http://2012.igem.org/wiki/index.php?title=Special:Contributions&feed=atom&limit=50&target=JaraO&year=&month=2012.igem.org - User contributions [en]2024-03-28T10:00:44ZFrom 2012.igem.orgMediaWiki 1.16.0http://2012.igem.org/Team:TU_Munich/Human_Practice/SurveyTeam:TU Munich/Human Practice/Survey2012-10-26T21:08:01Z<p>JaraO: /* Answers */</p>
<hr />
<div>{{Team:TU_Munich/Header}}<br />
<br />
= Survey =<br />
<hr/><br />
===Background===<br />
The survey was carried out by the [http://portal.mytum.de/jungeakademie/index_html/document_view?/ TUM young academy], which planned to install a [http://www.genial-info.de/ website] to inform the public about genetic engineering. They made an '''online questionnaire''' and informed munich people with a print out version of the project.<br />
As a whole '''1183''' people participated (979 people online and 204 people on the street) from which were 597 male people and 570 female people. It needs to consider, that the group of the 15 till 24 aged is drastically over represented (70% of all participants).<br />
<br/><br />
This team allowed us to use their data for our project as well. As they had 33 questions, we chose only the ones directly relied to genetic engineering.<br />
<br />
===Basic Data===<br />
*Participants: 1183<br />
*Online participants: 979<br />
*Street participants: 204<br />
*Male: 597 <br />
*Female: 570<br />
*Age: mainly between 15-24 (70%)<br />
<br />
===Questions===<br />
====Evaluated questions====<br />
*How do you feel about genetic engineering in general?<br />
*How good do you think is your knowledge about genetic engineering?<br />
*Are you concerned about genetically modified food?<br />
*Do you think genetic engineering is reasonable in medicine?<br />
*Do you think genetic engineering is reasonable in farming?<br />
*Do you think genetic engineering is reasonable in food?<br />
*Do you think the commentatorship in Germany is neutral?<br />
*Would you eat a genetically modified apple if it was healthier?<br />
*Would you eat a genetically modified apple if it tasted better?<br />
<br />
====Answers====<br />
<br/><br />
[[File:TUM12_Overall.png|thumb|left|400px|'''Fig. 1: Overall impression about genetic engineering.''']] <br />
[[File:TUM12_Knowledge.png|thumb|right|400px|'''Fig. 2: Knowledge about genetic engineering.''']]<br />
[[File:TUM12_Concerns about food.png|thumb|left|400px|'''Fig. 3: Concerns about genetically modified food.''']]<br />
[[File:TUM12_Genetic engineering in farming.png|thumb|right|400px|'''Fig. 4: Acceptance of genetic engineering in farming.''']]<br />
[[File:TUM12_Genetic engineering in medicine.png|thumb|left|400px|'''Fig. 5: Acceptance of genetic engineering in medicine.''']]<br />
[[File:TUM12_Media.png|thumb|right|400px|'''Fig. 6: Neutrality of the German media from the point of view of the public.''']]<br />
[[File:TUM12_Healthier apple.png|thumb|left|400px|'''Fig. 7: Decision to eat an genetically modified apple if it was healthier.''']]<br />
[[File:TUM12_Tastier apple.png|thumb|right|400px|'''Fig. 8: Decision to eat an genetically modified apple if it was tastier.''']]<br />
<br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br />
<br />
==Conclusion==<br />
In contrary to our first impression, about half of the questioned people said that they have a "normal" or better amount of knowledge. In our former human practice events we often realized that the discussions are rather emotional than scientific. This difference could arise from the mostly younger people who participated. Because in Germany genetic engineering started to play a bigger role in schools and in the media, although as mentioned below, the media is not always neutral.<br />
The overall impression of genetically modified organisms is not that bad, but it needs to be differentiated where genetic engineering is used. Most people agree that it is appropriate to use it in medicine.<br />
<br/><br />
In contrast 633 people (food) and 573 people (farming) of 1183 participants did not accept genetic engineering in food and agriculture. As already mentioned on our [https://2012.igem.org/Team:TU_Munich/Human_Practice/Overview overview page] people do not accept GMOs in their food. In Germany great skepticism persists.<br />
The fact that people (533) would not eat genetically modified apples even if they would taste similar, shows the low acceptance as well. If genetic engineering would be used for production of healthier aliment, the renunciation decreases. <br />
<br/><br/><br />
The advantages of green biotechnology are not present in people's minds and many people think that they are not informed impartial. This leads to a negative point of view and therefore neutral reporting is essential.<br />
<br />
All in all red biotechnology is widely accepted in Germany whereas green biotechnology is discussed controversial. Additionally some Germans think that media does not report objective about GMOs. Furthermore people wish that genetic engineering plays a bigger role in politics.</div>JaraOhttp://2012.igem.org/File:TUM12_Knowledge.pngFile:TUM12 Knowledge.png2012-10-26T21:03:44Z<p>JaraO: uploaded a new version of &quot;File:TUM12 Knowledge.png&quot;</p>
<hr />
<div></div>JaraOhttp://2012.igem.org/Team:TU_Munich/Human_Practice/SurveyTeam:TU Munich/Human Practice/Survey2012-10-26T20:58:16Z<p>JaraO: /* Answers */</p>
<hr />
<div>{{Team:TU_Munich/Header}}<br />
<br />
= Survey =<br />
<hr/><br />
===Background===<br />
The survey was carried out by the [http://portal.mytum.de/jungeakademie/index_html/document_view?/ TUM young academy], which planned to install a [http://www.genial-info.de/ website] to inform the public about genetic engineering. They made an '''online questionnaire''' and informed munich people with a print out version of the project.<br />
As a whole '''1183''' people participated (979 people online and 204 people on the street) from which were 597 male people and 570 female people. It needs to consider, that the group of the 15 till 24 aged is drastically over represented (70% of all participants).<br />
<br/><br />
This team allowed us to use their data for our project as well. As they had 33 questions, we chose only the ones directly relied to genetic engineering.<br />
<br />
===Basic Data===<br />
*Participants: 1183<br />
*Online participants: 979<br />
*Street participants: 204<br />
*Male: 597 <br />
*Female: 570<br />
*Age: mainly between 15-24 (70%)<br />
<br />
===Questions===<br />
====Evaluated questions====<br />
*How do you feel about genetic engineering in general?<br />
*How good do you think is your knowledge about genetic engineering?<br />
*Are you concerned about genetically modified food?<br />
*Do you think genetic engineering is reasonable in medicine?<br />
*Do you think genetic engineering is reasonable in farming?<br />
*Do you think genetic engineering is reasonable in food?<br />
*Do you think the commentatorship in Germany is neutral?<br />
*Would you eat a genetically modified apple if it was healthier?<br />
*Would you eat a genetically modified apple if it tasted better?<br />
<br />
====Answers====<br />
<br/><br />
[[File:TUM12_Overall.png|thumb|left|400px|'''Fig. 1: Overall impression about genetic engineering''']] <br />
[[File:TUM12_Knowledge.png|thumb|right|400px|'''Fig. 2: Knowledge about genetic engineering''']]<br />
[[File:TUM12_Concerns about food.png|thumb|left|400px|'''Fig. 3: Concerns about genetically modified food''']]<br />
[[File:TUM12_Genetic engineering in farming.png|thumb|right|400px|'''Fig. 4: Acceptance of genetic engineering in farming''']]<br />
[[File:TUM12_Genetic engineering in medicine.png|thumb|left|400px|'''Fig. 5: Acceptance of genetic engineering in medicine''']]<br />
[[File:TUM12_Media.png|thumb|right|400px|'''Fig. 6: Neutrality of the German media from the point of view of the public''']]<br />
[[File:TUM12_Healthier apple.png|thumb|left|400px|'''Fig. 7: Decision to eat an genetically modified apple if it was healthier''']]<br />
[[File:TUM12_Tastier apple.png|thumb|right|400px|'''Fig. 8: Decision to eat an genetically modified apple if it was tastier''']]<br />
<br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br />
<br />
==Conclusion==<br />
