Difference between revisions of "Part:BBa K1921000"
| Line 31: | Line 31: | ||
The bacteria were cultured in 5mL LB liquid medium with ampicillin in 37℃ overnight. After taking samples, we transfer them into 1L LB medium with ampicillin.<br> | The bacteria were cultured in 5mL LB liquid medium with ampicillin in 37℃ overnight. After taking samples, we transfer them into 1L LB medium with ampicillin.<br> | ||
| − | + | Cultured in bottles:<br> | |
After 4 hours culturing in 37℃ in bottles, we used 500μM IPTG induced in 16℃ for 8-12h. <br> | After 4 hours culturing in 37℃ in bottles, we used 500μM IPTG induced in 16℃ for 8-12h. <br> | ||
| − | + | Ni-sepharose purification:<br> | |
The supernatant was applied to the His-Accept nickel column. After washing unbound proteins with the lysis buffer(50 mM Tris-HCl, pH 7.5, 300 mM NaCl, 20 mM imidazole), the bound proteins were eluted with elution buffer (50 mM Tris-HCl, pH 7.5, 300 mM NaCl, 250 mM imidazole).<br> | The supernatant was applied to the His-Accept nickel column. After washing unbound proteins with the lysis buffer(50 mM Tris-HCl, pH 7.5, 300 mM NaCl, 20 mM imidazole), the bound proteins were eluted with elution buffer (50 mM Tris-HCl, pH 7.5, 300 mM NaCl, 250 mM imidazole).<br> | ||
| − | + | Ultrafiltration:<br> | |
To reduce the salt concentration, we use evaporating pipe to reduce the liquid volume to 1/6 and then add 5/6 A liquid. By using ultrafiltration at the speed of 3500rpm, we finally get 5mL protein solution.<br> | To reduce the salt concentration, we use evaporating pipe to reduce the liquid volume to 1/6 and then add 5/6 A liquid. By using ultrafiltration at the speed of 3500rpm, we finally get 5mL protein solution.<br> | ||
| − | + | Cation exchange column:<br> | |
Use Hitrap SP HP 5mL column to go through AKTA system to get the protein with certain pI.<br> | Use Hitrap SP HP 5mL column to go through AKTA system to get the protein with certain pI.<br> | ||
<p style="text-align: center;"> | <p style="text-align: center;"> | ||
| Line 50: | Line 50: | ||
| − | + | Ultrafiltration:<br> | |
Use the evaporating pipe to concentrate the solution to 0.5mL.<br> | Use the evaporating pipe to concentrate the solution to 0.5mL.<br> | ||
| Line 59: | Line 59: | ||
https://static.igem.org/mediawiki/igem.org/4/4e/TJUSLS.3.jpg<br> | https://static.igem.org/mediawiki/igem.org/4/4e/TJUSLS.3.jpg<br> | ||
</p> | </p> | ||
| − | + | Crystalization: | |
Using the way of vapor diffusion and sitting drop to grow good crystal to do X Ray diffraction.<br> | Using the way of vapor diffusion and sitting drop to grow good crystal to do X Ray diffraction.<br> | ||
| Line 66: | Line 66: | ||
https://static.igem.org/mediawiki/igem.org/5/53/ProofTJU2.jpg<br> | https://static.igem.org/mediawiki/igem.org/5/53/ProofTJU2.jpg<br> | ||
</p> | </p> | ||
| − | Figure 1. PETase’s self-degrading condition in different temperature. We take the quantity of PETase in the day the experiment begines as 100%. We can now see when stored in low temperature, the protein was degraded slowly, but in room temperature, the protein degrades rapidly. The Quantity of PETase left was measured by protein gel and analysised by a computer program called GEL-PRO. | + | Figure 1. PETase’s self-degrading condition in different temperature. We take the quantity of PETase in the day the experiment begines as 100%. We can now see when stored in low temperature, the protein was degraded slowly, but in room temperature, the protein degrades rapidly. The Quantity of PETase left was measured by protein gel and analysised by a computer program called GEL-PRO.<br> |
| + | |||
| + | Mutation: | ||
| + | <p style="text-align: center;"> | ||
| + | https://static.igem.org/mediawiki/igem.org/2/22/ProofTJU1.jpg<br> | ||
| + | </p> | ||
| + | Figure 2. The Comparison of the enzyme activity between PETase and three kinds of mutated PETase. The reaction condition is 100μL solution,pH 9.0(bicine-NaOH), 40 degree, 18h, the substrate is a round with a diameter of 2mm. The results are detected by Hplc. The y-axis stands for the area of the peak of MHET, the main product of the PETase’s degrading of PET. The x-axis stands for the concentration of the protein. | ||
Revision as of 06:32, 16 October 2016
PETase
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Usage
PETase is dedicated to the role of PET degradation. Our subject of the competition for this year is to modify PETase and developing cell surface display.The protagonists of our project, which are PETase and the surface display technology, will act in three following aspects.
