Difference between revisions of "Part:BBa K1921010"

 
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<partinfo>BBa_K1921010 short</partinfo>
 
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<partinfo>BBa_K1921010 parameters</partinfo>
 
<partinfo>BBa_K1921010 parameters</partinfo>
 
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 +
===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. <br>
 +
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. <br>
 +
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.
 +
<p style="text-align: center;">
 +
    https://static.igem.org/mediawiki/igem.org/c/cd/TJUSLS_4.jpg<br>
 +
'''Figure 1.''' 16℃ Pre-expression
 +
</p>
 +
<p style="text-align: center;">
 +
    https://static.igem.org/mediawiki/igem.org/3/3c/TJUSLS_5.jpg<br>
 +
'''Figure 2.''' 25℃ Pre-expression
 +
</p>
 +
<p style="text-align: center;">
 +
    https://static.igem.org/mediawiki/igem.org/9/9d/TJUSLS_6.jpg<br>
 +
'''Figure 3.''' 37℃ Pre-expression
 +
</p>
 +
 +
----
 +
 +
 +
===HPLC result===
 +
 +
----
 +
 +
 +
===Western Blot result===
 +
<p style="text-align: center;">
 +
    https://static.igem.org/mediawiki/igem.org/9/9e/TJUSLS_7.jpg<br>
 +
</p>
 +
 +
----
 +
 +
 +
===Immunofluorescence assay Result===

Revision as of 15:25, 12 October 2016


SP.a+PETase+Histag.a+AIDA


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 2237
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE 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.

TJUSLS_4.jpg
Figure 1. 16℃ Pre-expression

TJUSLS_5.jpg
Figure 2. 25℃ Pre-expression

TJUSLS_6.jpg
Figure 3. 37℃ Pre-expression


HPLC result


Western Blot result

TJUSLS_7.jpg


Immunofluorescence assay Result