Difference between revisions of "Part:BBa K1921000"
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===Protein Expression=== | ===Protein Expression=== | ||
| − | Pre-expression:<br> | + | =Pre-expression:=<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> | 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> | + | =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> | + | =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> | + | =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> | + | =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 47: | Line 47: | ||
| − | Ultrafiltration:<br> | + | =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 56: | Line 56: | ||
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：<br> | + | =Crystalization：=<br> |
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> | ||
| + | |||
| + | ===HPLC Result=== | ||
| + | <p style="text-align: center;"> | ||
| + | https://static.igem.org/mediawiki/igem.org/5/53/ProofTJU2.jpg<br> | ||
| + | </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. | ||
Revision as of 06:14, 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.
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.