Firstly, we find the enzyme catalytic center and its binding center by analyzing the structure of PETase with Protein crystallography and X ray diffraction technique, as well as choosing the mutation site under its character direction in order to carry out the directional mutation to improve the degradation efficiency and thermal stability.
Moreover, using the prokaryotic (E. coli) and eukaryotic (Pichia Pastoris) surface display for whole cell catalysis.
Thirdly, fusing the PETase and hydrophobic protein then expressing the fusion protein in Pichia Pastoris, which will take advantage of hydrophobic protein in hydrophobicity to give a hydrophobic environment for a better degradation efficiency. Meanwhile, the co-display combining PETase and hydrophobic protein in Pichia Pastoris will change the character of cells' surfaces so that cells can adapt the extreme environment, then the whole-cell biocatalyst might have higher catalytic efficiency as well as break the limitation set by reaction condition. According to that, the PETase degradation reaction conditions will be broaden which means it can be applied in industry easier.
Biology
PETase was found from a kind of microorganism（Ideonella sakaiensis 201-F6） living on PET as the main carbon source. It can degrade macromolecular polymers into monomers.PETase is the only enzyme found in bacteria which can degrade PET. Compare to the other enzyme found in fungi like LCC, TfH, FsC, PETase is much more active under low temperature environment, which means its reaction conditions is feasible in practical application than the others'.Additionally, PETase has been shown to have a degrading efficiency 120 times greater than alternative enzymes.
Reference
[1] Yoshida S, Hiraga K, Takehana T, et al. A bacterium that degrades and assimilates poly(ethylene terephthalate).[J]. Science, 2016, 351(6278):1196-1199.
Protein Expression
=Pre-expression:=
The bacteria were cultured in 5mL LB liquid medium with ampicillin in 37℃ overnight. After taking samples, we transfer them into 1L LB medium with ampicillin.
Cultured in bottles:
After 4 hours culturing in 37℃ in bottles, we used 500μM IPTG induced in 16℃ for 8-12h.
Ni-sepharose purification:
The supernatant was applied to the His-Accept nickel column. After washing unbound proteins with the lysis buffer(50 mM Tris-HCl, pH 7.5, 300 mM NaCl, 20 mM imidazole), the bound proteins were eluted with elution buffer (50 mM Tris-HCl, pH 7.5, 300 mM NaCl, 250 mM imidazole).
Ultrafiltration:
To reduce the salt concentration, we use evaporating pipe to reduce the liquid volume to 1/6 and then add 5/6 A liquid. By using ultrafiltration at the speed of 3500rpm, we finally get 5mL protein solution.
Cation exchange column:
Use Hitrap SP HP 5mL column to go through AKTA system to get the protein with certain pI.
Ultrafiltration:
Use the evaporating pipe to concentrate the solution to 0.5mL.
Gel filtration chromatography:
Use AKTA system and superdex75 gel filtration chromatography to separate proteins with different molecular weight.
Crystalization:
Using the way of vapor diffusion and sitting drop to grow good crystal to do X Ray diffraction.
HPLC Result
Figure 1. PETase’s self-degrading condition in different temperature. We take the quantity of PETase in the day the experiment begines as 100%. We can now see when stored in low temperature, the protein was degraded slowly, but in room temperature, the protein degrades rapidly. The Quantity of PETase left was measured by protein gel and analysised by a computer program called GEL-PRO.
Mutation:
Figure 2. The Comparison of the enzyme activity between PETase and three kinds of mutated PETase. The reaction condition is 100μL solution,pH 9.0(bicine-NaOH), 40 degree, 18h, the substrate is a round with a diameter of 2mm. The results are detected by Hplc. The y-axis stands for the area of the peak of MHET, the main product of the PETase’s degrading of PET. The x-axis stands for the concentration of the protein.