the standard errors of the mean. To compare run durations up and down (the gradient), a paired one-side t -test has been applied. A nonpaired t -test was used to compare run durations toward MeAsp for different strains. Significance marks stand for: P ≤ 0.05 (*), P ≤ 0.01 (**), P ≤ 0.001 (***), P > 0.5 (ns).

Article Snippet: Statistical significance values were calculated with a two-sample t -test (ttest2) in MATLAB.

Techniques: Produced, Purification

Journal: ACS Applied Materials & Interfaces

Article Title: Motile and Chemotactic Minicells and Minicell-Driven Biohybrids Engineered for Active Cargo Delivery

doi: 10.1021/acsami.5c04638

Figure Lengend Snippet: Motile and chemotactic minicell-based biohybrids enable cargo delivery toward a chemoattractant source. (A) A schematic of the attachment mechanism of streptavidin-coated fluorescent particles to the surface of minicells mediated by biotinylated autotransporter antigen 43 (Ag43). (B) Attachment of Δ min Δ ycgR + flhDC minicells (expressing GFP and modified Ag43 shown in green) to 1.4 μm fluorescent streptavidin-coated particles (red). The scale bar is 2 μm. (C) A scanning electron microscopy (SEM) image of a 1.4 μm streptavidin-coated particle with four attached minicells. The scale bar is 1 μm. (D) A time-lapse wide-field fluorescence series with a minicell (expressing GFP and modified Ag43, shown in green) carrying a 400 nm bead (shown in red) (see Movie S7 ). The scale bar is 5 μm; timestamps are shown. (E) Swimming speed of the minicells with attached 400 nm particles (minibots) in the absence and presence of 15% iodixanol. Total number of minibots included in the distributions: 450 (without iodixanol) and 1348 (with iodixanol). A two-sample t -test was used to calculate a significance value between the two conditions: P = 0.002 (**). (F) A schematic of the chemotactic chamber for the accumulation assays. Two wells are connected through a channel containing a porous agarose membrane; the lengths of both arms of the channel are shown. To establish a gradient, an attractant is added into one well while the minibots are loaded into the other one. Accumulation of fluorescent particles assessed by recording snapshots in the proximity of the attractant source (shown in the zoom-in panel). (G) Chemotactic accumulation of minibots in the presence of 1 mM MeAsp. The snapshots were taken after 30 min and 24 h after loading into the microfluidic device. The chip was kept at 22.5 °C during the entire experiment. The scale bar is 5 μm. (H) Quantification of the accumulated fluorescent particles performed with single particle counting. The particles attached to elongated parental cells, which accidentally appeared in the field of view (∼1%), were excluded from the quantification. The data represent the average across seven measurements per each condition for minibots and five measurements for the beads only control; the error bars are the standard deviations. A paired one-side t -test was used to calculate significance values for the measurements at t = 0.5 h and t = 24 h. A nonpaired t -test was used to compare the accumulation in the presence and in the absence of the attractant. Significance marks stand for: P ≤ 0.05 (*), P ≤ 0.01 (**), P ≤ 0.001 (***), P > 0.5 (ns).

Article Snippet: Statistical significance values were calculated with a two-sample t -test (ttest2) in MATLAB.

Techniques: Expressing, Modification, Electron Microscopy, Fluorescence, Membrane, Single Particle, Control