Team:Marburg/Project:Ptricornutum

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Phaectory: Good reasons for a new chassis
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PHAECTORY: ''Phaeodactylum tricornutum'' <html><a href="https://2013.igem.org/Team:Marburg/Project:Milestones"><img src="https://static.igem.org/mediawiki/2013/7/71/Mr-igem-next-arrow.png" style="float:right;margin-left:5px !important;" alt="Next"></a>&nbsp;<a href="https://2013.igem.org/Team:Marburg/Project:Challenge"><img src="https://static.igem.org/mediawiki/2013/1/13/Mr-igem-previous-arrow.png" alt="Previous" style="float:right;"></a></html>
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{{:Team:Marburg/Template:ContentStartNav}}Diatoms are of great ecological relevance because they are responsible for up to 20% of the global CO2 fixation and generate about 40 % of the marine biomass of primary producers (Falkowski et al., 1998, Field et al., 1998). Diatoms also represent an important source of lipids and silicate. This makes them attractive for various biotechnological applications e.g. in biofuel industry, food industry and bioplastics. The widely spread diatom ''Phaeodactylum tricornutum'' is particularly interesting. It is robust and exists in three different morphotypes: Oval, triradial and fusiform whereupon the latter one is the most common appearance. Its entire genome has been sequenced, an easy transfection medthod (Apt et al., 1996) is well established as well as protocols for the cultivation are available. Taking this into account it appeared to us as the perfect organism to produce complex proteins for the iGEM competition.
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<html><img src="https://static.igem.org/mediawiki/2013/d/d8/Phaeo-bild.png" width="115" alt="P. tricornutum" style="float: right; margin-left:10px !important; margin-top:5px !important;" title="© Ansgar Gruber, University Konstanz "/></html>Diatoms are of great ecological relevance because they are responsible for up to 20 % of the global carbon dioxide fixation and generate about 40 % of the marine biomass of primary producers (Falkowski ''et al.'', 1998, Science and Field ''et al.'', 1998, Science). Diatoms also represent an important source of lipids and silicate. This makes them attractive for various biotechnological applications e.g. in biofuel industry, food industry and bioplastic production. The widely spread diatom ''Phaeodactylum tricornutum'' is particularly interesting. It is robust and exists in three different morphotypes: Oval, triradial and fusiform whereupon the latter one is the most common appearance. Its entire genome has been sequenced, an easy transfection method (Apt ''et al.'', 1996, Mol Gen Genet) is well established as well as protocols for the cultivation are available. Taking this into account it appeared to us as the perfect organism to produce complex proteins for the iGEM competition.
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<html><img src="https://static.igem.org/mediawiki/2013/8/80/Phaeo-picture.png" alt="P. tricornutum pictures" /></html>
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<html><h2>Antibody secretion in PHAECTORY</h2></html>
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We challenged [[Team:Marburg/Project|PHAECTORY]] as a green system for the production of antibodies which are directly secreted into the pure surrounding medium. The secretion of the antibodies is mediated via the regulated secretory pathway. Antibodies or other substances like hormones or neurotransmitter are translated into the endoplasmic reticulum (ER) and afterwards transported to the plasma membrane via the trans Golgi network.
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The genes for the Hepatitis B antibody produced in PHAECTORY are encoded by the nuclear genome where transcription takes place. The produced messenger RNA of the Hepatitis B antibody contains an amino terminal signal peptide which is recognized and bound through a signal peptide recognition particle. Posttranslational modifications like the removal of the signal peptide through a signal peptidase are initially performed in the ER.
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<html><!--<a href="https://static.igem.org/mediawiki/2013/8/83/Mr-secretion.png" title="Secretion"><img src="https://static.igem.org/mediawiki/2013/b/bd/Mr-secretion-thumb.png" alt="Secretion" style="float:right;margin-left: 10px !important; margin-bottom: 5px !important;" /></a>--></html>
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<html><center><img src="https://static.igem.org/mediawiki/2013/8/83/Mr-secretion.png" width="450" alt="Secretion" /></center></html>
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The antibodies enter the ER as nascent proteins and are folded in the ER lumen with the aid of chaperones. Before the fully assembled antibodies leave the ER the N-glycosylation is conducted. Afterwards the antibodies are transported (anterograde traffic) to the Golgi apparatus via COP (coat protein complex) II vesicles. Therefore, the proteins exit the ER via ER exit sites and are packaged into COPII coated vesicles which perform vesicle budding. The COPII carrier shuttles the cargo from the ER to the Golgi network driven by motor proteins along the cytoskeleton in the cell. Reaching the Golgi network the v-SNAREs of the COPII vesicles bind to the t-SNAREs of the Golgi leading to a fusion. The antibodies enter the Golgi apparatus which consists of several cisternae (cis, medial and trans). Additional posttranslational modifications like the O-glycosylation and the further processing of ER-derived sugar chains are executed in the Golgi apparatus before leaving the compartment.
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Thereafter the posttranslational modified antibodies are packaged into secretory vesicles, which are directed to the cytoplasmic membrane. Where they fuse with the membrane leading to the release of the antibodies. However, the exact mechanism of the transport to the plasma membrane via secretory vesicles is not yet well characterized in plants.
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'''References'''
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* Mechanisms of regulated unconventional protein secretion, Nickel and Rabouille, 2009, Nature <br />
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* Organization of the ER-Golgi interface for membrane traffic control, Brandizzi and Barlowe, 2013, Nature
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<html><!--<img src="https://static.igem.org/mediawiki/2013/0/06/Phaeo-03.jpg" alt="P. tricornutum pictures" />--></html>
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Latest revision as of 22:48, 28 October 2013

