Difference between revisions of "Part:BBa K1921009"
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===Usage=== | ===Usage=== | ||
| − | + | The discovery of the autotransporter family has provided a mechanism for surface expression of proteins in laboratory strains of Escherichia coli. AIDA is one of the members of autotransporter family. We call it one of an anchor proteins for it can immobilize on the outer membrane of Escherichia coli. It is very useful because we can insert other proteins’ sequence into the AIDA sequence, and then the protein will be immobilized on the outer membrane. We can use it to do whole cell catalysis. In addition, it also have a broad range of applications in molecular biology, biochemistry, biotechnology, microbiology and vaccinology . Today we use this part to display our PETase on E.coli’s surface. Compared with the eukaryotic surface display system, display system with surface expression in prokaryotes cycle is short. 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. <br> | |
===Biology=== | ===Biology=== | ||
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===Reference=== | ===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 | [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 | ||
| + | |||
| + | ===Pre-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.<br> | ||
| + | <p style="text-align: center;"> | ||
| + | https://static.igem.org/mediawiki/igem.org/f/f6/Tjuresults53.jpg<br> | ||
| + | '''Figure 1.'''This is the pre-expression using E.coli BL21 at 16 ℃.<br> | ||
| + | </p> | ||
| + | <p style="text-align: center;"> | ||
| + | https://static.igem.org/mediawiki/igem.org/e/ed/Tjuresults54.jpg<br> | ||
| + | '''Figure 2.''' This is the pre-expression using E.coli BL21 at 25 ℃. <br> | ||
| + | </p> | ||
| + | <p style="text-align: center;"> | ||
| + | https://static.igem.org/mediawiki/igem.org/0/04/Tjuresults55.jpg<br> | ||
| + | '''Figure 3.''' This is the pre-expression using E.coli BL21 at 37 ℃. <br> | ||
| + | </p> | ||
| + | |||
| + | ===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
AIDA
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 1280
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Usage
The discovery of the autotransporter family has provided a mechanism for surface expression of proteins in laboratory strains of Escherichia coli. AIDA is one of the members of autotransporter family. We call it one of an anchor proteins for it can immobilize on the outer membrane of Escherichia coli. It is very useful because we can insert other proteins’ sequence into the AIDA sequence, and then the protein will be immobilized on the outer membrane. We can use it to do whole cell catalysis. In addition, it also have a broad range of applications in molecular biology, biochemistry, biotechnology, microbiology and vaccinology . Today we use this part to display our PETase on E.coli’s surface. Compared with the eukaryotic surface display system, display system with surface expression in prokaryotes cycle is short. 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. AIDA is one of an anchor proteins which belongs to Escherichia coli(Escherichia coli strain 2787). We find its sequence from NCBI. Surface display using AIDA contains three parts: signal peptide, passenger domain and anchor protein AIDAc. So this is a C-terminal anchoring. AIDAc protein structure has been analyzed. It is a transmembrane protein across the 12 membrane and its shape is just like a β-barrel. Theβ-barrel can be anchored on the outer membrane so that the special protein can be displayed on the surface.
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
Pre-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.This is the pre-expression using E.coli BL21 at 16 ℃.
Figure 2. This is the pre-expression using E.coli BL21 at 25 ℃.
Figure 3. This is the pre-expression using E.coli BL21 at 37 ℃.
Surface display HPLC results
Figure 4. Reletive enzyme activity of engineering bacteria E.coli(BL21)/pET22b(+) ap at 16 ℃.