In contrary to our first impression, about half of the questioned people said that they have a "normal" or better amount of knowledge. In our former human practice events we often realized that the discussions are rather emotional than scientific. This difference could arise from the mostly younger people who participated. Because in Germany genetic engineering started to play a bigger role in schools and in the media, although as mentioned below, the media is not always neutral.<br />
The overall impression of genetically modified organisms is not that bad, but it needs to be differentiated where genetic engineering is used. Most people agree that it is appropriate to use it in medicine.<br />
<br/><br />
In contrast 633 people (food) and 573 people (farming) of 1183 participants did not accept genetic engineering in food and agriculture. As already mentioned on our [https://2012.igem.org/Team:TU_Munich/Human_Practice/Overview overview page] people do not accept GMOs in their food. In Germany great skepticism persists.<br />
The fact that people (533) would not eat genetically modified apples even if they would taste similar, shows the low acceptance as well. If genetic engineering would be used for production of healthier aliment, the renunciation decreases. <br />
<br/><br/><br />
The advantages of green biotechnology are not present in people's minds and many people think that they are not informed impartial. This leads to a negative point of view and therefore neutral reporting is essential.<br />
<br />
All in all red biotechnology is widely accepted in Germany whereas green biotechnology is discussed controversial. Additionally some Germans think that media does not report objective about GMOs. Furthermore people wish that genetic engineering plays a bigger role in politics.</div>JaraOhttp://2012.igem.org/Team:TU_Munich/Human_Practice/SurveyTeam:TU Munich/Human Practice/Survey2012-10-26T20:58:02Z<p>JaraO: /* Answers */</p>
<hr />
<div>{{Team:TU_Munich/Header}}<br />
<br />
= Survey =<br />
<hr/><br />
===Background===<br />
The survey was carried out by the [http://portal.mytum.de/jungeakademie/index_html/document_view?/ TUM young academy], which planned to install a [http://www.genial-info.de/ website] to inform the public about genetic engineering. They made an '''online questionnaire''' and informed munich people with a print out version of the project.<br />
As a whole '''1183''' people participated (979 people online and 204 people on the street) from which were 597 male people and 570 female people. It needs to consider, that the group of the 15 till 24 aged is drastically over represented (70% of all participants).<br />
<br/><br />
This team allowed us to use their data for our project as well. As they had 33 questions, we chose only the ones directly relied to genetic engineering.<br />
<br />
===Basic Data===<br />
*Participants: 1183<br />
*Online participants: 979<br />
*Street participants: 204<br />
*Male: 597 <br />
*Female: 570<br />
*Age: mainly between 15-24 (70%)<br />
<br />
===Questions===<br />
====Evaluated questions====<br />
*How do you feel about genetic engineering in general?<br />
*How good do you think is your knowledge about genetic engineering?<br />
*Are you concerned about genetically modified food?<br />
*Do you think genetic engineering is reasonable in medicine?<br />
*Do you think genetic engineering is reasonable in farming?<br />
*Do you think genetic engineering is reasonable in food?<br />
*Do you think the commentatorship in Germany is neutral?<br />
*Would you eat a genetically modified apple if it was healthier?<br />
*Would you eat a genetically modified apple if it tasted better?<br />
<br />
====Answers====<br />
<br/><br />
[[File:TUM12_Overall.png|thumb|left|400px|'''Fig. 1: Overall impression about genetic engineering''']] <br />
<br/><br />
[[File:TUM12_Knowledge.png|thumb|right|400px|'''Fig. 2: Knowledge about genetic engineering''']]<br />
[[File:TUM12_Concerns about food.png|thumb|left|400px|'''Fig. 3: Concerns about genetically modified food''']]<br />
[[File:TUM12_Genetic engineering in farming.png|thumb|right|400px|'''Fig. 4: Acceptance of genetic engineering in farming''']]<br />
[[File:TUM12_Genetic engineering in medicine.png|thumb|left|400px|'''Fig. 5: Acceptance of genetic engineering in medicine''']]<br />
[[File:TUM12_Media.png|thumb|right|400px|'''Fig. 6: Neutrality of the German media from the point of view of the public''']]<br />
[[File:TUM12_Healthier apple.png|thumb|left|400px|'''Fig. 7: Decision to eat an genetically modified apple if it was healthier''']]<br />
[[File:TUM12_Tastier apple.png|thumb|right|400px|'''Fig. 8: Decision to eat an genetically modified apple if it was tastier''']]<br />
<br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br />
<br />
==Conclusion==<br />
In contrary to our first impression, about half of the questioned people said that they have a "normal" or better amount of knowledge. In our former human practice events we often realized that the discussions are rather emotional than scientific. This difference could arise from the mostly younger people who participated. Because in Germany genetic engineering started to play a bigger role in schools and in the media, although as mentioned below, the media is not always neutral.<br />
The overall impression of genetically modified organisms is not that bad, but it needs to be differentiated where genetic engineering is used. Most people agree that it is appropriate to use it in medicine.<br />
<br/><br />
In contrast 633 people (food) and 573 people (farming) of 1183 participants did not accept genetic engineering in food and agriculture. As already mentioned on our [https://2012.igem.org/Team:TU_Munich/Human_Practice/Overview overview page] people do not accept GMOs in their food. In Germany great skepticism persists.<br />
The fact that people (533) would not eat genetically modified apples even if they would taste similar, shows the low acceptance as well. If genetic engineering would be used for production of healthier aliment, the renunciation decreases. <br />
<br/><br/><br />
The advantages of green biotechnology are not present in people's minds and many people think that they are not informed impartial. This leads to a negative point of view and therefore neutral reporting is essential.<br />
<br />
All in all red biotechnology is widely accepted in Germany whereas green biotechnology is discussed controversial. Additionally some Germans think that media does not report objective about GMOs. Furthermore people wish that genetic engineering plays a bigger role in politics.</div>JaraOhttp://2012.igem.org/Team:TU_Munich/Human_Practice/SurveyTeam:TU Munich/Human Practice/Survey2012-10-26T20:57:45Z<p>JaraO: /* Answers */</p>
<hr />
<div>{{Team:TU_Munich/Header}}<br />
<br />
= Survey =<br />
<hr/><br />
===Background===<br />
The survey was carried out by the [http://portal.mytum.de/jungeakademie/index_html/document_view?/ TUM young academy], which planned to install a [http://www.genial-info.de/ website] to inform the public about genetic engineering. They made an '''online questionnaire''' and informed munich people with a print out version of the project.<br />
As a whole '''1183''' people participated (979 people online and 204 people on the street) from which were 597 male people and 570 female people. It needs to consider, that the group of the 15 till 24 aged is drastically over represented (70% of all participants).<br />
<br/><br />
This team allowed us to use their data for our project as well. As they had 33 questions, we chose only the ones directly relied to genetic engineering.<br />
<br />
===Basic Data===<br />
*Participants: 1183<br />
*Online participants: 979<br />
*Street participants: 204<br />
*Male: 597 <br />
*Female: 570<br />
*Age: mainly between 15-24 (70%)<br />
<br />
===Questions===<br />
====Evaluated questions====<br />
*How do you feel about genetic engineering in general?<br />
*How good do you think is your knowledge about genetic engineering?<br />
*Are you concerned about genetically modified food?<br />
*Do you think genetic engineering is reasonable in medicine?<br />
*Do you think genetic engineering is reasonable in farming?<br />
*Do you think genetic engineering is reasonable in food?<br />
