Supporting Information Cationic Conjugated Polymers-Induced ...
Supporting Information
Cationic Conjugated Polymers-Induced Quorum Sensing of Bacteria Cells Pengbo Zhang, Huan Lu, Hui Chen, Jiangyan Zhang, Libing Liu*, Fengting Lv, and Shu Wang* Beijing National Laboratory for Molecular Science, Key Laboratory of Organic Solids, Institute of Chemistry, Chinese Academy of Sciences, Beijing, 100190, P. R. China. E-mail: [email protected]; [email protected]
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Table of content 1. Materials and Methods 2. The bioluminescence and growth curve of V. harveyi 3. Preparation of cell-free culture fluids 4. Detection of AI-2 using V. harveyi bioassay 5. Confocal laser scanning microscopy (CLSM) characterization 6. Zeta potential measurements 7. Drug-resistance experiments 8. Biological compatibility of PFP-G2 9. Biofilm formation assay 10. Figure S1-2 and Table S1-2
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1. Materials and Methods E. coli MG1655 was obtained from Agricultural Culture Collection of China (ACCC). V. harveyi BB170 (BAA-1117) were purchased from American Type Culture Collection (ATCC). 2216E Agar and Liquid Medium were purchased from Qingdao Hope Bio-Technology Co., Ltd. AmpicillinNa (AMP) was bought from Xinjingke Biotechnology Co., Ltd (Beijing, China). 96-well cell culture plates and 96-well black microplates (for fluorescence assays) were purchased from Corning Inc. The liquid medium and double distilled water (ddH2O) were autoclaved at 120 °C for 20 min. The absorbance for bacteria analysis and the luminescence signals were read on a microplate reader (BIO-TEK Synergy HT, USA). Zeta potentials were measured on a Nano ZS 90 system. Bright-field and fluorescence images were taken with a confocal laser scanning microscope (FV1200-IX81, Olympus, Japan). All other reagents were of analytical reagent grade and used as purchased without further purification.
2. The bioluminescence and growth curve of V. harveyi A single colony of V. harveyi on a solid 2216E agar plate was transferred to 10 mL of 2216E liquid culture medium and grown at 30 °C with aeration overnight. Then dilute the culture 1:1000 into fresh autoinducer bioassay (AB) medium. The recipe for AB medium has been reported previously.1 180 µL of the inoculated medium were placed in each of the wells of a 96 well plate and 96-well black microplate and combined with 20 µL of AB medium. Light production and OD600 were recorded at 30 ºC every 30 min with aeration. The experiments were carried out in triplicate and the plotted curves were derived from the mean value.
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3. Preparation of cell-free culture fluids
A single colony of E. coli grown on a solid LB agar plate was used to inoculate 10 mL LB medium at 37 °C with aeration overnight. Then 10 µL of the overnight-grown cultures were added into 10 mL of fresh LB medium. Aliquots of this culture were then mixed with known volumes of stock solutions of PFP-G2 to make a final concentration of 100 µM. The mixtures were grown for various times ranging from 1 h to 8 h at 37 °C with aeration. Cell-free culture fluids were prepared by removing the cells from the growth medium by centrifugation at 15, 000 rpm for 5 min in a microcentrifuge. Cell-free culture fluids were used immediately or frozen to -20 ºC for storage.
4. Detection of AI-2 using V. harveyi bioassay
A single colony of V. harveyi on a solid 2216E agar plate was transferred to 10 mL of 2216E liquid culture medium and grown at 30 °C with aeration overnight. Then dilute the culture 1:1000 with fresh AB medium. 180 µL of the inoculated medium were placed in each of the wells of a 96-well black microplate and combined with 20 µL of the cell-free culture fluids mentioned above. Light production was recorded at 30 ºC every 30 min with aeration.
5. Confocal laser scanning microscopy (CLSM) characterization
E. coli with 100 µM PFP-G2 was incubated at 37 °C for 30 min, and then the mixtures were mounted on a glass slide with a cover slip on top and examined by confocal laser scanning microscopy using a 405 nm laser (FV5&LAMAR). The fluorescence of PFP-G2 was highlighted in blue. S4
6. Zeta potential measurements
E. coli with 100 µM PFP-G2 was incubated at 37 °C for 30 min, and then the unbounded PFP-G2 was removed by centrifugation at 7100 rpm for 5 min. The bacteria were washed with ddH2O or 500 mM NaCl once. After centrifugation at 7100 rpm for 5 min to remove the supernatant, the bacteria were resuspended in ddH2O and the suspensions were kept on ice for zeta potential measurements. As negative controls, untreated bacteria (without PFP-G2) were also incubated under exactly the same conditions.
7. Drug-resistance experiments
E. coli with 100 µM PFP-G2 was incubated at 37 °C for 5 h and 6 h with aeration and then diluted 10-fold with LB medium. 100 µL of the diluted bacteria were added to a 96-well microplate and combined with AMP (5 mg/mL). OD600 were recorded at 37 ºC every 30 min using the microplate reader. Blank was incubated under exactly the same conditions without PFP-G2. The survival rate is defined as OD600 produced by the mixture after addition of AMP divided by OD600 produced by the mixture in the absence of AMP at the same time interval.
