Difference between revisions of "Part:BBa K1921010"
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===Protein Expression=== | ===Protein Expression=== | ||
| − | The bacteria were cultured in 5mL LB liquid medium with ampicillin in 37℃ until the OD600 value between 0.6~1.2 . Then we put the E.coli into deferent shakers which their temperature are 16℃、25℃ and 37℃ to reduce the temperature. About 30 minutes, add gradient IPTG to the medium. Then do sampling regularly. | + | The bacteria were cultured in 5mL LB liquid medium with ampicillin in 37℃ until the OD600 value between 0.6~1.2 . Then we put the E.coli into deferent shakers which their temperature are 16℃、25℃ and 37℃ to reduce the temperature. About 30 minutes, add gradient IPTG to the medium. Then do sampling regularly. <br> |
<p style="text-align: center;"> | <p style="text-align: center;"> | ||
https://static.igem.org/mediawiki/igem.org/b/b9/TJUSLS..4.jpg<br> | https://static.igem.org/mediawiki/igem.org/b/b9/TJUSLS..4.jpg<br> | ||
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| − | ===HPLC | + | ===Surface display HPLC results=== |
| + | <p style="text-align: center;"> | ||
| + | https://static.igem.org/mediawiki/igem.org/d/d5/ProofTJU10.jpg<br> | ||
| + | '''Figure 4.''' Reletive enzyme activity of engineering bacteria E.coli(BL21)/pET22b(+) ap at 16 ℃. <br> | ||
| + | </p> | ||
Latest revision as of 07:02, 20 October 2016
SP.a+PETase+Histag.a+AIDA
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 2237
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Usage
This part is an original application using anchor protein AIDA. The whole sequence contains signal peptide, PETase and AIDAc. PETase is a new protein enzyme found in bacteria which can decompose PET. Through this method, we can anchor PETase on Escherichia coli’s outer membrane, then we can use this typical Escherichia coli to decompose PET. This is a way called whole cell catalysis. Using this method, we don’t need to purify the protein. In addition, prokaryote surface display system method is simple and mature. AIDAc is the autotransporter adhesin involved in diffuse adherence, so it can anchor stably on the membrane.
Biology
Surface expression of recombinant proteins was first described more than 30 years ago.
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.
The gram-negative bacterium Escherichia coli has historically been one of the most extensively used hosts for recombinant protein production. Since there is extensive documented knowledge regarding the genetics, growth and protein production of E. coli, it is an attractive platform also for surface expression applications. AIDA protein is from Escherichia coli strain 2787. Based on these, we use E.coli as our host.
Reference
[1] Jarmander, Johan; Gustavsson, Martin; Thi-Huyen Do: A dual tag system for facilitated detection of surface expressed proteins in Escherichia coli. MICROBIAL CELL FACTORIES 2012,11 [2] Charbonneau, Marie-Eve;Janvore, Julie ;Mourez, Michael:Autoprocessing of the Escherichia coli AIDA-I Autotransporter A NEW MECHANISM INVOLVING ACIDIC RESIDUES IN THE JUNCTION REGION. JOURNAL OF BIOLOGICAL CHEMISTRY 2009, 284: 17340-17351
Protein Expression
The bacteria were cultured in 5mL LB liquid medium with ampicillin in 37℃ until the OD600 value between 0.6~1.2 . Then we put the E.coli into deferent shakers which their temperature are 16℃、25℃ and 37℃ to reduce the temperature. About 30 minutes, add gradient IPTG to the medium. Then do sampling regularly.
Figure 1. 16℃ Pre-expression
Figure 2. 25℃ Pre-expression
Figure 3. 37℃ Pre-expression
Surface display HPLC results
Figure 4. Reletive enzyme activity of engineering bacteria E.coli(BL21)/pET22b(+) ap at 16 ℃.