Gain hmgb1-FL DNA fragment and pET28a plasmid
Gain hmgb1-FL, -AB box, -A box, -B box primer. Gene fragments were amplified using PCR. And the linearized carrier was prepared. Plasmids were constructed using homologous recombination.
Plasmids were expanded in DH5α and the plasmids were sequenced.
Plasmids with the correct sequence were recovered and stored in a -20 ° C refrigerator.
Gain pslg DNA fragment and primer.
Gene fragments were amplified using PCR. And the linearized carrier was prepared. Plasmids were constructed using homologous recombination.
Plasmids were expanded in DH5α and the plasmids were sequenced.
Plasmids with the correct sequence were recovered and stored in a -20 ° C refrigerator.
Plasmid “pET28a-6×His-hmgb1-FL” with ampicillin resistance was transferred into BL21. And BL21 was coated on LB plates containing ampicillin.
Single colonies were selected on LB plates and inoculated into 10mL of LB liquid medium containing ampicillin for initial enrichment culture.
10 mL of LB liquid medium containing enriched BL21 was poured into 1L of LB liquid medium containing ampicillin and incubated for 4 h at 37 ° C at 180 rpm on a shaker. And then IPTG was added to 100 μM to induce protein expression. And incubate it for 16 h at 16 ° C at 180 rpm on a shaker.
The bacteria that fully expressed the target protein were recovered and stored in a -80 ° C refrigerator for further use.
Plasmid “pET28a-6×His-hmgb1-AB” with ampicillin resistance was transferred into BL21. And BL21 was coated on LB plates containing ampicillin.
Single colonies were selected on LB plates and inoculated into 10mL of LB liquid medium containing ampicillin for initial enrichment culture.
10 mL of LB liquid medium containing enriched BL21 was poured into 1L of LB liquid medium containing ampicillin and incubated for 4 h at 37 ° C at 180 rpm on a shaker. And then IPTG was added to 100 μM to induce protein expression. And incubate it for 16 h at 16 ° C at 180 rpm on a shaker.
The bacteria that fully expressed the target protein were recovered and stored in a -80 ° C refrigerator for further use.
Plasmid “pET28a-6×His-hmgb1-A” with ampicillin resistance was transferred into BL21 was transferred into BL21. And BL21 was coated on LB plates containing ampicillin.
Single colonies were selected on LB plates and inoculated into 10mL of LB liquid medium containing ampicillin for initial enrichment culture.
10 mL of LB liquid medium containing enriched BL21 was poured into 1L of LB liquid medium containing ampicillin and incubated for 4 h at 37 ° C at 180 rpm on a shaker. And then IPTG was added to 100 μM to induce protein expression. And incubate it for 16 h at 16 ° C at 180 rpm on a shaker.
The bacteria that fully expressed the target protein were recovered and stored in a -80 ° C refrigerator for further use.
Plasmid “pET28a-6×His-hmgb1-B” with ampicillin resistance was transferred into BL21. And BL21 was coated on LB plates containing ampicillin.
Single colonies were selected on LB plates and inoculated into 10mL of LB liquid medium containing ampicillin for initial enrichment culture.
10 mL of LB liquid medium containing enriched BL21 was poured into 1L of LB liquid medium containing ampicillin and incubated for 4 h at 37 ° C at 180 rpm on a shaker. And then IPTG was added to 100 μM to induce protein expression. And incubate it for 16 h at 16 ° C at 180 rpm on a shaker.Thaw and recover the HT-29 cells, seed them in a 60 mm petri dish, and begin cell culturing.
The bacteria that fully expressed the target protein were recovered and stored in a -80 ° C refrigerator for further use.
Subculture cells at the 1:2 ratio.Filter the LPS solution through a 0.22 μm syringe filter, and dilute the original solution to 1.0 mg/mL for experiment use. Store it at -20℃.
Plasmid “pET28a-6×His-pslg” with ampicillin resistance was transferred into BL21 was transferred into BL21. And BL21 was coated on LB plates containing ampicillin.Subculture again, and adjust the subculture ratio to 1:4.
Single colonies were selected on LB plates and inoculated into 10mL of LB liquid medium containing ampicillin for initial enrichment culture.
10 mL of LB liquid medium containing enriched BL21 was poured into 1L of LB liquid medium containing ampicillin and incubated for 4 h at 37 ° C at 180 rpm on a shaker. And then IPTG was added to 100 μM to induce protein expression. And incubate it for 16 h at 16 ° C at 180 rpm on a shaker.Subculture the cells at the 1:4 ratio into 2 dishes.