*Do you think the commentatorship in Germany is neutral?<br />
*Would you eat a genetically modified apple if it was healthier?<br />
*Would you eat a genetically modified apple if it tasted better?<br />
<br />
====Answers====<br />
<br/><br />
[[File:TUM12_Overall.png|thumb|left|400px|'''Fig. 1: Overall impression about genetic engineering''']] <br />
<br/><br />
[[File:TUM12_Knowledge.png|thumb|right|400px|'''Fig. 2: Knowledge about genetic engineering''']]<br />
<br/><br />
[[File:TUM12_Concerns about food.png|thumb|left|400px|'''Fig. 3: Concerns about genetically modified food''']]<br />
[[File:TUM12_Genetic engineering in farming.png|thumb|right|400px|'''Fig. 4: Acceptance of genetic engineering in farming''']]<br />
[[File:TUM12_Genetic engineering in medicine.png|thumb|left|400px|'''Fig. 5: Acceptance of genetic engineering in medicine''']]<br />
[[File:TUM12_Media.png|thumb|right|400px|'''Fig. 6: Neutrality of the German media from the point of view of the public''']]<br />
[[File:TUM12_Healthier apple.png|thumb|left|400px|'''Fig. 7: Decision to eat an genetically modified apple if it was healthier''']]<br />
[[File:TUM12_Tastier apple.png|thumb|right|400px|'''Fig. 8: Decision to eat an genetically modified apple if it was tastier''']]<br />
<br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br />
<br />
==Conclusion==<br />
In contrary to our first impression, about half of the questioned people said that they have a "normal" or better amount of knowledge. In our former human practice events we often realized that the discussions are rather emotional than scientific. This difference could arise from the mostly younger people who participated. Because in Germany genetic engineering started to play a bigger role in schools and in the media, although as mentioned below, the media is not always neutral.<br />
The overall impression of genetically modified organisms is not that bad, but it needs to be differentiated where genetic engineering is used. Most people agree that it is appropriate to use it in medicine.<br />
<br/><br />
In contrast 633 people (food) and 573 people (farming) of 1183 participants did not accept genetic engineering in food and agriculture. As already mentioned on our [https://2012.igem.org/Team:TU_Munich/Human_Practice/Overview overview page] people do not accept GMOs in their food. In Germany great skepticism persists.<br />
The fact that people (533) would not eat genetically modified apples even if they would taste similar, shows the low acceptance as well. If genetic engineering would be used for production of healthier aliment, the renunciation decreases. <br />
<br/><br/><br />
The advantages of green biotechnology are not present in people's minds and many people think that they are not informed impartial. This leads to a negative point of view and therefore neutral reporting is essential.<br />
<br />
All in all red biotechnology is widely accepted in Germany whereas green biotechnology is discussed controversial. Additionally some Germans think that media does not report objective about GMOs. Furthermore people wish that genetic engineering plays a bigger role in politics.</div>JaraOhttp://2012.igem.org/Team:TU_Munich/Human_Practice/SurveyTeam:TU Munich/Human Practice/Survey2012-10-26T20:57:09Z<p>JaraO: /* Answers */</p>
<hr />
<div>{{Team:TU_Munich/Header}}<br />
<br />
= Survey =<br />
<hr/><br />
===Background===<br />
The survey was carried out by the [http://portal.mytum.de/jungeakademie/index_html/document_view?/ TUM young academy], which planned to install a [http://www.genial-info.de/ website] to inform the public about genetic engineering. They made an '''online questionnaire''' and informed munich people with a print out version of the project.<br />
As a whole '''1183''' people participated (979 people online and 204 people on the street) from which were 597 male people and 570 female people. It needs to consider, that the group of the 15 till 24 aged is drastically over represented (70% of all participants).<br />
<br/><br />
This team allowed us to use their data for our project as well. As they had 33 questions, we chose only the ones directly relied to genetic engineering.<br />
<br />
===Basic Data===<br />
*Participants: 1183<br />
*Online participants: 979<br />
*Street participants: 204<br />
*Male: 597 <br />
*Female: 570<br />
*Age: mainly between 15-24 (70%)<br />
<br />
===Questions===<br />
====Evaluated questions====<br />
*How do you feel about genetic engineering in general?<br />
*How good do you think is your knowledge about genetic engineering?<br />
*Are you concerned about genetically modified food?<br />
*Do you think genetic engineering is reasonable in medicine?<br />
*Do you think genetic engineering is reasonable in farming?<br />
*Do you think genetic engineering is reasonable in food?<br />
*Do you think the commentatorship in Germany is neutral?<br />
*Would you eat a genetically modified apple if it was healthier?<br />
*Would you eat a genetically modified apple if it tasted better?<br />
<br />
====Answers====<br />
[[File:TUM12_Overall.png|thumb|left|400px|'''Fig. 1: Overall impression about genetic engineering''']] <br />
<br/><br />
[[File:TUM12_Knowledge.png|thumb|right|400px|'''Fig. 2: Knowledge about genetic engineering''']]<br />
<br/><br />
[[File:TUM12_Concerns about food.png|thumb|left|400px|'''Fig. 3: Concerns about genetically modified food''']]<br />
[[File:TUM12_Genetic engineering in farming.png|thumb|right|400px|'''Fig. 4: Acceptance of genetic engineering in farming''']]<br />
[[File:TUM12_Genetic engineering in medicine.png|thumb|left|400px|'''Fig. 5: Acceptance of genetic engineering in medicine''']]<br />
[[File:TUM12_Media.png|thumb|right|400px|'''Fig. 6: Neutrality of the German media from the point of view of the public''']]<br />
[[File:TUM12_Healthier apple.png|thumb|left|400px|'''Fig. 7: Decision to eat an genetically modified apple if it was healthier''']]<br />
[[File:TUM12_Tastier apple.png|thumb|right|400px|'''Fig. 8: Decision to eat an genetically modified apple if it was tastier''']]<br />
<br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br />
<br />
==Conclusion==<br />
In contrary to our first impression, about half of the questioned people said that they have a "normal" or better amount of knowledge. In our former human practice events we often realized that the discussions are rather emotional than scientific. This difference could arise from the mostly younger people who participated. Because in Germany genetic engineering started to play a bigger role in schools and in the media, although as mentioned below, the media is not always neutral.<br />
The overall impression of genetically modified organisms is not that bad, but it needs to be differentiated where genetic engineering is used. Most people agree that it is appropriate to use it in medicine.<br />
<br/><br />
In contrast 633 people (food) and 573 people (farming) of 1183 participants did not accept genetic engineering in food and agriculture. As already mentioned on our [https://2012.igem.org/Team:TU_Munich/Human_Practice/Overview overview page] people do not accept GMOs in their food. In Germany great skepticism persists.<br />
The fact that people (533) would not eat genetically modified apples even if they would taste similar, shows the low acceptance as well. If genetic engineering would be used for production of healthier aliment, the renunciation decreases. <br />
<br/><br/><br />
The advantages of green biotechnology are not present in people's minds and many people think that they are not informed impartial. This leads to a negative point of view and therefore neutral reporting is essential.<br />
<br />
All in all red biotechnology is widely accepted in Germany whereas green biotechnology is discussed controversial. Additionally some Germans think that media does not report objective about GMOs. Furthermore people wish that genetic engineering plays a bigger role in politics.</div>JaraOhttp://2012.igem.org/File:Taste1.pngFile:Taste1.png2012-10-26T20:54:59Z<p>JaraO: </p>
<hr />
<div></div>JaraOhttp://2012.igem.org/File:TUM12_Tastier_apple.pngFile:TUM12 Tastier apple.png2012-10-26T20:52:38Z<p>JaraO: uploaded a new version of &quot;File:TUM12 Tastier apple.png&quot;</p>
