Difference between revisions of "Part:BBa K1071004"
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Promoter for nitrate reductase in ''Phaeodactylum tricornutum''. The protein nitrate reductase catalyses the reduction from nitrate to nitrite. Nitrate is important nitrogen source for plants, therefore is the NR promotor tightly regulated. Only if nitrate is the only nitrogen source, the promotor will be active. | Promoter for nitrate reductase in ''Phaeodactylum tricornutum''. The protein nitrate reductase catalyses the reduction from nitrate to nitrite. Nitrate is important nitrogen source for plants, therefore is the NR promotor tightly regulated. Only if nitrate is the only nitrogen source, the promotor will be active. | ||
| − | In the lab is the promotor used to express | + | In the lab is the promotor used to express recombinant proteinsin ''P. tricornutum''. The expression can be regulated by the amount of ammonia in the medium. |
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===Usage and Biology=== | ===Usage and Biology=== | ||
| − | < | + | To challenge PHAECTORY for the efficient antibody production and secretion we constructed an expression vector consisting of the following parts: <html><a href="http://2013.igem.org/Team:Marburg/Parts">BBa_K1071001-BBa_K1071008</a></html> and transfected them into the algae ''Phaeodactylum tricornutum''. A gene, which encoded 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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| + | 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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| + | <html> | ||
| + | <img src="https://static.igem.org/mediawiki/2013/7/79/Paectory.png" alt="Secretion" style="text-align:center;"/> | ||
| + | </html> | ||
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<span class='h3bb'>Sequence and Features</span> | <span class='h3bb'>Sequence and Features</span> | ||
<partinfo>BBa_K1071004 SequenceAndFeatures</partinfo> | <partinfo>BBa_K1071004 SequenceAndFeatures</partinfo> | ||
Latest revision as of 17:59, 12 October 2013
P NR
Promoter for nitrate reductase in Phaeodactylum tricornutum. The protein nitrate reductase catalyses the reduction from nitrate to nitrite. Nitrate is important nitrogen source for plants, therefore is the NR promotor tightly regulated. Only if nitrate is the only nitrogen source, the promotor will be active. In the lab is the promotor used to express recombinant proteinsin P. tricornutum. The expression can be regulated by the amount of ammonia in the medium.
Usage and Biology
To challenge PHAECTORY for the efficient antibody production and secretion we constructed an expression vector consisting of the following parts: BBa_K1071001-BBa_K1071008 and transfected them into the algae Phaeodactylum tricornutum. A gene, which encoded 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.
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.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 374
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]