8. Biological compatibility of PFP-G2
E. coli with 100 µM PFP-G2 was incubated at 37 °C for 30 min. Then the bacteria were diluted 5 × 104 fold in LB medium. A volume of 100 µL of the diluted bacteria was spread on the solid LB agar plate, and the colonies formed after 12 h incubation at 37 °C. Relative colony-forming units (cfu) of E. coli in the absence of PFP-G2 were normalized to 100%.
9．Biofilm formation assay Overnight cultured E. coli was diluted 1:100 into fresh LB medium. 100 µL of the inoculated medium combined with 100 µM PFP-G2 were placed in the wells of a 96 well plate and incubate S5
at 37 °C for 24 h. Discard the medium and wash the wells with PBS three times to remove planktonic cells. Bacteria attached on the bottom were fixed with 95% ethanol for 15 min and then stained with 0.1% crystal violet for 15 min. After washing three times with water, 10% acetic acid was added and absorbance at 590 nm was recorded. The experiments were carried out in triplicate.
Figure S1. Growth curve of V. harveyi in response to cell-free culture fluids extracted from E. coli incubated with 0 (a) and 100 µM (b) PFP-G2 for 1-8 hours. Growth curve (c) and luminescence spectra (d) of V. harveyi in response to AB medium (control), LB medium, LB medium with PFP-G2 after 6 h incubation, cell-free culture fluids extracted from E. coli after 6 h incubation and cell-free culture fluids extracted from E. coli with PFP-G2 after 6 h incubation. S6
Figure S2. Survival rate of E. coli incubated with 100 µM PFP-G2 for 5 h (a) and 6 h (b) against different concentrations of AMP after 1 h incubation.
Table S1. Cfu of E. coli in the presence and absence of PFP-G2 E. coli without PFP-G2
E. coli with PFP-G2
Average cfu
1574±13
1450±26
Relative cfu (%)
100
92.12±1.65
Table S2. Effect of PFP-G2 on biofilm formation of E. coli E. coli
E. coli with PFP-G2
increment
1
0.206
0.349
69.41%
2
0.240
0.355
47.91%
3
0.215
0.321
49.30%
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average
55.54±12.03%
References (1) E. P. Greenberg, J. W. Hastings, S. Ulitzur, Arch. Microbiol. 1979, 120, 87.
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Cationic Conjugated Polymers-Induced Quorum Sensing of Bacteria Cells Pengbo Zhang, Huan Lu, Hui Chen, Jiangyan Zhang, Libing Liu*, Fengting Lv, and Shu Wang* Beijing National Laboratory for Molecular Science, Key Laboratory of Organic Solids, Institute of Chemistry, Chinese Academy of Sciences, Beijing, 100190, P. R. China. E-mail: [email protected]; [email protected]
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Table of content 1. Materials and Methods 2. The bioluminescence and growth curve of V. harveyi 3. Preparation of cell-free culture fluids 4. Detection of AI-2 using V. harveyi bioassay 5. Confocal laser scanning microscopy (CLSM) characterization 6. Zeta potential measurements 7. Drug-resistance experiments 8. Biological compatibility of PFP-G2 9. Biofilm formation assay 10. Figure S1-2 and Table S1-2
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1. Materials and Methods E. coli MG1655 was obtained from Agricultural Culture Collection of China (ACCC). V. harveyi BB170 (BAA-1117) were purchased from American Type Culture Collection (ATCC). 2216E Agar and Liquid Medium were purchased from Qingdao Hope Bio-Technology Co., Ltd. AmpicillinNa (AMP) was bought from Xinjingke Biotechnology Co., Ltd (Beijing, China). 96-well cell culture plates and 96-well black microplates (for fluorescence assays) were purchased from Corning Inc. The liquid medium and double distilled water (ddH2O) were autoclaved at 120 °C for 20 min. The absorbance for bacteria analysis and the luminescence signals were read on a microplate reader (BIO-TEK Synergy HT, USA). Zeta potentials were measured on a Nano ZS 90 system. Bright-field and fluorescence images were taken with a confocal laser scanning microscope (FV1200-IX81, Olympus, Japan). All other reagents were of analytical reagent grade and used as purchased without further purification.
2. The bioluminescence and growth curve of V. harveyi A single colony of V. harveyi on a solid 2216E agar plate was transferred to 10 mL of 2216E liquid culture medium and grown at 30 °C with aeration overnight. Then dilute the culture 1:1000 into fresh autoinducer bioassay (AB) medium. The recipe for AB medium has been reported previously.1 180 µL of the inoculated medium were placed in each of the wells of a 96 well plate and 96-well black microplate and combined with 20 µL of AB medium. Light production and OD600 were recorded at 30 ºC every 30 min with aeration. The experiments were carried out in triplicate and the plotted curves were derived from the mean value.
