Team:Marburg/Project

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PHAECTORY: An elegant tool for production and secretion of Hepatitis B antibodies
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PHAECTORY: Good reasons for a new chassis
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{{:Team:Marburg/Template:ContentStartNav}}To challenge PHAECTORY for the efficient antibody production and secretion we constructed an expression vector consisting of the following parts: <html><a href="https://2013.igem.org/Team:Marburg/Parts">BBa_K1071001-BBa_K1071008</a></html> and transfected them into the algae ''Phaeodactylum tricornutum''. Two gene, which encode for the Hepatitis B antibody, was placed under the control of a nitrate induciable promoter. Five different clones of PHAECTORY were grown to an optical density of 0.4 in a nitrate-containing medium (Figure). In a next step, we wanted to analyse how much Hepatitis B antibody was secreted from the algae into the surrounding medium. Therefore, intact cells were separated from the surrounding medium by centrifugation. Both, cell pellets and supernatant were analysed for the presence of Hepatitis B antibodies by Western blot analysis.
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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. These features make 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 ballistic method for transfection 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 <html><a href="https://igem.org/" target="_blank">iGEM competition<a></html>.
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The Western blot analysis (Figure) shows that huge amounts of Hepatitis B antibodies were secreted into the medium. The positive signal in the cell pellet shows that antibody production by PHAECTORY was still in progress. Taken together, the amount of Hepatitis B antibodies in supernatant was significantly higher in the supernatant then in the cell pellet. This is the ‘proof of concept’ that PHAECTORY has not only the ability to produce high-value proteins (e.g. antibodies), but also secrets them in huge amounts into the surrounding medium. Therefore, PHAECTORY allows direct secretion of high-value proteins into the medium, and allows their easy purification with low effort and costs.
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"Classical" biotechnology is applied in bacteria, yeast and other fungi. Both need to be fed with carbon sources and the purification of the desired products e.g. high-value proteins is cost intensive, very time consuming and difficult. This is due to the fact that the cells need to be disrupted hence proteins/metabolites have to be removed from the medium.
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By using a green cell factory of plants or algae it is possible to produce all kinds of products even high-value proteins like human antibodies in a large-scale background. Looking at previous iGEM-projects we recognized that no practical green chassis was introduced into the competition although these organisms perform photosynthesis, which enables a carbon dioxide neutral handling. In the photosynthetic process light energy is converted into chemical energy, which can be used to fuel different cellular reactions in the cell. Concerning the problem of global warming these organisms offer the possibility to reduce the emission of green house gases and work against the extensive use of fossil fuels. Moreover, it is not necessary to feed green algal cell factories with carbohydrates. The world population is continuously growing resulting in a scarceness of nutrition. The iGEM team Marburg aims at introducing a green cell factory, which is driven by sunlight, produces no carbon dioxide and above all does not compete with the nutrition-producing sector.
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The microalga ''P. tricornutum'' is a green organism incorporating all above-mentioned advantages. This fast-growing organism lives with CO2 and light and is able to produce substances like bioplastic, biofuels, spider silk and other complex proteins <html><!--(XX add citation)--></html>. This year, the iGEM team Marburg decided to produce a human antibody against Hepatitis B virus (PHAECTORY). An important feature of PHAECTORY is that the antibody is directly secreted into the medium, which enormously simplifies purification. In general, Microalgae secret only very few additional substances and therefore all products produced in PHAECTORY are already present in a high purity (Hempel ''et al.'', 2011).
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In order to introduce this organism as a chassis to the iGEM community, we build a toolbox, which contains expression vectors, promoters, selection markers, reporter proteins and signal peptides for ''P. tricornutum''. This allows every future iGEM team to produce their own complex proteins, which can easily be processed.
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Revision as of 23:38, 26 October 2013

PHAECTORY: Good reasons for a new chassis

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. These features make 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 ballistic method for transfection 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.

"Classical" biotechnology is applied in bacteria, yeast and other fungi. Both need to be fed with carbon sources and the purification of the desired products e.g. high-value proteins is cost intensive, very time consuming and difficult. This is due to the fact that the cells need to be disrupted hence proteins/metabolites have to be removed from the medium.

By using a green cell factory of plants or algae it is possible to produce all kinds of products even high-value proteins like human antibodies in a large-scale background. Looking at previous iGEM-projects we recognized that no practical green chassis was introduced into the competition although these organisms perform photosynthesis, which enables a carbon dioxide neutral handling. In the photosynthetic process light energy is converted into chemical energy, which can be used to fuel different cellular reactions in the cell. Concerning the problem of global warming these organisms offer the possibility to reduce the emission of green house gases and work against the extensive use of fossil fuels. Moreover, it is not necessary to feed green algal cell factories with carbohydrates. The world population is continuously growing resulting in a scarceness of nutrition. The iGEM team Marburg aims at introducing a green cell factory, which is driven by sunlight, produces no carbon dioxide and above all does not compete with the nutrition-producing sector.

The microalga P. tricornutum is a green organism incorporating all above-mentioned advantages. This fast-growing organism lives with CO2 and light and is able to produce substances like bioplastic, biofuels, spider silk and other complex proteins . This year, the iGEM team Marburg decided to produce a human antibody against Hepatitis B virus (PHAECTORY). An important feature of PHAECTORY is that the antibody is directly secreted into the medium, which enormously simplifies purification. In general, Microalgae secret only very few additional substances and therefore all products produced in PHAECTORY are already present in a high purity (Hempel et al., 2011). In order to introduce this organism as a chassis to the iGEM community, we build a toolbox, which contains expression vectors, promoters, selection markers, reporter proteins and signal peptides for P. tricornutum. This allows every future iGEM team to produce their own complex proteins, which can easily be processed.

Secretion

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