Team:Saint Joseph/Demonstrate
Project Demonstration
The demonstration of our project is to take wastewater from a textile dyeing factory and to decolorize it. For this aim, we expressed the laccase enzyme in wastewater so that it could be decolorized. We used POLYSTONE YL for our demonstration. By this way, we can prove that our system works under realistic conditions. Here are some photos of it
| Number | Name | dsb | pET26b | Buffer | DNA Ligase | Nfw |
|---|---|---|---|---|---|---|
| 1 | pET26b+dsb (heavy) | 2μL | 2μL | 2μL | 1μL | 15μL |
| 2 | ppET26b+dsb (light) | 1.71μL | 1μL | 2μL | 1μL | 14.29μL |
| 3 | pET26b+wt (light) | 1μL | 1μL | 2μL | 1μL | 15μL |
| 4 | pET26b+wt (heavy) | 1,7μL | 2μL | 2μL | 1μL | 14.3μL |
| 5 | pET26b+pelB (heavy) | 5.2μL | 1μL | 2μL | 1μL | 10.8μL |
| 6 | pET26b+pelB (light) | 5.2μL | 1μL | 2μL | 1μL | 10.8μL |
Decolorization efficiency: pelB+laccase > dsb+laccase > wt laccase
Polystone Textile dye incubated with three different types of laccase (pelb signalled, dsb added and wild type) for 2 hours in NaOAc buffer (pH 5.0). Then centrifuged at 8,000g. The activity of enzymes detected visually according to colour difference. The wastewater is decolorized.
Enzyme activity is at it’s highest in the first two hours. After 2 hours, it begins to decrease.
dsb+laccase
pelB+labccasee
wt laccas
control
The Protocol
Method 2: Direct Lysis of Bacterial Cell Cultures
- We added dd 110μl of FastBreakTM Cell Lysis Reagent, 10X, (1/10 volume) directly to 1ml of fresh bacterial culture.
- We resuspended lyophilized DNase I as indicated on the vial, and added 1μl per milliliter of original culture volume. We stored the resuspended DNase I in aliquots at –20°C for long term or at 4°C for up to one week.
- We incubated with shaking for 10–20 minutes at room temperature on a rotary mixer or shaking platform.
C. Purification of Polyhistidine- or HQ-Tagged Proteins from 1ml of Bacterial Culture Using MagneHisTM Ni-Particles
- We added 500mM NaCl to HQ-tagged protein lysate (i.e., 0.03g NaCl per 1.0ml of lysate) to improve binding to MagneHisTM Ni-Particles.
- We vortexed the MagneHisTM Ni-Particles to a uniform suspension.
- We added 30μl of MagneHisTM Ni-Particles either to cell pellet resuspended in 1X FastBreakTM Cell Lysis Reagent or to 1.1ml of cell lysate. Note: You may need to increase the amount of MagneHisTM Ni-Particles used for high-expressing proteins.
- We inverted tube to mix (approximately 10 times), and incubated for 2 minutes at room temperature. It was made sure that the MagneHisTM Ni-Particles are well mixed.
- We placed the tube in the appropriate magnetic stand for approximately 30 seconds to capture the MagneHisTM Ni-Particles. Using a pipette, we carefully removed the supernatant.
- We removed the tube from the magnetic stand, added 150μl of MagneHisTM Binding/Wash Buffer to the MagneHisTM Ni-Particles and pipet to mix. If NaCl was added for binding, also use NaCl during washing. Make sure that particles are resuspended well.
- We placed the tube in the appropriate magnetic stand for approximately 30 seconds to capture the MagneHisTM Ni-Particles. Using a pipette, we carefully removed the supernatant.
- We repeated the wash step 2 times for a total of 3 washes.
- We removed the tube from the magnetic stand and added 100μl of MagneHisTM Elution Buffer, pipet to mix. Note: HQ-tagged proteins may elute at a lower concentration of imidazole (50–100mM) compared to polyhistidine-tagged proteins. The MagneHisTM Elution Buffer, which contains 500mM imidazole, can be diluted with MagneHisTM Binding/Wash Buffer or water, if required.
- We incubated for 1–2 minutes at room temperature, placed in a magnetic stand to capture the MagneHisTM Ni-Particles. Using a pipette, we removed the supernatant containing the purified protein. We analyzed the samples by SDS-PAGE or by functional assay.
The results were positive and we achieved what we wanted to succeed! The wastewater is decolorized!
Reference: Promega.