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3. Preparation of cell-free culture fluids
A single colony of E. coli grown on a solid LB agar plate was used to inoculate 10 mL LB medium at 37 °C with aeration overnight. Then 10 µL of the overnight-grown cultures were added into 10 mL of fresh LB medium. Aliquots of this culture were then mixed with known volumes of stock solutions of PFP-G2 to make a final concentration of 100 µM. The mixtures were grown for various times ranging from 1 h to 8 h at 37 °C with aeration. Cell-free culture fluids were prepared by removing the cells from the growth medium by centrifugation at 15, 000 rpm for 5 min in a microcentrifuge. Cell-free culture fluids were used immediately or frozen to -20 ºC for storage.
4. Detection of AI-2 using V. harveyi bioassay
A single colony of V. harveyi on a solid 2216E agar plate was transferred to 10 mL of 2216E liquid culture medium and grown at 30 °C with aeration overnight. Then dilute the culture 1:1000 with fresh AB medium. 180 µL of the inoculated medium were placed in each of the wells of a 96-well black microplate and combined with 20 µL of the cell-free culture fluids mentioned above. Light production was recorded at 30 ºC every 30 min with aeration.
5. Confocal laser scanning microscopy (CLSM) characterization
E. coli with 100 µM PFP-G2 was incubated at 37 °C for 30 min, and then the mixtures were mounted on a glass slide with a cover slip on top and examined by confocal laser scanning microscopy using a 405 nm laser (FV5&LAMAR). The fluorescence of PFP-G2 was highlighted in blue. S4
6. Zeta potential measurements
E. coli with 100 µM PFP-G2 was incubated at 37 °C for 30 min, and then the unbounded PFP-G2 was removed by centrifugation at 7100 rpm for 5 min. The bacteria were washed with ddH2O or 500 mM NaCl once. After centrifugation at 7100 rpm for 5 min to remove the supernatant, the bacteria were resuspended in ddH2O and the suspensions were kept on ice for zeta potential measurements. As negative controls, untreated bacteria (without PFP-G2) were also incubated under exactly the same conditions.
7. Drug-resistance experiments
E. coli with 100 µM PFP-G2 was incubated at 37 °C for 5 h and 6 h with aeration and then diluted 10-fold with LB medium. 100 µL of the diluted bacteria were added to a 96-well microplate and combined with AMP (5 mg/mL). OD600 were recorded at 37 ºC every 30 min using the microplate reader. Blank was incubated under exactly the same conditions without PFP-G2. The survival rate is defined as OD600 produced by the mixture after addition of AMP divided by OD600 produced by the mixture in the absence of AMP at the same time interval.
8. Biological compatibility of PFP-G2
E. coli with 100 µM PFP-G2 was incubated at 37 °C for 30 min. Then the bacteria were diluted 5 × 104 fold in LB medium. A volume of 100 µL of the diluted bacteria was spread on the solid LB agar plate, and the colonies formed after 12 h incubation at 37 °C. Relative colony-forming units (cfu) of E. coli in the absence of PFP-G2 were normalized to 100%.
9．Biofilm formation assay Overnight cultured E. coli was diluted 1:100 into fresh LB medium. 100 µL of the inoculated medium combined with 100 µM PFP-G2 were placed in the wells of a 96 well plate and incubate S5
at 37 °C for 24 h. Discard the medium and wash the wells with PBS three times to remove planktonic cells. Bacteria attached on the bottom were fixed with 95% ethanol for 15 min and then stained with 0.1% crystal violet for 15 min. After washing three times with water, 10% acetic acid was added and absorbance at 590 nm was recorded. The experiments were carried out in triplicate.
Figure S1. Growth curve of V. harveyi in response to cell-free culture fluids extracted from E. coli incubated with 0 (a) and 100 µM (b) PFP-G2 for 1-8 hours. Growth curve (c) and luminescence spectra (d) of V. harveyi in response to AB medium (control), LB medium, LB medium with PFP-G2 after 6 h incubation, cell-free culture fluids extracted from E. coli after 6 h incubation and cell-free culture fluids extracted from E. coli with PFP-G2 after 6 h incubation. S6
Figure S2. Survival rate of E. coli incubated with 100 µM PFP-G2 for 5 h (a) and 6 h (b) against different concentrations of AMP after 1 h incubation.
Table S1. Cfu of E. coli in the presence and absence of PFP-G2 E. coli without PFP-G2
E. coli with PFP-G2
Average cfu
1574±13
1450±26
Relative cfu (%)
100
92.12±1.65
Table S2. Effect of PFP-G2 on biofilm formation of E. coli E. coli
E. coli with PFP-G2
increment
1
0.206
0.349
69.41%
2
0.240
0.355
47.91%
3
0.215
0.321
49.30%
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average
55.54±12.03%
References (1) E. P. Greenberg, J. W. Hastings, S. Ulitzur, Arch. Microbiol. 1979, 120, 87.
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