The bacteria that fully expressed the target protein were recovered and stored in a -80 ° C refrigerator for further use.
The BL21 containing HMGB1-FL and HMGB1-AB box was broken, and the supernatant was collected after ultra-high speed centrifugation. The target proteins HMGB1-FL and HMGB1-AB box were obtained after the supernatant was purified by Ni beads.Subculture the cells in one dish at the 1:4 ratio. Freeze cells in the other dish, and preserve it at -80℃ first, then store in liquid nitrogen.
The BL21 containing HMGB1-A box and HMGB1-B box was broken, and the supernatant was collected after ultra-high speed centrifugation. The target proteins HMGB1-FL and HMGB1-AB box were obtained after the supernatant was purified by Ni beads.
The BL21 containing PslG was broken, and the supernatant was collected after ultra-high speed centrifugation. The target proteins HMGB1-FL and HMGB1-AB box were obtained after the supernatant was purified by Ni beads. Observed many floating dead cells, and cells grew slowly at the same time.Wash with PBS twice, then subculture the cells at the 1:4 ratio.
The phenomenon remained still, so it was assumed contaminated by mycoplasmas. Wash with PBS twice, then add Mycoplasma Elimination Reagent (C0292, Beyotime) to the culture medium and subculture the cells at the 1:4 ratio. Resuscitate PAO1.
Dead cells became less, but its growth rate was still slow. Subculture the cells at the 1:4 ratio. Do inhibition of biofilm growth experiment using FL. Collect the data of 0h, 6h and 12h. Add different concentrations of 3OC12HSL to the medium in the 96-well plate and incubate at 37℃, detecting fluorescence every 10 minutes, lasting 3 hours.
Few dead cells existed, and the growth rate gradually recovered. Subculture the cells at the 1:4 ratio. Do inhibition of biofilm growth experiment using FL. Collect the data of 24h. Add different concentrations (adjusted after the first experiment) of 3OC12HSL to the medium in the 96-well plate and incubate at 37℃, detecting fluorescence every 10 minutes, lasting 3 hours.
Cells returned normal. Subculture the cells at the 1:4 ratio. Resuscitate PAO1. Repeat the Quorum Sensing experiment. Shake before each detection.
Add 100 ng/mL LPS into one cell culture (positive control) and an equal amount of PBS to the other (negative control), then incubate for an hour. Extract RNA, and conduct reverse transcription followed by qPCR. Store RNA at -20℃. Trypsinize cells from one cell culture to make the cell suspension for cytotoxicity test (control). Stain with trypan blue and calculate survival rate. Subculture the cells at the 1:4 ratio. Inoculate PAO1 into 96-well plates (100ul per well), culture at 37℃ for 24h. Repeat the Quorum Sensing experiment.
Use the remaining extracted RNA again, and adjust the dilution factor of products of reverse transcription to twofold. Then rerun qPCR. Do biofilm disassembly experiment using PslG and FL.
Subculture the cells at the 1:4 ratio. Prepare M9 medium.
Resuscitate PAO1. Pseudomonas aeruginosa were resuscitated and spread onto LB solid medium.
Add 2,000 ng/mL PslG, 500 ng/mL and 2,000 ng/mL HMGB1 (fl) into 3 cell cultures respectively, then incubate for an hour. Add 100 ng/mL LPS, 2,000 ng/mL PslG, 500 ng/mL and 2,000 ng/mL HMGB1 (fl) into 4 cell cultures respectively, then incubate for 2 hours. Extract RNA, and conduct reverse transcription followed by qPCR using the twofold dilution factor. Store RNA at -20℃. Repeat the cytotoxicity test for the control group. Add 1 - 6 μg/mL LPS into 6 cell cultures and incubate for 24 hours for cytotoxicity test. Subculture the cells at the 1:4 ratio. Inoculate PAO1 into 96-well plates (100ul per well), culture at 37℃ for 24h.
Trypsinize cells from cell cultures to make the cell suspension for cytotoxicity test (1 - 6 μg/mL LPS). Stain with trypan blue and calculate survival rate. Do biofilm disassembly experiment using FL, AB, A, B, PslG.
Subculture the cells at the 1:4 ratio. Repeat the Quorum Sensing experiment.
