Part 2: Key Experiments
Growth Condition
Bacteria were inoculated into Luria Bertani (LB) medium at 37°C, 25 °C, 20°C or 16°C, with shaking at 160 rpm.
When required, ampicillin was added to a final concentration of 100 μg/mL.
Cultures grown overnight were diluted to give OD600=0.1 and when they reached OD600=0.6 expression of the recombinant fragment was induced with isopropyl β-d-1-thiogalactopyranoside (IPTG) at various final concentrations. (0.1mM, 0.5mM, 1 mM, etc.)
The time of induction was one hours.
Western Blot
1. Inoculate the bacteria in LB medium containing kanamycin and incubate overnight at 37°C with shaking at 250 rpm.
2. Dilute the overnight culture 1:50 in fresh LB medium containing kanamycin. Continue incubating at 37°C with shaking at 250 rpm until OD600 reaches 0.6-0.8.
3. Add IPTG to a final concentration of 1 mM and incubate at 37°C or 25°C with shaking at 250 rpm for 1 hour.
4. Take 1-1.5 mL of the bacterial culture, centrifuge at 12,000 rpm for 1 minute to remove the supernatant, and resuspend the pellet in 40 µL ddH2O. Add 10 µL of 5X SDS loading buffer and mix well. Heat at 102°C in a metal bath for 10 minutes, then immediately place on ice.
5. Load the prepared samples into the wells of an SDS-PAGE gel. Run at 80V for 20 minutes until the blue dye front is compressed at the interface of the stacking and separating gels, then increase the voltage to 130V and continue running for 40 minutes.
6. Assemble the transfer sandwich in the order of Cathode - Filter paper - Gel - Nitrocellulose membrane - Filter paper - Anode, and transfer at 300 mA for 40 minutes.
7. Place the nitrocellulose membrane in an incubation box. Wash with 1X TBST at room temperature with shaking for 5 minutes, repeating three times.
8. Add 5% non-fat milk prepared in TBST and incubate at room temperature with shaking for 1 hour.
9. Wash the membrane with 1X TBST at room temperature with shaking for 5 minutes, repeating three times.
10. Incubate with the primary antibody (1:3000) overnight at 4°C with shaking.
11. Wash the membrane with 1X TBST at room temperature with shaking for 5 minutes, repeating three times.
12. Incubate with the secondary antibody (1:3000) at room temperature with shaking for 1 hour.
13. Wash the membrane with 1X TBST at room temperature with shaking for 5 minutes, repeating three times.
14. Mix the developing solution by combining equal parts of Solution A and Solution B. Add 200 µL to the membrane, protect from light, and wait for 2 minutes. Then develop using the imaging system.
Immunofluoroscene Assay
Strains were grown as described in the section “Growth conditions”.
Slide pretreatment
Coverslips were dipped in 70% ethanol and kept in the flame using forceps for approximately 15-20 s and directly put inside the well of a sterile tissue culture plate.
The slides were pre-treated with L-polylysine(50μg/mL) to encourage cell attachment. The slides were then air-dried, followed by fixation.
Approximately 108 bacterial cells were collected by centrifugation (10,000*g for 5 min).
Bacteria were washed with 300 µL of PBS and fluorescently labelled with Oregon Green 488 Carboxylic Acid, Succinimidyl Ester, 6-isomer diluted 1:1000 in PBS.
Stock solution of Oregon Green: dissolve 5mg of the dye into 0.5mL DMSO. When using the dye, dilute 1:1000 in PBS. Wrap it in tin foil to avoid light exposure.
Bacteria were washed twice with 300 µL of sterile PBS and then resuspended in 100 µL of PBS containing 2% (v/v) formaldehyde.
Samples were spotted onto the polylysine-treated slide and incubated for 10-15 min.
Bacteria were washed with 100 µL of PBS and then incubated for 40 min at room temperature with the primary antibody diluted.1:500 In PBS.
Bacteria were washed with 300 µL of PBS and incubated for 20-30 min at RT in the dark with 100 µL of PBS containing a ABflo® 594-conjugated Goat anti-Mouse IgG (H+L) diluted 1:250.
After two washes with 300 μL of PBS, a droplet of DAPI was applied. Incubate 5 min at RT in the dark.