<hr />
<div></div>JaraOhttp://2012.igem.org/File:TUM12_Healthier_apple.pngFile:TUM12 Healthier apple.png2012-10-26T20:22:57Z<p>JaraO: uploaded a new version of &quot;File:TUM12 Healthier apple.png&quot;</p>
<hr />
<div></div>JaraOhttp://2012.igem.org/File:TUM12_Media.pngFile:TUM12 Media.png2012-10-26T20:19:48Z<p>JaraO: uploaded a new version of &quot;File:TUM12 Media.png&quot;</p>
<hr />
<div></div>JaraOhttp://2012.igem.org/File:TUM12_Genetic_engineering_in_medicine.pngFile:TUM12 Genetic engineering in medicine.png2012-10-26T20:18:17Z<p>JaraO: uploaded a new version of &quot;File:TUM12 Genetic engineering in medicine.png&quot;</p>
<hr />
<div></div>JaraOhttp://2012.igem.org/File:TUM12_Genetic_engineering_in_farming.pngFile:TUM12 Genetic engineering in farming.png2012-10-26T20:17:30Z<p>JaraO: uploaded a new version of &quot;File:TUM12 Genetic engineering in farming.png&quot;</p>
<hr />
<div></div>JaraOhttp://2012.igem.org/File:TUM12_Concerns_about_food.pngFile:TUM12 Concerns about food.png2012-10-26T20:16:24Z<p>JaraO: uploaded a new version of &quot;File:TUM12 Concerns about food.png&quot;</p>
<hr />
<div></div>JaraOhttp://2012.igem.org/File:TUM12_Knowledge.pngFile:TUM12 Knowledge.png2012-10-26T20:15:34Z<p>JaraO: uploaded a new version of &quot;File:TUM12 Knowledge.png&quot;</p>
<hr />
<div></div>JaraOhttp://2012.igem.org/File:TUM12_Healthier_apple.pngFile:TUM12 Healthier apple.png2012-10-26T20:14:31Z<p>JaraO: uploaded a new version of &quot;File:TUM12 Healthier apple.png&quot;</p>
<hr />
<div></div>JaraOhttp://2012.igem.org/File:TUM12_Healthier_apple.pngFile:TUM12 Healthier apple.png2012-10-26T20:09:21Z<p>JaraO: uploaded a new version of &quot;File:TUM12 Healthier apple.png&quot;</p>
<hr />
<div></div>JaraOhttp://2012.igem.org/File:TUM12_Overall.pngFile:TUM12 Overall.png2012-10-26T20:07:25Z<p>JaraO: uploaded a new version of &quot;File:TUM12 Overall.png&quot;: Reverted to version as of 20:04, 26 October 2012</p>
<hr />
<div></div>JaraOhttp://2012.igem.org/File:TUM12_Overall.pngFile:TUM12 Overall.png2012-10-26T20:05:51Z<p>JaraO: uploaded a new version of &quot;File:TUM12 Overall.png&quot;</p>
<hr />
<div></div>JaraOhttp://2012.igem.org/File:TUM12_Overall.pngFile:TUM12 Overall.png2012-10-26T20:04:49Z<p>JaraO: uploaded a new version of &quot;File:TUM12 Overall.png&quot;</p>
<hr />
<div></div>JaraOhttp://2012.igem.org/File:TUM12_Overall.pngFile:TUM12 Overall.png2012-10-26T20:03:07Z<p>JaraO: uploaded a new version of &quot;File:TUM12 Overall.png&quot;</p>
<hr />
<div></div>JaraOhttp://2012.igem.org/Team:TU_MunichTeam:TU Munich2012-10-26T20:01:21Z<p>JaraO: /* Vision */</p>
<hr />
<div>{{Team:TU_Munich/Header}}<br />
__NOTOC__<br />
= TUM-Brew: iGEM's first and finest SynBio Beer =<br />
<hr/><br />
<br />
[[File:TUM12_AE120612001.jpg|300px||right||]]<br />
<br />
The TU Munich iGEM Team engineers ''Saccharomyces cerevisiae'', also known as baker's yeast, in order to lay the foundations for a new generation of functional foods with nutritionally valuable ingredients.<br />
<br />
As an example, for iGEM’s first “SynBio Beer” the compounds [[Team:TU_Munich/Project/Xanthohumol|'''xanthohumol''']] (anticancerogenic), [[Team:TU_Munich/Project/Limonene|'''limonene''']] (limeflavor), [[Team:TU_Munich/Project/Caffeine|'''caffeine''']] (CNS-stimulant) as well as the [[Team:TU_Munich/Project/Thaumatin|'''thaumatin''']] (protein sweetener) were chosen to demonstrate the spectrum of possibilities to complement traditional foods or beverages.<br />
<br />
The metabolic pathways for these substances were converted to genetic BioBricks. Using the shuttle vector [[Team:TU_Munich/Project/Vector_Design|'''pTUM100''']], which was adapted to the iGEM standard, transient transfection and expression in yeast were achieved. The gene products were subsequently characterized and their biosynthetic activities investigated.<br />
<br />
[[Team:TU_Munich/Project/Constitutive_Promoter|'''Constitutive''']], [[Team:TU_Munich/Project/Ethanol_Inducible_Promoter|'''ethanol-inducible''']] and [[Team:TU_Munich/Project/Light_Switchable_Promoter|'''light-switchable''']] promoter systems were developed to individually regulate the expression of these gene cassettes. By combining these BioBricks our team has been able to brew iGEM’s first and finest [[Team:TU_Munich/Project/Brewing|'''SynBio Beer''']].<br />
<br />
<br />
== Vision ==<br />
----<br />
<div><br />
<br />
We, TU Munich’s 2012 iGEM team, strive to catalyze the diffusion process of knowledge about genetic engineering and synthetic biology among the general public. Using the example of iGEM’s first and finest SynBio Beer we involve, interest and inspire people to reconsider preconceived ideas and encourage them to openly engage in a broad discussion weighing pros and cons of genetic engineering in foodstuff. We sketch a future where new technology can be applied in a meaningful way to complement traditional foods or beverages.<br />
</div><br />
<br />
<html><iframe style="box-shadow: 1px 1px 2px rgba(0, 0, 0, 0.2);padding: 5px;margin: 5px;background-color: white;" src="http://player.vimeo.com/video/51804324?color=d92540" width="800" height="450" frameborder="0" webkitAllowFullScreen mozallowfullscreen allowFullScreen></iframe></html><br />
<br />
== Overview ==<br />
----<br />
<html><br />
<script src="https://2012.igem.org/Team:TU_Munich/jquery.li-scroller.1.0.js?action=raw&ctype=text/js"></script><br />
</html><br />
<center><br />
<div class="ui-corner-all" id="ticker"><br />
<ul id="ticker01" ><br />
<li><span><b>878</b> Documented experiments in our labjournal</span></li><br />
<li><span><b>923</b> Plasmids</span></li><br />
<li><span><b>49</b> Annotated BioBricks</span></li><br />
<li><span><b>107</b> Days in the lab</span></li><br />
<li><span><b>283</b> Sequencing orders</span></li><br />
<li><span><b>13</b> Subprojects</span></li><br />
<li><span><b>19</b> Students</span></li><br />
<li><span>Over <b>10.000.000</b> systems of differential equations solved</span></li><br />
<li><span><b>878</b> Documented experiments in our labjournal</span></li><br />
<li><span><b>923</b> Plasmids</span></li><br />
<li><span><b>49</b> Annotated BioBricks</span></li><br />
<li><span><b>107</b> Days in the lab</span></li><br />
<li><span><b>283</b> Sequencing orders</span></li><br />
<li><span><b>13</b> Subprojects</span></li><br />
<li><span><b>19</b> Students</span></li><br />
<li><span>Over <b>10.000.000</b> systems of differential equations solved</span></li><br />
<li><span><b>878</b> Documented experiments in our labjournal</span></li><br />
<li><span><b>923</b> Plasmids</span></li><br />
<li><span><b>49</b> Annotated BioBricks</span></li><br />
<li><span><b>107</b> Days in the lab</span></li><br />
<li><span><b>283</b> Sequencing orders</span></li><br />
<li><span><b>13</b> Subprojects</span></li><br />
<li><span><b>19</b> Students</span></li><br />
<li><span>Over <b>10.000.000</b> systems of differential equations solved</span></li><br />
</ul><br />
</div><br />
</center><br />
<html><br />
<script><br />
$(document).ready(function(){<br />
$('ul#ticker01').liScroll({travelvelocity: 0.03});<br />
});<br />
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<br />
{{Team:TU_Munich/Overview}}<br />
<br />
== Reference ==<br />
----<br />
<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/6327079 Edens et al., 1984]] Edens, L., Bom, I., Ledeboer, A. M., Maat, J., Toonen, M. Y., Visser, C., and Verrips, C. T. (1984). Synthesis and processing of the plant protein thaumatin in yeast. ''Cell'', 37(2):629–33.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/10724480 Mazzoni et al., 2000]] Mazzoni, C., Santori, F., Saliola, M., and Falcone, C. (2000). Molecular analysis of uas(e), a cis element containing stress response elements responsible for ethanol induction of the kladh4 gene of ''Kluyveromyces lactis''. ''Res Microbiol'', 151(1):19–28.<br />
<br />
== Sponsors ==<br />
----<br />
<br />
<html><br />
<center class="noborder"><br />
<a href="http://www.applichem.com"><img src="https://static.igem.org/mediawiki/2012/f/f5/TUM_Applichem.jpg" width="200px"></a><br />
<a href="http://www.biomers.net"><img src="https://static.igem.org/mediawiki/2012/3/37/TUM_Biomers.png" width="200px"></a><br />
