HomeMicrobiologyNon-electrostatic interactions related to mixture formation between polyallylamine and Escherichia coli

Non-electrostatic interactions related to mixture formation between polyallylamine and Escherichia coli


PAA (M.W. = 1600) was bought from Nittobo Medical Co., Ltd. (Fukushima, Japan). Methyl glycolate and sodium hydroxide have been bought from Tokyo Chemical Business Co., Ltd. (Tokyo, Japan). Ampicillin, arabinose, acetate, sodium acetate, glycine, and dimethylformamide have been bought from FUJIFILM Wako Pure Chemical Co., Ltd. (Osaka, Japan). HEPES was bought from Dojin Chemical Analysis Institute (Kumamoto, Japan).

Micro organism strains and progress circumstances

The pressure E. coli BL21 (DE3) was reworked by pGEX 4T-2 vector (Cytiva, Ma, USA) and grown in LB broth containing 100 μg/mL of ampicillin at 37 °C. The GFP-expressed E. coli (pGLO™ Bacterial Transformation Equipment; Bio-Rad Laboratories, CA, USA) was used for the bacterial aggregation assay, and grown in LB broth containing 1% arabinose and 100 μg/mL of ampicillin at 37 °C. On the finish of the exponential progress part, these micro organism have been centrifuged and washed three-times with 100 mM sodium acetate (pH 4.0) or 100 mM glycine sodium hydroxide (pH 10). The bacterial suspension was adjusted by measuring the optical density at a wavelength of 600 nm (OD600).

Isothermal titration calorimetry

ITC experiments have been carried out with a calorimeter (MicroCal VP-ITC, Malvern Devices Ltd., UK) at 25 °C. PAA (0.02%, w/w, 125 μM) or glycolylated PAA (19.7% G-PAA: 0.032%, w/w, 156 μM; 37.7% G-PAA: 0.037%, w/w, 157 μM; 59.0% G-PAA: 0.043%, w/w, 157 μM) in a syringe was titrated into E. coli resolution (OD600 = 1.0) within the cell. The amount was 10 μL for every injection, and the cell was constantly stirred at 90 rpm. The corresponding warmth of dilution of PAA titrated into the buffer was used to appropriate the information. The thermodynamic parameters have been evaluated utilizing the one-set of unbiased binding websites mannequin equipped by MicroCal Origin 7.0 software program, which every binding web site is assumed to indicate the similar affinity and enthalpy change25. The molarity of micro organism was assumed to be calculated utilizing the equation (variety of E. coli in resolution)/(6.02 × 1023)32.

Synthesis of G-PAA

The amino group of PAA was substituted with methyl glycolate (Supplementary Figs. S1B and S5)33 as follows. Methyl glycolate was reacted to PAA resolution with stirring in a single day and the resultant combination was added sodium hydroxide resolution, evaporated on a rotary evaporator to take away methanol, and dialyzed to take away by-product sodium glycolate. The diploma of substitution of the first amine group was modified by the response situation of methyl glycolate, PAA resolution, and sodium hydroxide resolution (element situation was described within the legend of Supplementary Fig.S5.).

Labeling of PAA

The Cy5.5 labeling of PAA was carried out utilizing Cy5.5 NHS ester (Lumiprobe Company, MD, USA). PAA (0.1%, w/w, 625 μM) in 100 mM HEPES buffer (pH 7.0, 270 μL) was combined with Cy5.5 NHS in dimethylformamide (6.25 mM, 30 μL). The combination was stirred at room temperature for 3 h then 800 μL of 100 mM glycine sodium hydroxide (pH 10) was added to the combination to cease the response for the aggregation assay.

Bacterial aggregation assay

The suspension of GFP-expressed E. coli (OD600 = 1.0, 1400 μL) was incubated with PAA (0.0025%, 0.025%, and 0.25%, w/w, 300 μL) or Cy5.5-labeled PAA (0.025%, w/w, 300 μL) in every buffer at room temperature for 3 min. The combination was noticed utilizing fluorescence microscopy (BZ-X710, Keyence, Osaka, Japan).

Dynamic mild scattering

The dimensions distribution of the polymer was measured by DLS (Zetasizer Nano ZS, Malvern Devices Ltd., UK) at 25 °C. The polymer was diluted in 100 mM sodium acetate (pH 4.0) or 100 mM glycine sodium hydroxide (pH 10).



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