Latest revision as of 18:40, 14 November 2013

PHAECTORY: An elegant tool for production and secretion of Hepatitis B antibodies

To challenge PHAECTORY for the efficient antibody production and secretion we constructed a series of expression vectors consisting of the following parts: BBa_K1071001-BBa_K1071008. The alga Phaeodactylum tricornutum was transfected with these vectors. Two genes, which encode for the Hepatitis B antibody, were under the control of nitrate-inducible promoters. Five different clones of PHAECTORY were grown to an optical density of 0.4 (OD at 600nm) in a nitrate-containing medium (Figure). In a next step, we analysed how much Hepatitis B antibody was secreted from the algae into the surrounding medium. For this purpose, intact cells were separated from the surrounding medium by centrifugation. Both, cell pellets and supernatants were examined 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 than 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.

Secretion

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 PHAECTORY: An elegant tool for production and secretion of Hepatitis B antibodies <html><a href="https://2013.igem.org/Team:Marburg/Project:RFP"><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:Milestones"><img src="https://static.igem.org/mediawiki/2013/1/13/Mr-igem-previous-arrow.png" alt="Previous" style="float:right;"></a></html>
-{{:Team:Marburg/Template:ContentStartNav}}
+{{:Team:Marburg/Template:ContentStartNav}}To challenge PHAECTORY for the efficient antibody production and secretion we constructed a series of expression vectors consisting of the following parts: <html><a href="https://2013.igem.org/Team:Marburg/Parts">BBa_K1071001-BBa_K1071008</a></html>. The alga ''Phaeodactylum tricornutum'' was transfected with these vectors. Two genes, which encode for the Hepatitis B antibody, were under the control of nitrate-inducible promoters. Five different clones of PHAECTORY were grown to an optical density of 0.4 (OD at 600nm) in a nitrate-containing medium (Figure). In a next step, we analysed how much Hepatitis B antibody was secreted from the algae into the surrounding medium. For this purpose, intact cells were separated from the surrounding medium by centrifugation. Both, cell pellets and supernatants were examined for the presence of Hepatitis B antibodies by Western blot analysis.
-To challenge PHAECTORY for the efficient antibody production and secretion we constructed a series of expression vectors consisting of the following parts: <html><a href="https://2013.igem.org/Team:Marburg/Parts">BBa_K1071001-BBa_K1071008</a></html>. The alga ''Phaeodactylum tricornutum'' was transfected with these vectors. Two genes, which encode for the Hepatitis B antibody, were placed under the control of nitrate-inducible promoters. Five different clones of PHAECTORY were grown to an optical density of 0.4 (OD at 600nm) in a nitrate-containing medium (Figure). In a next step, we analysed how much Hepatitis B antibody was secreted from the algae into the surrounding medium. For this purpose, intact cells were separated from the surrounding medium by centrifugation. Both, cell pellets and supernatants 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 than 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.
-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 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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Team:Marburg/Results

From 2013.igem.org

Latest revision as of 18:40, 14 November 2013

PHAECTORY: An elegant tool for production and secretion of Hepatitis B antibodies