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Experiments

Stock Solution Preparation

  1. 50 mg/ml Kanamycin Stock Reagent

    1. 1 g kanamycin powder (Beyotime: ST101) was weighed and added to a 50 ml centrifuge tube. Subsequently, 20 ml of sterilized ddH2O (Sangon Biotech: B541017) was added. The mixture was vortexed to homogenize, allowed to stand for 3 minutes, and then sterilized by filtration using a 0.22 μm sterile membrane filter (Merck Millex™-GS Sterile Syringe Filter Unit, MCE). The resulting 20 ml of 50 mg/ml kanamycin stock solution was aliquoted into 2 ml EP tubes for storage.

  2. 50 mg/ml Streptomycin Stock Reagent

    1. 500 mg streptomycin powder (Beyotime: ST2722-1g) was weighed and transferred into a 50 ml centrifuge tube. A volume of 10 ml of sterilized ddH2O (Sangon Biotech: B541017) was then added. The mixture was vortexed, allowed to stand for 3 minutes, and subsequently sterilized by filtration using a 0.22 μm sterile membrane filter (Merck Millex™-GS Sterile Syringe Filter Unit, MCE). The resulting 10 ml of 50 mg/ml streptomycin stock solution was aliquoted into 2 ml EP tubes for storage.

  3. 100 mM IPTG Stock Reagent

    • A 1.2 g quantity of IPTG powder (Beyotime: ST098) was weighed and added to a 50 ml centrifuge tube. The tube was then filled with 50 ml of sterilized ddH2O (Sangon Biotech: B541017). The solution was vortexed, allowed to stand for 3 minutes, and filtered for sterilization using a 0.22 μm sterile membrane filter (Merck Millex™-GS Sterile Syringe Filter Unit, MCE). The resulting 50 ml of 100 mM IPTG stock solution was aliquoted into 2 ml EP tubes for storage.

  4. 10 mg/ml Congo Red Stock Reagent

    1. 0.5 g quantity of Congo Red powder (Aladdin: C196465) was weighed and transferred into a 50 ml centrifuge tube. The tube was filled with 50 ml of sterilized ddH2O (Sangon Biotech: B541017). The solution was vortexed to mix thoroughly, allowed to stand for 3 minutes, and sterilized by filtration using a 0.22 μm sterile membrane filter (Merck Millex™-GS Sterile Syringe Filter Unit, MCE). The resulting 50 ml of 10 mg/ml Congo Red stock solution was aliquoted into 2 ml EP tubes for storage.

  5. 10 mg/ml Coomassie Brilliant Blue Stock Reagent

    1. 0.5 g quantity of Coomassie Brilliant Blue G-250 powder (Beyotime: ST1119) was weighed and transferred into a 50 ml centrifuge tube. The tube was filled with 50 ml of sterilized ddH2O (Sangon Biotech: B541017). The mixture was vortexed, allowed to stand for 3 minutes, and sterilized by filtration using a 0.22 μm sterile membrane filter (Merck Millex™-GS Sterile Syringe Filter Unit, MCE). The resulting 50 ml of 10 mg/ml Coomassie Brilliant Blue stock solution was aliquoted into 2 ml EP tubes for storage.

LB Medium Preparation

  1. A bottle of BeyoPure™ LB Broth (Catalog No. ST156) was opened and the entire contents were added to a 1L blue-capped bottle. Ultrapure water from the Milli-Q® Type 1 Ultrapure Water System was added to a total volume of 500 ml. The mixture was shaken well, autoclave tape was applied, the bottle cap was loosened, and the solution was sterilized at 121°C for 30 minutes.

LB Agar Plate Preparation

  1. A bottle of BeyoPure™ LB Broth with Agar (Catalog No. ST158) was opened and the entire contents were added to a 1L blue-capped bottle. Ultrapure water from the Milli-Q® Type 1 Ultrapure Water System was added to a final volume of 500 ml. The solution was shaken thoroughly, autoclave tape was applied, the bottle cap was loosened, and the solution was sterilized at 121°C for 30 minutes. After cooling to approximately 60°C, the corresponding antibiotic was added.