Add 1 - 6 μg/mL HMGB1 (fl) and 1 - 6 μg/mL PslG into cell cultures and incubate for 24 hours for cytotoxicity test. Subculture the cells at the 1:4 ratio. Resuscitate PAO1, prepare 300nM imidazole.
Trypsinize cells from cell cultures to make the cell suspension for cytotoxicity test (1 - 6 μg/mL HMGB1 (fl) and 1 - 6 μg/mL PslG). Stain with trypan blue and calculate survival rate. Do inhibition of biofilm growth experiment using FL, AB, A, B, PslG. Collect the data of 0h, 6h and 12h.
Subculture the cells at the 1:4 ratio. Do inhibition of biofilm growth experiment using FL, AB, A, B, PslG. Collect the data of 24h. Pseudomonas aeruginosa were resuscitated and spread onto LB solid medium.
Single colonies were picked on LB plates, inoculated into LB liquid medium, and incubated overnight at 37 ° C in a shaker.
Repeat inflammation verification for the negative control group, the positive control group (1 h and 2 h), 500 ng/mL and 2,000 ng/mL HMGB1 (fl) group (1 h and 2 h), and 2,000 ng/mL PslG group (1 h and 2 h)R epeat cytotoxicity test for the LPS groups (1 - 6 μg/mL LPS). Subculture the cells at the 1:4 ratio. Prepare GB buffer. Resuscitate PAO1. The medium was diluted and inoculated into cell culture dishes and incubated for 48 hours in a constant temperature incubator at 37 ° C. Repeat the Quorum Sensing experiment. Add bacteria sample as representative of 3OC12HSL near the pathogenic bacteria.
Trypsinize cells from cell cultures to make the cell suspension for cytotoxicity test (1 - 6 μg/mL LPS). Stain with trypan blue and calculate survival rate. Do inhibition of biofilm growth experiment using FL, AB, A, B, PslG. Collect the data of 0h, 6h and 12h. HMGB1_A box and PslG proteins were also added and treated for 4 hours in a constant temperature incubator at 37 ° C. After treatment, staining was performed, followed by imaging under a confocal microscope.
Subculture the cells at the 1:4 ratio. Do inhibition of biofilm growth experiment using FL, AB, A, B, PslG. Collect the data of 24h. Repeat the Quorum Sensing experiment.
Resuscitate PAO1.
Repeat inflammation verification for the negative control group, the positive control group (1 h and 2 h), 500 ng/mL and 2,000 ng/mL HMGB1 (fl) group (1 h and 2 h), and 2,000 ng/mL PslG group (1 h and 2 h). Repeat cytotoxicity test for the HMGB1 (fl) groups (1 - 6 μg/mL HMGB1 (fl)). Subculture the cells at the 1:4 ratio. Inoculate PAO1 into 96-well plates (100ul per well), culture at 37℃ for 24h. Repeat the Quorum Sensing experiment to revise some extreme outliers.
Trypsinize cells from cell cultures to make the cell suspension for cytotoxicity test (1 - 6 μg/mL HMGB1 (fl)). Stain with trypan blue and calculate survival rate. Do biofilm disassembly experiment using FL, AB, A, B, PslG. Resuscitate PAO1.
Subculture the cells at the 1:4 ratio. Do inhibition of biofilm growth experiment using FL, AB, A, B, PslG. Collect the data of 0h, 6h and 12h. Repeat the Quorum Sensing experiment to revise some extreme outliers.
Do inhibition of biofilm growth experiment using FL, AB, A, B, PslG. Collect the data of 24h.
Repeat cytotoxicity test for the LPS groups (1 - 6 μg/mL LPS), and PslG groups (1 - 6 μg/mL PslG). Subculture the cells at the 1:4 ratio. Resuscitate PAO1.
Trypsinize cells from cell cultures to make the cell suspension for cytotoxicity test (1 - 6 μg/mL LPS and 1 - 6 μg/mL PslG). Stain with trypan blue and calculate survival rate. Inoculate PAO1 into 96-well plates (100ul per well), culture at 37℃ for 24h.
Subculture the cells at the 1:4 ratio. Do biofilm disassembly experiment using FL, AB, A, B, PslG.
Repeat cytotoxicity test for the PslG groups (1 - 6 μg/mL PslG), and HMGB1 (fl) groups (1 - 6 μg/mL HMGB1 (fl)). Subculture the cells at the 1:4 ratio.
Trypsinize cells from cell cultures to make the cell suspension for cytotoxicity test (1 - 6 μg/mL PslG, and 1 - 6 μg/mL HMGB1 (fl)). Stain with trypan blue and calculate survival rate.