PHAECTORY: Phaeodactylum tricornutum Next Previous

P. tricornutumDiatoms are of great ecological relevance because they are responsible for up to 20 % of the global carbon dioxide fixation and generate about 40 % of the marine biomass of primary producers (Falkowski et al., 1998, Science and Field et al., 1998, Science). Diatoms also represent an important source of lipids and silicate. This makes them attractive for various biotechnological applications e.g. in biofuel industry, food industry and bioplastic production. The widely spread diatom Phaeodactylum tricornutum is particularly interesting. It is robust and exists in three different morphotypes: Oval, triradial and fusiform whereupon the latter one is the most common appearance. Its entire genome has been sequenced, an easy transfection method (Apt et al., 1996, Mol Gen Genet) is well established as well as protocols for the cultivation are available. Taking this into account it appeared to us as the perfect organism to produce complex proteins for the iGEM competition.

Antibody secretion in PHAECTORY

We challenged PHAECTORY as a green system for the production of antibodies which are directly secreted into the pure surrounding medium. The secretion of the antibodies is mediated via the regulated secretory pathway. Antibodies or other substances like hormones or neurotransmitter are translated into the endoplasmic reticulum (ER) and afterwards transported to the plasma membrane via the trans Golgi network.

The genes for the Hepatitis B antibody produced in PHAECTORY are encoded by the nuclear genome where transcription takes place. The produced messenger RNA of the Hepatitis B antibody contains an amino terminal signal peptide which is recognized and bound through a signal peptide recognition particle. Posttranslational modifications like the removal of the signal peptide through a signal peptidase are initially performed in the ER.

Secretion

The antibodies enter the ER as nascent proteins and are folded in the ER lumen with the aid of chaperones. Before the fully assembled antibodies leave the ER the N-glycosylation is conducted. Afterwards the antibodies are transported (anterograde traffic) to the Golgi apparatus via COP (coat protein complex) II vesicles. Therefore, the proteins exit the ER via ER exit sites and are packaged into COPII coated vesicles which perform vesicle budding. The COPII carrier shuttles the cargo from the ER to the Golgi network driven by motor proteins along the cytoskeleton in the cell. Reaching the Golgi network the v-SNAREs of the COPII vesicles bind to the t-SNAREs of the Golgi leading to a fusion. The antibodies enter the Golgi apparatus which consists of several cisternae (cis, medial and trans). Additional posttranslational modifications like the O-glycosylation and the further processing of ER-derived sugar chains are executed in the Golgi apparatus before leaving the compartment.

Thereafter the posttranslational modified antibodies are packaged into secretory vesicles, which are directed to the cytoplasmic membrane. Where they fuse with the membrane leading to the release of the antibodies. However, the exact mechanism of the transport to the plasma membrane via secretory vesicles is not yet well characterized in plants.

References

  • Mechanisms of regulated unconventional protein secretion, Nickel and Rabouille, 2009, Nature
  • Organization of the ER-Golgi interface for membrane traffic control, Brandizzi and Barlowe, 2013, Nature

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