Congo Red Plate Preparation

  1. Eight bottles of BeyoPure™ LB Broth with Agar (Catalog No. ST158) were opened and added to eight 1L blue-capped bottles. Ultrapure water from the Milli-Q® Type 1 Ultrapure Water System was added to a final volume of 500 ml per bottle. The solutions were shaken well, autoclave tape was applied, the bottle caps were loosened, and the solutions were sterilized at 121°C for 30 minutes. After cooling to approximately 60°C, the corresponding antibiotic, Congo Red, Coomassie Brilliant Blue, and IPTG stock solutions were added as follows:
Group 10 mg/ml Congo Red Stock 10 mg/ml Coomassie Brilliant Blue Stock 50 mg/ml Kanamycin Stock 100 mM IPTG Stock
Group 1 2.5 ml 50 µl 1 ml 0 ml
Group 2 2.5 ml 50 µl 1 ml 0.5 ml
Group 3 2.5 ml 50 µl 1 ml 2.5 ml
Group 4 2.5 ml 50 µl 1 ml 5 ml
Group 5 0 ml 0 ml 1 ml 0 ml
Group 6 0 ml 0 ml 1 ml 0.5 ml
Group 7 0 ml 0 ml 1 ml 2.5 ml
Group 8 0 ml 0 ml 1 ml 5 ml

Bacterial Transformation

  1. Trans BL21(DE3) Chemically Competent Cells (TransGene CD601-02) were retrieved from the -80°C freezer and allowed to thaw on ice for 10 minutes.
  2. 10 ng of the target plasmid was added to the thawed competent cells, gently mixed, and incubated on ice for 30 minutes. Meanwhile, a water bath was prepared at 42°C.
  3. The competent cell-plasmid mixture was subjected to heat shock at 42°C for 45 seconds, followed by an immediate transfer to ice for 2 minutes without agitation of the centrifuge tube.
  4. Subsequently, 500 µl of sterile SOC medium was added to each tube, mixed thoroughly, and incubated at 37°C with shaking at 200 rpm for 1 hour to facilitate bacterial recovery. Meanwhile, LB plates with the appropriate antibiotic were prepared and placed in the 37°C incubator.
  5. Transformed competent cells were plated onto LB agar plates containing the appropriate antibiotic and spread evenly. The plates were incubated at 37°C until the liquid was absorbed, then inverted and incubated overnight at 37°C.

A glimpse of our wet-lab experiments 1

Protein Expression

  1. POI (Protein of Interest) Expression

    1. 3 ml volume of LB medium with the appropriate antibiotic was added to a 12 ml culture tube.
    2. Three single colonies were selected from the plate and inoculated into the 3 ml LB medium with the appropriate antibiotic.
    3. The culture was incubated overnight in a shaker at 220 rpm and 37°C.

  2. Curlis Fiber Expression

    1. 3 ml volume of LB medium with the appropriate antibiotic was added to a 12 ml culture tube.
    2. Three single colonies were selected from the plate and inoculated into the 3 ml LB medium with the appropriate antibiotic.
    3. The culture was incubated overnight in a shaker at 220 rpm and 37°C.
    4. After overnight incubation, 40 µl overnight culture was transferred into 4 ml of LB medium with the appropriate antibiotic and incubated in a shaker at 220 rpm and 37°C for 2 hours. 40 µl 100mM IPTG was added to a final concentration of 1 mM, and the culture was incubated at 220 rpm and 30°C for 36 hours.
    5. After overnight incubation, filamentous precipitates were observed in the culture medium. The precipitates were collected using an inoculating loop and dissolved in 4M guanidine hydrochloride.