<a href="http://www.biozym.com"><img src="https://static.igem.org/mediawiki/2012/d/d8/TUM_Biozym.jpg" width="200px"></a><br />
<a href="http://www.cipsm.de"><img src="https://static.igem.org/mediawiki/2012/8/88/TUM_Cipsm.jpg" width="200px"></a><br />
<a href="http://www.eurofins.de"><img src="https://static.igem.org/mediawiki/2012/b/bc/TUM_Eurofins.png" width="200px"></a><br />
<a href="http://www.idt.com"><img src="https://static.igem.org/mediawiki/2012/0/0b/TUM_IDT.jpg" width="200px"></a><br />
<a href="http://www.metabion.com"><img src="https://static.igem.org/mediawiki/2012/9/93/TUM_Metabion.png" width="200px"></a><br />
<a href="http://www.geneious.com/"><img src="https://static.igem.org/mediawiki/2012/8/82/TUM_Geneious.png" width="200px"></a><br />
<a href="http://www.roche.de/"><img src="https://static.igem.org/mediawiki/2012/a/a4/TUM_Roche.jpg" width="200px"></a><br />
<a href="http://www.daad.de/promos/"><img src="https://static.igem.org/mediawiki/2012/7/77/TUM_DAAD.gif" width="200px"></a><br />
<a href="http://www.qiagen.com"><img src="https://static.igem.org/mediawiki/2012/3/39/TUM_Qiuagen.jpg" width="100px"></a><br />
<a href="http://www.carlroth.com"><img src="https://static.igem.org/mediawiki/2012/6/63/TUM_Roth.gif" width="100px"></a><br />
<a href="http://www.bund-der-freunde.tum.de/"><img src="https://static.igem.org/mediawiki/2012/1/1d/TUM_Bund.jpg" width="200px"></a><br />
</center><br />
</html><br />
<br />
<html><br />
<center class="noborder"><br />
<a href="http://biologische-chemie.userweb.mwn.de/"><img src="https://static.igem.org/mediawiki/2012/1/1f/TUM_TUM.png" width="400px"></a><br />
</center><br />
</html><br />
<br />
<center>http://www4.clustrmaps.com/stats/maps-no_clusters/2012.igem.org-Team-TU_Munich-thumb.jpg</center></div>JaraOhttp://2012.igem.org/Team:TU_MunichTeam:TU Munich2012-10-26T19:59:28Z<p>JaraO: /* Vision */</p>
<hr />
<div>{{Team:TU_Munich/Header}}<br />
__NOTOC__<br />
= TUM-Brew: iGEM's first and finest SynBio Beer =<br />
<hr/><br />
<br />
[[File:TUM12_AE120612001.jpg|300px||right||]]<br />
<br />
The TU Munich iGEM Team engineers ''Saccharomyces cerevisiae'', also known as baker's yeast, in order to lay the foundations for a new generation of functional foods with nutritionally valuable ingredients.<br />
<br />
As an example, for iGEM’s first “SynBio Beer” the compounds [[Team:TU_Munich/Project/Xanthohumol|'''xanthohumol''']] (anticancerogenic), [[Team:TU_Munich/Project/Limonene|'''limonene''']] (limeflavor), [[Team:TU_Munich/Project/Caffeine|'''caffeine''']] (CNS-stimulant) as well as the [[Team:TU_Munich/Project/Thaumatin|'''thaumatin''']] (protein sweetener) were chosen to demonstrate the spectrum of possibilities to complement traditional foods or beverages.<br />
<br />
The metabolic pathways for these substances were converted to genetic BioBricks. Using the shuttle vector [[Team:TU_Munich/Project/Vector_Design|'''pTUM100''']], which was adapted to the iGEM standard, transient transfection and expression in yeast were achieved. The gene products were subsequently characterized and their biosynthetic activities investigated.<br />
<br />
[[Team:TU_Munich/Project/Constitutive_Promoter|'''Constitutive''']], [[Team:TU_Munich/Project/Ethanol_Inducible_Promoter|'''ethanol-inducible''']] and [[Team:TU_Munich/Project/Light_Switchable_Promoter|'''light-switchable''']] promoter systems were developed to individually regulate the expression of these gene cassettes. By combining these BioBricks our team has been able to brew iGEM’s first and finest [[Team:TU_Munich/Project/Brewing|'''SynBio Beer''']].<br />
<br />
<br />
== Vision ==<br />
----<br />
<div><br />
<br />
We, TU Munich’s 2012 iGEM team, strive to catalyze the diffusion process of knowledge about genetic engineering and synthetic biology among the general public. Using the example of iGEM’s first and finest SynBio Beer we involve, interest and inspire people to reconsider preconceived ideas and encourage them to openly engage in a broad discussion weighing pros and cons of genetic engineering in foodstuff. We sketch a future where new technology can be applied in a meaningful way to complement traditional foods or beverages.<br />
</div><br />
<br />
<html><iframe style="box-shadow: 1px 1px 2px rgba(0, 0, 0, 0.2);padding: 5px;margin: 5px;background-color: white;" src="http://player.vimeo.com/video/51804324?color=d92540" width="720" height="405" frameborder="0" webkitAllowFullScreen mozallowfullscreen allowFullScreen></iframe></html><br />
<br />
== Overview ==<br />
----<br />
<html><br />
<script src="https://2012.igem.org/Team:TU_Munich/jquery.li-scroller.1.0.js?action=raw&ctype=text/js"></script><br />
</html><br />
<center><br />
<div class="ui-corner-all" id="ticker"><br />
<ul id="ticker01" ><br />
<li><span><b>878</b> Documented experiments in our labjournal</span></li><br />
<li><span><b>923</b> Plasmids</span></li><br />
<li><span><b>49</b> Annotated BioBricks</span></li><br />
<li><span><b>107</b> Days in the lab</span></li><br />
<li><span><b>283</b> Sequencing orders</span></li><br />
<li><span><b>13</b> Subprojects</span></li><br />
<li><span><b>19</b> Students</span></li><br />
<li><span>Over <b>10.000.000</b> systems of differential equations solved</span></li><br />
<li><span><b>878</b> Documented experiments in our labjournal</span></li><br />
<li><span><b>923</b> Plasmids</span></li><br />
<li><span><b>49</b> Annotated BioBricks</span></li><br />
<li><span><b>107</b> Days in the lab</span></li><br />
<li><span><b>283</b> Sequencing orders</span></li><br />
<li><span><b>13</b> Subprojects</span></li><br />
<li><span><b>19</b> Students</span></li><br />
<li><span>Over <b>10.000.000</b> systems of differential equations solved</span></li><br />
<li><span><b>878</b> Documented experiments in our labjournal</span></li><br />
<li><span><b>923</b> Plasmids</span></li><br />
<li><span><b>49</b> Annotated BioBricks</span></li><br />
<li><span><b>107</b> Days in the lab</span></li><br />
<li><span><b>283</b> Sequencing orders</span></li><br />
<li><span><b>13</b> Subprojects</span></li><br />
<li><span><b>19</b> Students</span></li><br />
<li><span>Over <b>10.000.000</b> systems of differential equations solved</span></li><br />
</ul><br />
</div><br />
</center><br />
<html><br />
<script><br />
$(document).ready(function(){<br />
$('ul#ticker01').liScroll({travelvelocity: 0.03});<br />
});<br />
</script><br />
</html><br />
<br />
{{Team:TU_Munich/Overview}}<br />
<br />
== Reference ==<br />
----<br />
<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/6327079 Edens et al., 1984]] Edens, L., Bom, I., Ledeboer, A. M., Maat, J., Toonen, M. Y., Visser, C., and Verrips, C. T. (1984). Synthesis and processing of the plant protein thaumatin in yeast. ''Cell'', 37(2):629–33.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/10724480 Mazzoni et al., 2000]] Mazzoni, C., Santori, F., Saliola, M., and Falcone, C. (2000). Molecular analysis of uas(e), a cis element containing stress response elements responsible for ethanol induction of the kladh4 gene of ''Kluyveromyces lactis''. ''Res Microbiol'', 151(1):19–28.<br />
<br />
== Sponsors ==<br />
----<br />
<br />
<html><br />
<center class="noborder"><br />
<a href="http://www.applichem.com"><img src="https://static.igem.org/mediawiki/2012/f/f5/TUM_Applichem.jpg" width="200px"></a><br />
<a href="http://www.biomers.net"><img src="https://static.igem.org/mediawiki/2012/3/37/TUM_Biomers.png" width="200px"></a><br />
<a href="http://www.biozym.com"><img src="https://static.igem.org/mediawiki/2012/d/d8/TUM_Biozym.jpg" width="200px"></a><br />
<a href="http://www.cipsm.de"><img src="https://static.igem.org/mediawiki/2012/8/88/TUM_Cipsm.jpg" width="200px"></a><br />
<a href="http://www.eurofins.de"><img src="https://static.igem.org/mediawiki/2012/b/bc/TUM_Eurofins.png" width="200px"></a><br />
<a href="http://www.idt.com"><img src="https://static.igem.org/mediawiki/2012/0/0b/TUM_IDT.jpg" width="200px"></a><br />
<a href="http://www.metabion.com"><img src="https://static.igem.org/mediawiki/2012/9/93/TUM_Metabion.png" width="200px"></a><br />
<a href="http://www.geneious.com/"><img src="https://static.igem.org/mediawiki/2012/8/82/TUM_Geneious.png" width="200px"></a><br />