Stock solution of DAPI: dissolve 1mg of the dye into 1mL DMSO. When using the dye, dilute 1:1000 in PBS. Wrap it in tin foil to avoid light exposure.
Analyse the samples with a fluorescence microscope.
Co-IP
Day 1
Strains were grown as described in the section “Growth conditions”.
Day 2
1. Take out the culture and collect 40 μL of the induced culture sample.
(Sample preparation: Take 1 mL of the culture (amount can vary), centrifuge at 12000 rpm for 1min, discard the supernatant, add 50 μL of 1x SDS Loading Buffer (containing β-mercaptoethanol), heat at 102°C for 10 min until the sample becomes clear and non-viscous; avoid it being sticky.)
2. Prepare an ice bath, pre-cool the centrifuge at 4°C, and thaw the two types of magnetic beads on ice.
3. Lysis and preparation of bacterial or yeast samples.
For 1 mL of culture, centrifuge in a large centrifuge at room temperature, 8000 rpm, for 5min. If necessary, wash once with PBS. Remove all residual liquid, add 100-200 μL of lysis buffer with inhibitors, gently resuspend, and transfer to another 1.5 mL EP tube.
Lyse on ice for 2-10 min, typically 10 min (during this time, prepare the magnetic beads as described in step 4).
After thorough lysis, centrifuge at 4°C, 10,000-14,000*g for 3-5 min, and collect the supernatant (remember to add control IgG).
4. Prepare magnetic beads.
Since magnetic beads are stored in a special protective solution, they need to be washed appropriately before adding the sample.
(1) Resuspend the magnetic beads with a pipette, prepare the appropriate amount of beads according to the ratio of 20 μL beads per 500 μL sample, and transfer the desired amount of beads to a clean centrifuge tube.
4 μL of control IgG beads is appropriate, requiring 50 + 50 μL of the supernatant from step 3.
(2) Add TBS to a final volume of approximately 500 μL.
(3) Gently resuspend the beads with a pipette. Place on a magnetic rack for 10 seconds to separate and discard the supernatant. Repeat the above steps twice.
(4) Resuspend the beads in TBS to the initial volume.
5. Immunoprecipitation (IP)
(1) Add the beads and incubate. Add the beads at a ratio of 20 μL beads per 500 μL protein sample (60 μL for 1500 μL) and incubate on a rotating mixer at room temperature for 2 hours or overnight at 4°C (tested 4°C for 7 hours).
(2) Magnetic separation. After incubation, place the supernatant on a magnetic rack for 10 seconds to separate it and discard it. Note: Some supernatants can be kept to detect the effectiveness of the immunoprecipitation. Keep 40 μL of the final Flag wash solution for best results.
(3) Washing. Add 0.5 mL of lysis buffer with inhibitors, gently resuspend the beads with a pipette, place on a magnetic rack for 10 seconds, and discard the supernatant. Repeat the washing with lysis buffer containing inhibitors three times. Note: Washing completeness can also be judged by measuring the OD280 of the wash liquid. If OD280 is greater than 0.05, increase the number of washes as needed.
(4) Elution: SDS-PAGE Sample Buffer Elution Method:
(a) For each 20 μL of original bead volume, add 100 μL of SDS-PAGE Sample Loading Buffer (1X), and heat at 95°C for 5 minutes.
(b) Place on a magnetic rack for 10 seconds to separate and collect the supernatant for SDS-PAGE electrophoresis or Western blot detection.
FACS
1. Target strain is induced with 1mM of IPTG in 25℃ incubator shaking at 250rpm for 1h
2. Approximately 108 bacteria were collected by centrifugation (10,000 g for 5 min).
3. Bacteria were resuspended and fixed for 20 min at 4 °C with 100uL PBS containing 4% (v/v) formaldehyde following centrifugation
4. The fixed bacteria were then washed with 500uL PBS containing 1% BSA (w/v) and centrifuged
5. Bacteria were resuspended in 100 µL of a solution containing mouse anti-DDDDK antibody diluted 1:500 in PBS containing 1% BSA and incubated at approximately 100rpm for 2h at room temperature.
6. Bacteria were washed with 500 µL of PBS + 1% BSA. Bacteria were then resuspended in 100 µL of a FITC-conjugated goat anti mouse antibody diluted 1:250 in PBS + 1% BSA and incubated at approximately 100rpm for 1h at room temperature in the dark.