Subculture the cells at the 1:4 ratio.
Repeat cytotoxicity test for the 2 μg/mL and 6 μg/mL HMGB1 (fl) groups, and 2 μg/mL, 5 μg/mL, and 6 μg/mL PslG groups to revise extreme data outliers. Repeat inflammation verification for the 2,000 ng/mL HMGB1 (fl) group (2 h), and 2,000 ng/mL PslG groups (1 h and 2 h) to revise extreme data outliers.
Trypsinize cells from cell cultures to make the cell suspension for cytotoxicity test (2 μg/mL and 6 μg/mL HMGB1 (fl), and 2 μg/mL, 5 μg/mL, and 6 μg/mL PslG). Stain with trypan blue and calculate survival rate.
construct the plasmid
construct the plasmid
transform into BL21, spread a plate.
Do the experiment: The growth curve induced by arabinose (M9&LB-ECN&BL21)
Do the experiment: The growth curve induced by arabinose (LB-BL21)
Do the experiment: DNA concentration detection
In order to verify the disassembly ability of PslG to biofilm and to find out the protein with the strongest biofilm disassembly ability among the four HMGB1-based modified proteins, we designed a crystal violet staining experiment with reference to an article on the disassembly of biofilm using PslG and selected Pseudomonas aeruginosa PAO1 (abbreviated to PAO1 in the following part), which is a gram-negative bacterium that can and easily form biofilm, as the experimental strain. The experiment was divided into two parts: inhibition of biofilm growth and disassembly of biofilm. In the experiment of inhibiting biofilm growth, we inoculated PAO1 and added the proteins to the mediums at the same time, and the ability of the proteins to disassemble the biofilm is reflected by measuring the growth status of the biofilm; In the biofilm disassembly experiment, we inoculated PAO1 and cultured it first, and then added proteins after it formed a sufficient amount of biofilm, so as to reflect the ability of protein disassembly of biofilm by measuring the biofilm disassembly status. The biomass of biofilm in both experiments was measured by crystal violet staining assay.
Inhibition of biofilm growth experiment: (1) Use LB medium to resuscitate PAO1; (2) The resuscitated PAO1 bacterial solution was diluted to OD600=0.001 using LB medium, and inoculated into 96-well plates (100ul per well). FL (final concentration 200nM) was added to the treatment group, while no protein was added to the control group, and cultured at 37℃; (3) When the culture time reaches 0h, 6h, 12h, 24h, the following operations are carried out: Add 150ul ddH2O for oscillatory cleaning and sucking out (twice), add 150ul 0.1% crystal violet solution for dark dyeing for 15min and sucking out, add 200ul ddH2O for oscillatory cleaning and sucking out (twice), add 200ul anhydrous ethanol for dark dissolution for 15min. The OD550 of the sample was detected with an enzyme-labeler and the data was recorded.
Note: Time course of FL effects on biofilm growth. The biofilm biomass was measured by CV assay.
Biofilm disassembly experiment: (1) PAO1 was resuscitated using LB medium; (2) The recovered PAO1 solution was diluted to OD600=0.001 using LB medium, inoculated into 96-well plates (100ul per well), and cultured at 37℃ for 24h; (3) In the treatment group, FL (final concentration 200nM) and PslG (final concentration 50nM) were added respectively and cultured at 37℃; (4) When the culture time reaches 0min, 1h, 2h, 4h, 8h, the following operations are carried out: Add 150ul ddH2O for oscillatory cleaning and sucking out (twice), add 150ul 0.1% crystal violet solution for dark dyeing for 15min and sucking out, add 200ul ddH2O for oscillatory cleaning and sucking out (twice), add 200ul anhydrous ethanol for dark dissolution for 15min. The OD550 of the sample was detected with an enzyme-labeler and the data was recorded.
Note: Time course of FL effects on biofilm disassembly. The biofilm biomass was measured by CV assay.
In the experiment of inhibiting biofilm growth, the absorbance of OD550 corresponding to biofilm biomass in the control group reached about 2.0 at 24h, while the absorbance of FL treatment group was only about 1.3 at the same time, which effectively inhibited the growth of biofilm. In the biofilm disassembly experiment, the FL or PslG treatment groups reduced the OD550 absorbance corresponding to the biofilm biomass from about 1.5 to about 0.5 in the first two hours. From the results of the two groups of experiments, it can be concluded that FL and PslG have significant disintegration effects on biofilm, which provides a theoretical basis for us to select these two proteins and their modified proteins to destroy biofilm.