<a href="http://www.roche.de/"><img src="https://static.igem.org/mediawiki/2012/a/a4/TUM_Roche.jpg" width="200px"></a><br />
<a href="http://www.daad.de/promos/"><img src="https://static.igem.org/mediawiki/2012/7/77/TUM_DAAD.gif" width="200px"></a><br />
<a href="http://www.qiagen.com"><img src="https://static.igem.org/mediawiki/2012/3/39/TUM_Qiuagen.jpg" width="100px"></a><br />
<a href="http://www.carlroth.com"><img src="https://static.igem.org/mediawiki/2012/6/63/TUM_Roth.gif" width="100px"></a><br />
<a href="http://www.bund-der-freunde.tum.de/"><img src="https://static.igem.org/mediawiki/2012/1/1d/TUM_Bund.jpg" width="200px"></a><br />
</center><br />
</html><br />
<br />
<html><br />
<center class="noborder"><br />
<a href="http://biologische-chemie.userweb.mwn.de/"><img src="https://static.igem.org/mediawiki/2012/1/1f/TUM_TUM.png" width="400px"></a><br />
</center><br />
</html><br />
<br />
<center>http://www4.clustrmaps.com/stats/maps-no_clusters/2012.igem.org-Team-TU_Munich-thumb.jpg</center></div>JaraOhttp://2012.igem.org/Team:TU_MunichTeam:TU Munich2012-10-26T19:59:02Z<p>JaraO: /* Vision */</p>
<hr />
<div>{{Team:TU_Munich/Header}}<br />
__NOTOC__<br />
= TUM-Brew: iGEM's first and finest SynBio Beer =<br />
<hr/><br />
<br />
[[File:TUM12_AE120612001.jpg|300px||right||]]<br />
<br />
The TU Munich iGEM Team engineers ''Saccharomyces cerevisiae'', also known as baker's yeast, in order to lay the foundations for a new generation of functional foods with nutritionally valuable ingredients.<br />
<br />
As an example, for iGEM’s first “SynBio Beer” the compounds [[Team:TU_Munich/Project/Xanthohumol|'''xanthohumol''']] (anticancerogenic), [[Team:TU_Munich/Project/Limonene|'''limonene''']] (limeflavor), [[Team:TU_Munich/Project/Caffeine|'''caffeine''']] (CNS-stimulant) as well as the [[Team:TU_Munich/Project/Thaumatin|'''thaumatin''']] (protein sweetener) were chosen to demonstrate the spectrum of possibilities to complement traditional foods or beverages.<br />
<br />
The metabolic pathways for these substances were converted to genetic BioBricks. Using the shuttle vector [[Team:TU_Munich/Project/Vector_Design|'''pTUM100''']], which was adapted to the iGEM standard, transient transfection and expression in yeast were achieved. The gene products were subsequently characterized and their biosynthetic activities investigated.<br />
<br />
[[Team:TU_Munich/Project/Constitutive_Promoter|'''Constitutive''']], [[Team:TU_Munich/Project/Ethanol_Inducible_Promoter|'''ethanol-inducible''']] and [[Team:TU_Munich/Project/Light_Switchable_Promoter|'''light-switchable''']] promoter systems were developed to individually regulate the expression of these gene cassettes. By combining these BioBricks our team has been able to brew iGEM’s first and finest [[Team:TU_Munich/Project/Brewing|'''SynBio Beer''']].<br />
<br />
<br />
== Vision ==<br />
----<br />
<div><br />
<br />
We, TU Munich’s 2012 iGEM team, strive to catalyze the diffusion process of knowledge about genetic engineering and synthetic biology among the general public. Using the example of iGEM’s first and finest SynBio Beer we involve, interest and inspire people to reconsider preconceived ideas and encourage them to openly engage in a broad discussion weighing pros and cons of genetic engineering in foodstuff. We sketch a future where new technology can be applied in a meaningful way to complement traditional foods or beverages.<br />
</div><br />
<br />
<html><iframe style="box-shadow: 1px 1px 2px rgba(0, 0, 0, 0.2);padding: 5px;margin: 5px;background-color: white;float:right;" src="http://player.vimeo.com/video/51804324?color=d92540" width="720" height="405" frameborder="0" webkitAllowFullScreen mozallowfullscreen allowFullScreen></iframe></html><br />
<br />
== Overview ==<br />
----<br />
<html><br />
<script src="https://2012.igem.org/Team:TU_Munich/jquery.li-scroller.1.0.js?action=raw&ctype=text/js"></script><br />
</html><br />
<center><br />
<div class="ui-corner-all" id="ticker"><br />
<ul id="ticker01" ><br />
<li><span><b>878</b> Documented experiments in our labjournal</span></li><br />
<li><span><b>923</b> Plasmids</span></li><br />
<li><span><b>49</b> Annotated BioBricks</span></li><br />
<li><span><b>107</b> Days in the lab</span></li><br />
<li><span><b>283</b> Sequencing orders</span></li><br />
<li><span><b>13</b> Subprojects</span></li><br />
<li><span><b>19</b> Students</span></li><br />
<li><span>Over <b>10.000.000</b> systems of differential equations solved</span></li><br />
<li><span><b>878</b> Documented experiments in our labjournal</span></li><br />
<li><span><b>923</b> Plasmids</span></li><br />
<li><span><b>49</b> Annotated BioBricks</span></li><br />
<li><span><b>107</b> Days in the lab</span></li><br />
<li><span><b>283</b> Sequencing orders</span></li><br />
<li><span><b>13</b> Subprojects</span></li><br />
<li><span><b>19</b> Students</span></li><br />
<li><span>Over <b>10.000.000</b> systems of differential equations solved</span></li><br />
<li><span><b>878</b> Documented experiments in our labjournal</span></li><br />
<li><span><b>923</b> Plasmids</span></li><br />
<li><span><b>49</b> Annotated BioBricks</span></li><br />
<li><span><b>107</b> Days in the lab</span></li><br />
<li><span><b>283</b> Sequencing orders</span></li><br />
<li><span><b>13</b> Subprojects</span></li><br />
<li><span><b>19</b> Students</span></li><br />
<li><span>Over <b>10.000.000</b> systems of differential equations solved</span></li><br />
</ul><br />
</div><br />
</center><br />
<html><br />
<script><br />
$(document).ready(function(){<br />
$('ul#ticker01').liScroll({travelvelocity: 0.03});<br />
});<br />
</script><br />
</html><br />
<br />
{{Team:TU_Munich/Overview}}<br />
<br />
== Reference ==<br />
----<br />
<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/6327079 Edens et al., 1984]] Edens, L., Bom, I., Ledeboer, A. M., Maat, J., Toonen, M. Y., Visser, C., and Verrips, C. T. (1984). Synthesis and processing of the plant protein thaumatin in yeast. ''Cell'', 37(2):629–33.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/10724480 Mazzoni et al., 2000]] Mazzoni, C., Santori, F., Saliola, M., and Falcone, C. (2000). Molecular analysis of uas(e), a cis element containing stress response elements responsible for ethanol induction of the kladh4 gene of ''Kluyveromyces lactis''. ''Res Microbiol'', 151(1):19–28.<br />
<br />
== Sponsors ==<br />
----<br />
<br />
<html><br />
<center class="noborder"><br />
<a href="http://www.applichem.com"><img src="https://static.igem.org/mediawiki/2012/f/f5/TUM_Applichem.jpg" width="200px"></a><br />
<a href="http://www.biomers.net"><img src="https://static.igem.org/mediawiki/2012/3/37/TUM_Biomers.png" width="200px"></a><br />
<a href="http://www.biozym.com"><img src="https://static.igem.org/mediawiki/2012/d/d8/TUM_Biozym.jpg" width="200px"></a><br />
<a href="http://www.cipsm.de"><img src="https://static.igem.org/mediawiki/2012/8/88/TUM_Cipsm.jpg" width="200px"></a><br />
<a href="http://www.eurofins.de"><img src="https://static.igem.org/mediawiki/2012/b/bc/TUM_Eurofins.png" width="200px"></a><br />
<a href="http://www.idt.com"><img src="https://static.igem.org/mediawiki/2012/0/0b/TUM_IDT.jpg" width="200px"></a><br />
<a href="http://www.metabion.com"><img src="https://static.igem.org/mediawiki/2012/9/93/TUM_Metabion.png" width="200px"></a><br />
<a href="http://www.geneious.com/"><img src="https://static.igem.org/mediawiki/2012/8/82/TUM_Geneious.png" width="200px"></a><br />
<a href="http://www.roche.de/"><img src="https://static.igem.org/mediawiki/2012/a/a4/TUM_Roche.jpg" width="200px"></a><br />
<a href="http://www.daad.de/promos/"><img src="https://static.igem.org/mediawiki/2012/7/77/TUM_DAAD.gif" width="200px"></a><br />
<a href="http://www.qiagen.com"><img src="https://static.igem.org/mediawiki/2012/3/39/TUM_Qiuagen.jpg" width="100px"></a><br />
<a href="http://www.carlroth.com"><img src="https://static.igem.org/mediawiki/2012/6/63/TUM_Roth.gif" width="100px"></a><br />
<a href="http://www.bund-der-freunde.tum.de/"><img src="https://static.igem.org/mediawiki/2012/1/1d/TUM_Bund.jpg" width="200px"></a><br />
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<center>http://www4.clustrmaps.com/stats/maps-no_clusters/2012.igem.org-Team-TU_Munich-thumb.jpg</center></div>JaraOhttp://2012.igem.org/Team:TU_MunichTeam:TU Munich2012-10-26T19:58:07Z<p>JaraO: /* Vision */</p>