7. After a final washing with 500uL PBS, the cells were resuspended in 200 μL of PBS.
8. All data were collected using a CytoFLEX S by acquiring 100,000 events, and the data analysed using the Flow-Jo software.
GFP Induction
1. Strains were grown as described in the section “Growth conditions”.
2. Induce Strains with 3OC12HSL and 3OC6HSL, measure RFU and OD600 under the following conditions, and use RFU/OD600 as the final result
3. RFU measurement requires the use of a Cytation3 multifunctional microplate reader.
(1) Incubate the bacterial solution until the OD600 reaches 0.6, then add 270 μL of bacterial solution and 30 μL of AHL solution to a 96-well plate
(2) Set up a total of 8 concentration gradients for the AHL solution, namely, 0, 10-10, 10-9, 10-8, 10-7, 10-6, 10-5, and 10-4 M, respectively
(3) Measure fluorescence using a black 96-well plate
(4) Measure OD600 and fluorescence every 10 minutes
CcdB Killing Verification Experiment
1.Incubate bacteria overnight (100uL into 5mL LB liquid medium with Kanamycin(100μg/mL), add chloromycetin (34μg/mL)[based on our experimental strain])
2.Dilute the bacteria solution with 200uL into 3ml LB liquid medium with Kanamycin, Additional chloromycetin is still
required. Shake the liquid for 5-6 hours till the OD600~0.6(OD~0.8 optimum)
3.Dilution of gradients-induced bacterial solution using IPTG for six samples. The first group was diluted ten times
the stock solution, the second group was diluted one hundred times the stock solution, and so on. We used a total
of six experimental groups. The specific operation was to add 270uL of bacterial solution to the wells, then add
30uL 0.1M IPTG to the first well, mix well, and then transfer 30uL from the first well to the second well, and so on.
In the control wells, we added 30uL ddH2O to 270uL of bacterial solution and aspirated it after mixing.
4.Prolonged assay using a fluorescent microplate reader: using a 96-well plate, 270ul of bacterial solution was
added to each well along with a gradient of IPTG. The machine was adjusted to measure the absorbance of
OD600 every twenty minutes, during which time the plate was incubated at 37° with shaking.
CcdA Detoxification Experiment
1.Incubate bacteria overnight (100uL into 5mL LB liquid medium with Kanamycin(100μg/mL), add chloromycetin (34μg/mL)[based on our experimental strain])
2.Dilute the bacteria solution with 200uL into 3ml LB liquid medium with Kanamycin, Additional chloromycetin is still required. Shake the liquid
for 5-6 hours till the OD600~0.6(OD~0.8 optimum)
3.Dilution of gradients-induced bacterial solution using L-arabinose for six samples.
The first group was diluted ten times the stock solution, the second group was diluted
one hundred times the stock solution, and so on. We used a total of six experimental groups.
The specific operation was to add 0.1M IPTG to the original solution to reach the concentration of 0.1 mM IPTG.
Then add 270uL of bacterial solution to the wells, then add 30uL 100g/L L-arabinose(with 0.1mM IPTG) to the first well,
mix well, and then transfer 30uL from the first well to the second well, and so on. In the control wells,
we added 30uL ddH2O to 270uL of bacterial solution and aspirated it after mixing.
4.Prolonged assay using a fluorescent microplate reader: using a 96-well plate, 270ul of bacterial solution was added to each well along with a gradient of IPTG. The machine was adjusted to measure the absorbance of OD600 every twenty minutes, during which time the plate was incubated at 37°C with shaking.
Cryo-EM
1.Bacterial Culture and Induction:
Inoculate 30 μL of an overnight bacterial culture into 3 ml of medium containing antibiotics. Incubate at 37°C in a shaker at 220 rpm until the OD600 reaches 0.6-0.8.
Add IPTG or arabinose to the desired concentration and induce at the target temperature in a shaker at 220 rpm for the desired duration.
Take 1 ml of the induced bacterial culture and centrifuge at 6000 rpm for 10 minutes.
2.Preparation of Cell Suspension:
Resuspend bacterial cells in 1 ml of 4% paraformaldehyde solution.