Since the purpose of our experiment was only to illustrate and measure the disassembly ability of various proteins to the biofilm, protein concentration gradient was not established, and the concentration used in the literature was directly adopted. These two sets of results show that both proteins can produce significant effects at the concentrations provided in the literature, so we will continue to use these two concentrations in subsequent experiments.
In the following experiments, we need to further compare the ability of the four modified proteins of HMGB1 (FL, AB, A, B) to disassemble the biofilm, and select the most functional protein as the secreted protein of the engineered bacteria. Considering that LB medium is relatively rich in nutrients, which may not be conducive to the formation of biofilm, we plan to replace LB medium with chemical defined M9 medium (with relatively less nutrients and closer to the intestinal environment) in subsequent experiments. In order to make the experiment more convincing, we also need to supplement the protein-free group in the biofilm disassembly experiment.
To further determine the function of the various proteins, we performed biofilm growth inhibition and biofilm disassembly experiments below in more detail.
Buffer replacement: (1) Prepare a new buffer GB (50mM Tris, 150mM NaCl); (2) Add the protein into a suitable type of concentration tube and centrifuge it to the limit volume by 2300g; (3) Discard the waste liquid, add 2ml buffer solution GB to the concentration tube, and centrifuge 2300g to the limit volume; (4) Repeat step 3 twice; (5) Add 2ml buffer GB to the concentration tube, mix with the protein, suck out and store.
Inhibition of biofilm growth experiment: (1) Use LB medium to resuscitate PAO1; (2) The resuscitated PAO1 bacterial solution was diluted to OD600=0.001 using M9 medium and inoculated into 96-well plates (100ul per well). FL (final concentration 200nM), AB (final concentration 200nM), A (final concentration 200nM), B (final concentration 200nM) were added to the treatment group, respectively. PslG (final concentration 50nM) was used to supplement the total volume of solution in each hole to 130ul using buffer GB (50mM Tris, 150mM NaCl), and the control group was directly added with 30ul buffer GB and cultured at 37℃. (3) When the culture time reaches 0h, 6h, 12h, 24h, the following operations are carried out: Add 150ul ddH2O for oscillatory cleaning and sucking out (twice), add 150ul 0.1% crystal violet solution for dark dyeing for 15min and sucking out, add 200ul ddH2O for oscillatory cleaning and sucking out (twice), add 200ul anhydrous ethanol for dark dissolution for 15min. The OD550 of the sample was detected with an enzyme-labeler and the data was recorded.
Note: Time course of PslG，FL，AB，A，B effect on biofilm growth. The biofilm biomass was measured by CV assay.
Biofilm disassembly experiment: (1) PAO1 was resuscitated using LB medium; (2) The recovered PAO1 solution was diluted to OD600=0.001 using M9 medium, inoculated into 96-well plates (100ul per well), and cultured at 37℃ for 24h; (3) FL (final concentration 200nM), AB (final concentration 200nM), A (final concentration 200nM), B (final concentration 200nM), PslG (final concentration 50nM) were added to the treatment group, and the buffer solution GB (50mM Tris) was used. 150mM NaCl) was used to supplement the total volume of solution in each hole to 130ul, and the control group was directly added with 30ul buffer GB and cultured at 37℃. (4) When the culture time reaches 0min, 30min, 1h, 2h, 4h, 8h, the following operations are carried out: Add 150ul ddH2O for oscillatory cleaning and sucking out (twice), add 150ul 0.1% crystal violet solution for dark dyeing for 15min and sucking out, add 200ul ddH2O for oscillatory cleaning and sucking out (twice), add 200ul anhydrous ethanol for dark dissolution for 15min. The OD550 of the sample was detected with an enzyme-labeler and the data was recorded.
Note: Time course of PslG，FL，AB，A，B effect on biofilm disassembly. The biofilm biomass was measured by CV assay.
In disassembly assay, the biomass of the biofilm decreases sharply in the first 30min and keep reducing for a few hours. PslG, HMGB1-B box, HMGB1-FL have better effect than others. In growth inhibition assay, all the protein treatment groups have almost the same result on growth inhibition. Through these assays, we proof PslG indeed have an effective ability to disassemble biofilm. We illustrated that the HMGB1-FL (full length) is the relatively efficient one among four HMGB1 modified proteins to disassemble biofilm. Those two proteins will be chosen to be our functional protein.