<hr />
<div>{{Team:TU_Munich/Header}}<br />
__NOTOC__<br />
= TUM-Brew: iGEM's first and finest SynBio Beer =<br />
<hr/><br />
<br />
[[File:TUM12_AE120612001.jpg|300px||right||]]<br />
<br />
The TU Munich iGEM Team engineers ''Saccharomyces cerevisiae'', also known as baker's yeast, in order to lay the foundations for a new generation of functional foods with nutritionally valuable ingredients.<br />
<br />
As an example, for iGEM’s first “SynBio Beer” the compounds [[Team:TU_Munich/Project/Xanthohumol|'''xanthohumol''']] (anticancerogenic), [[Team:TU_Munich/Project/Limonene|'''limonene''']] (limeflavor), [[Team:TU_Munich/Project/Caffeine|'''caffeine''']] (CNS-stimulant) as well as the [[Team:TU_Munich/Project/Thaumatin|'''thaumatin''']] (protein sweetener) were chosen to demonstrate the spectrum of possibilities to complement traditional foods or beverages.<br />
<br />
The metabolic pathways for these substances were converted to genetic BioBricks. Using the shuttle vector [[Team:TU_Munich/Project/Vector_Design|'''pTUM100''']], which was adapted to the iGEM standard, transient transfection and expression in yeast were achieved. The gene products were subsequently characterized and their biosynthetic activities investigated.<br />
<br />
[[Team:TU_Munich/Project/Constitutive_Promoter|'''Constitutive''']], [[Team:TU_Munich/Project/Ethanol_Inducible_Promoter|'''ethanol-inducible''']] and [[Team:TU_Munich/Project/Light_Switchable_Promoter|'''light-switchable''']] promoter systems were developed to individually regulate the expression of these gene cassettes. By combining these BioBricks our team has been able to brew iGEM’s first and finest [[Team:TU_Munich/Project/Brewing|'''SynBio Beer''']].<br />
<br />
<br />
== Vision ==<br />
----<br />
<div style="float:left;width:400px;"><br />
<br />
We, TU Munich’s 2012 iGEM team, strive to catalyze the diffusion process of knowledge about genetic engineering and synthetic biology among the general public. Using the example of iGEM’s first and finest SynBio Beer we involve, interest and inspire people to reconsider preconceived ideas and encourage them to openly engage in a broad discussion weighing pros and cons of genetic engineering in foodstuff. We sketch a future where new technology can be applied in a meaningful way to complement traditional foods or beverages.<br />
</div><br />
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<html><iframe style="box-shadow: 1px 1px 2px rgba(0, 0, 0, 0.2);padding: 5px;margin: 5px;background-color: white;float:right;" src="http://player.vimeo.com/video/51804324?color=d92540" width="720" height="405" frameborder="0" webkitAllowFullScreen mozallowfullscreen allowFullScreen></iframe></html><br />
<br />
== Overview ==<br />
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<li><span><b>878</b> Documented experiments in our labjournal</span></li><br />
<li><span><b>923</b> Plasmids</span></li><br />
<li><span><b>49</b> Annotated BioBricks</span></li><br />
<li><span><b>107</b> Days in the lab</span></li><br />
<li><span><b>283</b> Sequencing orders</span></li><br />
<li><span><b>13</b> Subprojects</span></li><br />
<li><span><b>19</b> Students</span></li><br />
<li><span>Over <b>10.000.000</b> systems of differential equations solved</span></li><br />
<li><span><b>878</b> Documented experiments in our labjournal</span></li><br />
<li><span><b>923</b> Plasmids</span></li><br />
<li><span><b>49</b> Annotated BioBricks</span></li><br />
<li><span><b>107</b> Days in the lab</span></li><br />
<li><span><b>283</b> Sequencing orders</span></li><br />
<li><span><b>13</b> Subprojects</span></li><br />
<li><span><b>19</b> Students</span></li><br />
<li><span>Over <b>10.000.000</b> systems of differential equations solved</span></li><br />
<li><span><b>878</b> Documented experiments in our labjournal</span></li><br />
<li><span><b>923</b> Plasmids</span></li><br />
<li><span><b>49</b> Annotated BioBricks</span></li><br />
<li><span><b>107</b> Days in the lab</span></li><br />
<li><span><b>283</b> Sequencing orders</span></li><br />
<li><span><b>13</b> Subprojects</span></li><br />
<li><span><b>19</b> Students</span></li><br />
<li><span>Over <b>10.000.000</b> systems of differential equations solved</span></li><br />
</ul><br />
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{{Team:TU_Munich/Overview}}<br />
<br />
== Reference ==<br />
----<br />
<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/6327079 Edens et al., 1984]] Edens, L., Bom, I., Ledeboer, A. M., Maat, J., Toonen, M. Y., Visser, C., and Verrips, C. T. (1984). Synthesis and processing of the plant protein thaumatin in yeast. ''Cell'', 37(2):629–33.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/10724480 Mazzoni et al., 2000]] Mazzoni, C., Santori, F., Saliola, M., and Falcone, C. (2000). Molecular analysis of uas(e), a cis element containing stress response elements responsible for ethanol induction of the kladh4 gene of ''Kluyveromyces lactis''. ''Res Microbiol'', 151(1):19–28.<br />
<br />
== Sponsors ==<br />
----<br />
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<html><br />
<center class="noborder"><br />
<a href="http://www.applichem.com"><img src="https://static.igem.org/mediawiki/2012/f/f5/TUM_Applichem.jpg" width="200px"></a><br />
<a href="http://www.biomers.net"><img src="https://static.igem.org/mediawiki/2012/3/37/TUM_Biomers.png" width="200px"></a><br />
<a href="http://www.biozym.com"><img src="https://static.igem.org/mediawiki/2012/d/d8/TUM_Biozym.jpg" width="200px"></a><br />
<a href="http://www.cipsm.de"><img src="https://static.igem.org/mediawiki/2012/8/88/TUM_Cipsm.jpg" width="200px"></a><br />
<a href="http://www.eurofins.de"><img src="https://static.igem.org/mediawiki/2012/b/bc/TUM_Eurofins.png" width="200px"></a><br />
<a href="http://www.idt.com"><img src="https://static.igem.org/mediawiki/2012/0/0b/TUM_IDT.jpg" width="200px"></a><br />
<a href="http://www.metabion.com"><img src="https://static.igem.org/mediawiki/2012/9/93/TUM_Metabion.png" width="200px"></a><br />
<a href="http://www.geneious.com/"><img src="https://static.igem.org/mediawiki/2012/8/82/TUM_Geneious.png" width="200px"></a><br />
<a href="http://www.roche.de/"><img src="https://static.igem.org/mediawiki/2012/a/a4/TUM_Roche.jpg" width="200px"></a><br />
<a href="http://www.daad.de/promos/"><img src="https://static.igem.org/mediawiki/2012/7/77/TUM_DAAD.gif" width="200px"></a><br />
<a href="http://www.qiagen.com"><img src="https://static.igem.org/mediawiki/2012/3/39/TUM_Qiuagen.jpg" width="100px"></a><br />
<a href="http://www.carlroth.com"><img src="https://static.igem.org/mediawiki/2012/6/63/TUM_Roth.gif" width="100px"></a><br />
<a href="http://www.bund-der-freunde.tum.de/"><img src="https://static.igem.org/mediawiki/2012/1/1d/TUM_Bund.jpg" width="200px"></a><br />
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</center><br />
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<center>http://www4.clustrmaps.com/stats/maps-no_clusters/2012.igem.org-Team-TU_Munich-thumb.jpg</center></div>JaraOhttp://2012.igem.org/File:TUM12_Overall.pngFile:TUM12 Overall.png2012-10-26T19:48:39Z<p>JaraO: uploaded a new version of &quot;File:TUM12 Overall.png&quot;</p>
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<div></div>JaraOhttp://2012.igem.org/File:TUM12_Overall.pngFile:TUM12 Overall.png2012-10-26T19:47:52Z<p>JaraO: uploaded a new version of &quot;File:TUM12 Overall.png&quot;</p>
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<div></div>JaraOhttp://2012.igem.org/IGEM_PublicityIGEM Publicity2012-10-24T15:25:01Z<p>JaraO: </p>
<hr />
<div>__NOTOC__<br />
<br />
<br />
<div style="color:#aaa; padding-bottom:20px;">(Members of the press, please see the [https://igem.org/Press_Kit | iGEM Press Kit])</div><br />
<br />
If you would like to share an article that was written about iGEM or your iGEM team, please link to it on this page. If you have multiple articles featuring your team, link to them all individually!<br />
<br />
Post your official team name, the title of your article, where it was featured, and provide a link to it. <br />
<br />
''Example'':<br> <br />
'''Team iGEM Headquarters''': ''Title of article'', Nature, [link]<br />