3.Incubation:
Incubate the cell suspension at room temperature (approximately 20-25°C) for 30 minutes. Ensure the temperature remains stable throughout the incubation process and avoid vigorous shaking or disturbing the suspension.
4.Washing:
Preparation of Washing Buffer: Use 1X PBS as the washing buffer, pre-prepare it, and keep it at room temperature.
First Wash: Centrifuge the cell suspension at 5000 rpm (or an appropriate speed) for 5 minutes to pellet the cells. Carefully remove the supernatant without disturbing the pellet. Add an appropriate amount of 1X PBS (typically 1-2 times the initial volume), resuspend the pelleted cells, and gently mix.
Second and Third Washes: Repeat the centrifugation and resuspension process from the first wash, washing the cells a total of three times. During each wash, ensure the cells are thoroughly mixed to remove excess fixative. After the final wash, resuspend the cells in 1 ml of PBS for subsequent experiments.
Note: Handle the cells gently throughout the procedure to avoid damaging their structure. Select an appropriate centrifugation speed and duration to ensure the cells are fully pelleted without being damaged.
Sample Loading:
(1).Preparation of Nickel Grids:
Use 200 or 300 mesh nickel grids.
(2).Cell Coating:
Take 5 μL of the fixed and washed cell suspension and apply it onto the nickel grid. Allow it to sit for a few minutes to let the cells adhere to the grid.
(3).Antibody Incubation:
Blocking: Apply 100 μL of PBS solution containing 1% BSA to the nickel grid and incubate at room temperature for 30 minutes to block nonspecific binding sites.
Primary Antibody Incubation: Prepare the primary antibody (e.g., mouse antibody) by diluting it with PBS at the recommended ratio. Transfer the blocked nickel grid to 50 μL of the primary antibody solution and incubate at room temperature for 1 hour. Wash the grid three times with PBS, using 100 μL each time, for 5 minutes.
Secondary Antibody Incubation: Prepare the gold-labeled secondary antibody (e.g., gold-labeled anti-mouse IgG) by diluting it with 1% BSA-PBS at the recommended ratio. Transfer the washed nickel grid to 50 μL of the secondary antibody solution and incubate at room temperature for 1 hour. Wash the grid three times with PBS, using 100 μL each time, for 5 minutes.
(4).Washing and Drying:
Final Wash: Wash the nickel grid three times with 100 μL of distilled water for 5 minutes each to remove PBS and BSA.
Drying: Place the nickel grid on clean filter paper and allow it to air dry naturally or dry it in a dust-free environment.
(5).Negative Staining:
Add 5 μL of 2% uranyl acetate stain and stain the sample for 30 seconds to 1 minute.
(6).Observation:
Examine the sample under a cryo-electron microscope (cryo-EM).
Enzymatic Cleavage Experiment
1.Activation: In E. coli BL21(DE3), spread on LB agar plates with antibiotic resistance, incubate overnight at 37°C, pick a single colony, and culture in LB broth until OD600 reaches 0.6-0.8.
2.Induction: Add 1mM final concentration IPTG.
Induction at 37°C, 16°C, all for 1h.
3.Collection: Centrifuge at 5000rpm for 10min, wash the pellet with PBS twice.
4.Cleavage: Add 25mM pH 8.0 Tris-HCl and 1-3U enterokinase (Enterokinase, Recombinant), and incubate overnight at 25°C (reaction volume according to the enzyme's instructions).
(1). Adjust the cleavage conditions according to the experimental arrangement.
(2). Note: The definition of activity units: At 25°C, in a reaction buffer of 50 mM Tris-HCl (pH 8.0), the amount of enzyme required to cleave 50ug (1mg/ml) of fusion protein by more than 95% in 16 hours is defined as one unit of activity U.
5.Detection: Centrifuge at 5000rpm for 10 minutes and take the supernatant.
Perform WB detection on proteins with GST/6xHis tags to check for the presence of proteins with GST/6xhis tags in the supernatant and check for proteins with FLAG tags on the bacterial surface proteins.
Protein Purification
1. Shake a bacterial culture of more than 10mL. Centrifuge at 5000rpm for 5 minutes to collect the bacteria. Following the protocol of the Beyotime P2226 His-Tagged Protein Purification Kit (Redox-Resistant Chelating Type), add non-denaturing lysis buffer at a ratio of 4mL per gram of wet bacterial weight. Resuspend the bacterial cells thoroughly by pipetting.