POI Purification and Characterization

  1. The cells were centrifuged at 10000 rpm for 5 minutes, and the supernatant was discarded.
  2. A volume of 8 ml of denaturing lysis buffer was added, and the mixture was homogenized.
  3. The lysate was subjected to ultrasonic disruption for 5 minutes under the following conditions:
    1. Power: 600W
    2. Sonication on-time: 5 seconds
    3. Sonication off-time: 5 seconds
  4. The lysate was centrifuged, and the supernatant was collected for further processing.
    1. Protein purification was carried out using the Beyotime His-tag Protein Purification Kit (Denaturant-resistant; P2229S) or Beyotime GST-tag Protein Purification Kit (Denaturant-resistant; P2262).
      1. The BeyoGold™ Purification Resin (denaturant-resistant) was loaded into the column, and the purification column was equilibrated twice with 1 ml of lysis buffer.
      2. The supernatant was loaded onto the column. To maximize yield, the flow-through was collected and reloaded onto the column 3-5 times to ensure complete binding of the His-tagged protein.
      3. The column was washed five times with 1 ml of denaturing lysis buffer.
      4. The column was then washed five times with 1 ml of washing buffer.
      5. The bound protein was eluted 6-10 times with 0.5 ml of elution buffer, and the final eluate was collected.

Curlis Fiber Purification and Characterization

  1. The filamentous precipitates were collected from the culture medium using an inoculating loop or pipette tip and dissolved in 4M guanidine hydrochloride. The mixture was homogenized until fully dissolved, serving as the supernatant for subsequent purification.
    1. Protein purification was carried out using the Beyotime His-tag Protein Purification Kit (Denaturant-resistant; P2229S).
      1. The BeyoGold™ His-tag Purification Resin (denaturant-resistant) was loaded into the column, and the purification column was equilibrated twice with 1 ml of lysis buffer.
      2. The supernatant was loaded onto the column. To maximize yield, the flow-through was collected and reloaded onto the column 3-5 times to ensure complete binding of the His-tagged protein.
      3. The column was washed five times with 1 ml of denaturing lysis buffer.
      4. The column was then washed five times with 1 ml of washing buffer.
      5. The bound protein was eluted 6-10 times with 0.5 ml of elution buffer, and the final eluate was collected.
      6. SDS-PAGE analysis was conducted to verify the purity of the eluted protein.

Congo Red Characterization of Curlis Fiber

  1. The correctly sequenced Curlis Fiber-pET28a and empty pET28a glycerol stocks were retrieved from the -80°C freezer, streaked onto kanamycin plates, and incubated overnight at 37°C. After overnight incubation, the plates were stored at 4°C for up to one week.
  2. Three single colonies were selected from the plate and inoculated into 3 ml of LB medium with kanamycin, followed by incubation at 220 rpm and 37°C overnight.
  3. 30 µl overnight culture was spotted onto Congo Red plates containing the appropriate antibiotic and different concentrations of IPTG. After the spots dried, the plates were inverted and incubated at 30°C in a controlled environment for 48 hours.
  4. Photographs were taken to document the results.

SDS-PAGE

  1. 20 µl aliquot of each sample was mixed with 5 µl of SDS-PAGE protein loading buffer (Boyetime P0015), vortexed, and incubated at 98°C in a water bath for 10 minutes to prepare the samples for loading.
  2. The samples were centrifuged at 10000 rpm for 1 minute.
  3. 1X MOPS-SDS running buffer was prepared by diluting 50 ml of 20X MOPS-SDS running buffer in 950 ml of ddH2O.
  4. YoungPAGE™ (Genscript: M00928) pre-cast gels were set up in a Biorad Mini-PROTEAN Tetra Vertical Electrophoresis Cell according to the manufacturer’s instructions, and the 1X MOPS-SDS running buffer was added.
  5. 10 µl of the prepared samples was loaded into the gel wells, and Broad Multi Color Pre-Stained Protein Standard (Genscript: M00624) was added as a protein molecular weight marker in the empty wells.
  6. Electrophoresis was performed at 200 mV for 20 minutes, with the electrophoresis time adjusted as needed based on the results.
  7. Upon completion of electrophoresis, the SDS gel was removed and subjected to staining and destaining procedures as per the manufacturer’s instructions.
  8. After destaining, the gel was placed in a ChemiDoc Imaging System (Biorad: 12003153) for imaging, and the images were uploaded.

Binding Assay Verification

  1. Equal volumes (50 µl) of curlis fiber and POI protein solutions were mixed and incubated overnight at 30°C with shaking at 80 rpm.
  2. SDS-PAGE was performed to verify the binding interactions.

A glimpse of our wet-lab experiments 2