Difference between revisions of "Part:BBa K1456006:Experience"

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==Chacarterisation==
 
==Chacarterisation==
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<center>[[File:Atoms aprotinin 4.png|400px|]]</center><br/>
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<center>[[File:Atoms aprotinin 4.png|600px|]]</center><br/>
 
<li>HEK 293T cells have been cotransfected with 1 µg, 2 µg, 3µg ve 4 µg Tet off – pTRE Aprotinin. The cells collected and protein isolation was performed to measure the serine protease inhibition. According to the positive control test, these results Show dose-dependent reduction and the production of aprotinin has increased according to the amount of cells successfully transfected.
 
<li>HEK 293T cells have been cotransfected with 1 µg, 2 µg, 3µg ve 4 µg Tet off – pTRE Aprotinin. The cells collected and protein isolation was performed to measure the serine protease inhibition. According to the positive control test, these results Show dose-dependent reduction and the production of aprotinin has increased according to the amount of cells successfully transfected.
  
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</br><h2>Combination of DsbA Signal Peptide with Aprotinin to Enhance Protein Purification (Lubbock_TTU iGEM 2016)</h2>
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The Aprotinin BioBrick by the ATOMS-Turkiye 2014 iGEM team was enhanced for better protein purification by using a DsbA signal tag in combination with the Aprotinin BioBrick. DsbA signal peptide carries Aprotinin to the periplasm where proper protein folding is enhanced. A protein extraction method known as osmotic shock creates pores in the outer membrane releasing the Aprotinin and other proteins in the periplasm into the supernatant. There should be fewer proteins in the solution compared with using a cell lysis method that releases all proteins within the cell into the solution. This results in cleaner protein purification of Aprotinin, while maintaining functionality.
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<center><br><img src="https://static.igem.org/mediawiki/parts/5/5d/Lubbock_TTU_aprotinin_growth_curve.png" height="" width="50%"></br><b>Fig 1.</b> In the above figure we see that IPTG induced origami cells containing the Aprotinin expression vector </br>exhibit the greatest fitness cost to proliferation rate, indicating that significant resources are being allocated to protein production. n=3</center>
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</br><h2>Simplification and E. coli optimization(Alma iGEM 2024)</h2>
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The Aprotinin BioBrick by the ATOMS-Turkiye 2014 was redesigned to be a more simplified part. This includes removing the DsbA signal peptide along with the histidine signal at the end of the protein. This fix removes the reading frame shift the part had. A codon optimization for E. coli was performed to make the final part <partinfo>BBa_K5324000</partinfo> .
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===Applications of BBa_K1456006===
 
===Applications of BBa_K1456006===

Latest revision as of 00:03, 26 September 2024

Aprotinin Assay

  • As Aprotinin is a serine protease inhibitor aprotinin activity can be measured using the correlation of serine protease inhibition the aprotinin inhibits.
    Atoms aprotinin 3.png

  • HEK 293T cell was transfected with 1 µg pTRE-Aprotinin and cells were obtained after 36 hours which continued with the isolation of proteins. Afterwards, the isolated protein was placed into a tube containing the serine protease and its activity was measured.
  • The graphic shows that serine protease inhibition was not observed in the isolated proteins of the cells transfected with the pTRE vectors and our negative controls only.
  • The inhibition values obtained by adding 6 mg/dl Bovine Aprotinin(sigmaA1153) samples obtained by cattles and the cotransfected Tet off – pTRE Aprotinin, show that the transfected HEK 293 T cells perform a lower amount of inhibition compared to our standard sample however that is a very minimal difference. As a result, our transfected aprotinin has accomplished more inhibition than our negative control and has proved that it can pe produced functionally. Afterwards the HEK 293 T cells cotransfected with Tet off – pTRE Aprotinin have been transfected in different amounts to characterise and measure its serine protease activity.

    Chacarterisation

    Atoms aprotinin 4.png

  • HEK 293T cells have been cotransfected with 1 µg, 2 µg, 3µg ve 4 µg Tet off – pTRE Aprotinin. The cells collected and protein isolation was performed to measure the serine protease inhibition. According to the positive control test, these results Show dose-dependent reduction and the production of aprotinin has increased according to the amount of cells successfully transfected.

    Combination of DsbA Signal Peptide with Aprotinin to Enhance Protein Purification (Lubbock_TTU iGEM 2016)

    The Aprotinin BioBrick by the ATOMS-Turkiye 2014 iGEM team was enhanced for better protein purification by using a DsbA signal tag in combination with the Aprotinin BioBrick. DsbA signal peptide carries Aprotinin to the periplasm where proper protein folding is enhanced. A protein extraction method known as osmotic shock creates pores in the outer membrane releasing the Aprotinin and other proteins in the periplasm into the supernatant. There should be fewer proteins in the solution compared with using a cell lysis method that releases all proteins within the cell into the solution. This results in cleaner protein purification of Aprotinin, while maintaining functionality.


    Fig 1. In the above figure we see that IPTG induced origami cells containing the Aprotinin expression vector
    exhibit the greatest fitness cost to proliferation rate, indicating that significant resources are being allocated to protein production. n=3

    Simplification and E. coli optimization(Alma iGEM 2024)

    The Aprotinin BioBrick by the ATOMS-Turkiye 2014 was redesigned to be a more simplified part. This includes removing the DsbA signal peptide along with the histidine signal at the end of the protein. This fix removes the reading frame shift the part had. A codon optimization for E. coli was performed to make the final part BBa_K5324000 .

    Applications of BBa_K1456006

    User Reviews

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