2. Add 24μL of 100mg/mL lysozyme. Mix well and place on ice for 30 minutes. Pre-cool a centrifuge to 4°C.
3. Transfer the bacterial suspension to a 10mL EP tube. Sonicate the bacteria on ice using a power setting of 40% (total power 650w), with each sonication pulse lasting 10s followed by a 10s interval, for a total of 6 pulses. After sonication, transfer the lysate to two 1.5mL EP tubes, approximately 1.0mL in each. Centrifuge at 4°C and 10,000g for 25 minutes. Collect the supernatant in a 10mL centrifuge tube. Reserve 20μL as a sample, labeled "Supernatant".
4. Take 1mL of well-mixed 50% BeyoGoldTM Purification Resin. Centrifuge at 4°C (1000gx10s) and discard the supernatant. Add 0.5mL of non-denaturing lysis buffer to the resin, mix well to equilibrate, and centrifuge again at 4°C (1000gx10s) before discarding the supernatant. Repeat this equilibration step twice. Since the bacterial lysate volume exceeds 1mL, mix approximately 500μL of the lysate supernatant with all the resin and inject the mixture into a 10mL EP tube containing the remaining lysate supernatant. Place the 10mL EP tube horizontally on a shaker at 4°C and shake gently for 60 minutes.
Load the mixture of lysate and resin into an empty affinity chromatography column provided with the kit.
5. Open the bottom cap of the purification column, then the top cap, and collect 1mL of flow-through. For subsequent operations, always close the top cap before the bottom cap, and when draining liquid, open the bottom cap first, followed by the top cap.
6. Wash the column 5 times, adding 700μL of non-denaturing wash buffer each time, and collecting approximately 0.5mL of wash buffer after each wash.
7. Elute the target protein 6 times, using 0.5mL of non-denaturing elution buffer for each elution.
8. Detect the protein by WB or Coomassie brilliant blue stain.
Membrane and Cytoplasm Protein Extraction
1. Thoroughly dissolve and mix the Membrane Protein Extraction Reagents A and B at room temperature, and immediately place them on ice after dissolution. Before use, add PMSF to the appropriate amount of A and B a few minutes prior, to achieve a final concentration of 1mM for PMSF.
2.Culture approximately 20 to 50 million cells, centrifuge to collect the cells, remove the supernatant, and retain the precipitate.
3.Wash the cells with an appropriate amount of ice-cold PBS (gently resuspend the precipitate), followed by centrifugation at 600g for 5 minutes at 4°C to sediment the cells.
4.Discard the supernatant, then centrifuge again at 600g for 1 minute at 4°C to sediment any residual liquid on the walls of the centrifuge tube and further sediment the cells. Try to aspirate as much residual liquid as possible.
5.Add 1mL of Membrane Protein Extraction Reagent A, with PMSF added immediately before use, to the cell precipitate from step 3. Gently and thoroughly resuspend the cells, and place on ice for 10-15 minutes.
6.Sonicate the cells to disrupt them.
7.Centrifuge at 700g for 10 minutes at 4°C, carefully collect the supernatant into a new centrifuge tube to remove cell nuclei and unbroken cells.
8.Centrifuge at 14,000g for 30 minutes at 4°C to sediment cell membrane fragments. Try to aspirate as much supernatant as possible. It is acceptable to gently touch the precipitate or even aspirate a very small amount of it.
9.Add 200μL of Membrane Protein Extraction Reagent B, vortex at the highest speed for 5 seconds to resuspend the precipitate, and place on ice for 5-10 minutes. Repeat the vortexing and ice incubation steps 1-2 times to thoroughly extract membrane proteins.
10.Centrifuge at 14,000g for 5 minutes at 4°C, and collect the supernatant, which is now the cell membrane protein solution. Using this product to lyse 50 million cells can yield 0.3-3mg of cell membrane proteins, with variations depending on the cell type.
Coomassie Brilliant Blue staining
1.After SDS-PAGE,transfer the gel into a container, soak the gel in Coomassie brilliant blue and shake it slowly on s shaker for 25min.
2. Wash the gel with ddH2O for several times. Photograph the gel under white light.