<br />
<br />
<span style="color:#1e90ff; font-size:150%">'''blogs </span><span style="color:#3cb371; font-size:150%">covering iGEM 2012'''</span><br><br />
'''Peking2012 iGEM''': ''Peking iGEM'', [http://page.renren.com/601444914] <br><br />
'''UANL Mexico''': ''Bio! iGEM Collection'', Blogspot, [http://biospot10.blogspot.de/2012/02/bio-about-igem.html]<br><br />
'''iGEM Valencia 2012''': ''Synth(ethic) Biology'', Wordpress, [http://igemvalencia2012.wordpress.com/]<br><br />
'''TU_Munich''': ''TUM iGEM 2012'', [http://tum-igem2012.blog.de/]<br><br />
<br />
<br><br />
<span style="color:#1e90ff; font-size:150%">'''video/radio</span><span style="color:#3cb371; font-size:150%"> about iGEM 2012'''</span><br><br />
'''TU_Munich''': LIVE radio interview, Kortex: TUM-Projektteam iGEM 2012 - Bier und synthetische Biologie, M94.5, soundcloud, [http://soundcloud.com/m945/kortex-tum-projektteam-igem]<br />
<br><br />
'''TU_Munich''': ''TUM Brew'', [http://vimeo.com/51804324]<br />
<br><br />
'''Peking2012 iGEM''': ''Human Practices ---"Sowing Tomorrow Synthetic Biologists"'', [http://youtu.be/cd7m5POAwRY] <br><br />
'''Peking2012 iGEM''': ''Human Practices --- Series Lectures --- Intro to Synthetic Biology'', [http://youtu.be/BpmCMeBqZuI]<br />
<br><br />
'''Peking2012 iGEM''': ''Human Practices --- Series Lectures --- Research guideline of Synthetic Biology'', [http://youtu.be/gfNFW6LQ1SQ]<br />
<br><br />
'''Peking2012 iGEM''': ''Human Practices --- Series Lectures --- iGEM HS-Division in a Nutshell'', [http://youtu.be/R3g0zVi0DgI]<br />
<br><br />
'''University College London''': ''#gemFM'', radioshow Mixcloud / Soundcloud, [https://2012.igem.org/Team:University_College_London/gemFM] <br><br />
'''Bielefeld-Germany''': ''LIVE radio interview'', Radio Hertz, [http://www.radiohertz.de/beta-site/2012/07/02/impuls-am-04-juli-2012/]<br><br />
'''SDU-Denmark''': ''Forsker i fritiden'', TV2 Fyn, [http://www.tv2fyn.dk/article/370281?autoplay=1&video_id=54463]<br><br />
'''University College London''':''London 'bio-hackers' catching the eye of professionals'', BBC News TV, [http://www.bbc.co.uk/news/uk-england-london-19964886]<br /><br />
<br><br />
<span style="color:#1e90ff; font-size:150%">'''news articles</span><span style="color:#3cb371; font-size:150%"> about iGEM 2012'''</span><br />
<br><br />
'''Peking2012 iGEM''': ''Lecture "Life as Machines --- Rational Design for Artificial Biological Systems"'', Beijing Teenager Science and Technology Club, [http://www.scitech-youth.org.cn/club_news/dt/4676.html] <br><br />
'''iGEM Leicester''', ''Google Campus hosts future gene-iuses'', University of Leicester press office, [http://www2.le.ac.uk/news/blog/2012/august/google-campus-hosts-future-gene-iuses]<br><br />
'''Lyon-INSA iGEM''': ''Ready, Set, Go for OSLI 2012 iGEM Teams'', Osli, [http://www.osli.ca/storybank/39/59/Ready-Set-Go-for-OSLI-2012-iGEM-Teams]<br />
'''TU_Munich''': ''TUM Brew'', [http://vimeo.com/51804324]<br><br />
<br />
====<font size=4><font color=dodgerblue>'''general'''</font></font>====<br />
<br />
'''Peking2012 iGEM''': ''Designing the Scientific Cradle for Quantitative Biologists'', ACS Synth. Biol., [http://pubs.acs.org/doi/abs/10.1021/sb3000386]<br />
<br><br />
'''iGEM Teams Germany''', ''iGEM: Elf Teams tüfteln für die Biokonstrukteurs-WM'', biotechnologie.de, [http://www.biotechnologie.de/BIO/Navigation/DE/root,did=153792.html]<br><br />
'''iGEM Teams France''', ''Site iGEM France'', [https://sites.google.com/site/igemfrance/]<br><br />
<br />
'''Buenos Aires 2012 iGEM Team''', Synthetic Communities, <br />
[http://blogs.scientificamerican.com/lab-rat/2012/09/09/igem-buenos-aires-synthetic-bacterial-communities/]<br><br />
'''SDU-Denmark''': ''iGEM – Verdens største konkurrence indenfor syntetisk biologi vender tilbage til SDU'', Sund & Hed, [http://sundoghed.dk/7038/igem-verdens-storste-konkurrence-indenfor-syntetisk-biologi-vender-tilbage-til-sdu/?fb_comment_id=fbc_365648506787388_4738817_366895283329377]<br><br />
<br />
'''Waterloo 2012 iGEM Team''', Educational Podcast on SynBio by Virtual Researcher On Call (Canadian Educational Resource Organization) [http://sns.vroc.ca/p.jsp?i=17]<br><br />
<br />
====<font size=4><font color=dodgerblue>'''team specific'''</font></font>====<br />
'''Arizona_State''': ''Students create low-cost biosensor to detect contaminated water in developing nations'',[https://asunews.asu.edu/20120906_waterbiosensor]<br/><br />
'''Potsdam-Bioware''': ''Hamsterzellen als Antikörperfabrik, biotechnologie.de'',[http://www.biotechnologie.de/BIO/Navigation/DE/root,did=153816.html]<br/><br />
'''Valencia (UPV,UCV)''' : ''Seminar at the Institute of marine science (CSIC, Barcelona)'',[http://igemvalencia2012.wordpress.com/2012/09/11/seminar-in-barcelona/] <br/><br />
'''Valencia (UPV,UCV)''' : ''Interview with D. Justo Aznar, an expert in Bioethics, Wordpress'',[http://igemvalencia2012.wordpress.com/2012/08/29/interview-with-justo-aznar-lucena-m-d/] <br/><br />
'''Valencia (UPV,UCV)''' : ''What do you know about Synthetic Biology?, Wordpress'',[http://igemvalencia2012.wordpress.com/2012/09/07/what-do-you-know-about-synthetic-biology/] <br/><br />
'''TU Delft''' : ''Our team gets featured in the national news paper'',[https://static.igem.org/mediawiki/igem.org/c/c7/ZomerrepoNRC7aug.pdf] <br/><br />
'''TU Delft''' : '' Team members reaching out to the general public at The Night of Art and Science in Groningen to spread awareness on Synthetic Biology '', [http://www.youtube.com/watch?v=j9t7-Qnho7o&feature=share] <br/><br />
'''TU Delft''' : ''Licht aan de bacterie - Our team member Sietske explains how cool and fascinating Synthetic Biology is for LowLabs!! '', [http://www.youtube.com/watch?v=4q2Ol89ekz4&feature=youtu.be] <br/><br />
'''Valencia (UPV,UCV)''': ''Biohacking: Do it yourself!'', Wordpress, [http://igemvalencia2012.wordpress.com/2012/08/21/biohacking-do-it-yourself/] <br/><br />
'''Valencia (UPV,UCV)''': ''Where will synthetic biology lead us?'', Wordpress, [http://igemvalencia2012.wordpress.com/2012/08/13/a-life-of-its-own-where-will-synthetic-biology-lead-us-by-michael-specter/] <br/><br />
'''Valencia (UPV,UCV)''': ''iGEM: Why of all this'', Wordpress, [http://igemvalencia2012.wordpress.com/2012/08/14/igem-the-why-of-all-this/] <br/><br />
'''University of Leicester''': ''Synthetic biology solution to polystyrene degradation'', Blogger, [http://uoleicesterigem2012.blogspot.co.uk/] <br/><br />
'''University College London''': ''Land Grab: Could Bioremediation Turn Pacific Garbage Patch Into Habitable Island?'', Good, [http://www.good.is/post/land-grab-could-bioremediation-turn-pacific-garbage-patch-into-habitable-island/] <br/><br />
'''University College London''': ''Synthetic Bacteria Could Turn Ocean Garbage into One Big Island'', Smithsonian, [http://blogs.smithsonianmag.com/smartnews/2012/07/synthetic-bacteria-could-turn-ocean-garbage-into-one-big-island/] <br/><br />
'''University College London''': ''Students synthesizing bacteria to create islands out of garbage'', DVICE, [http://dvice.com/archives/2012/07/students-synthe.php] <br/><br />
'''University College London''': ''Nanobots could turn 'Great Pacific Plastic Patch' into a floating island'', Wired, [http://www.wired.co.uk/news/archive/2012-07/18/nanobots-recycling-plastic] <br/><br />
'''University College London''': ''Synthetic Biology Speed Debate'', C-LAB, [http://c-lab.co.uk/events/synthetic-biology-speed-debate.html] <br/><br />
'''University College London''': ''From Pixels To DNA: The Next Frontier In Interaction Design'', Co.Design, [http://www.fastcodesign.com/1670825/from-pixels-to-dna-the-next-frontier-in-interaction-design#1]<br /><br />
'''University College London''': ''Biohacking, iGEM and the limits of citizen science'', Po Ve Sham, [http://povesham.wordpress.com/2012/10/05/biohacking-igem-and-the-limits-of-citizen-science/]<br /><br />
'''University College London''': ''Right or Risk? World's First Public BioBrick'', C-LAB, [http://c-lab.co.uk/events/right-or-risk-worlds-first-public-biobrick.html]<br /><br />
'''Queen's University''': ''Getting Biological over the Break'', Kingston This Week, [http://www.kingstonthisweek.com/2012/06/21/getting-biological-over-the-break] <br/><br />
'''Queen's University''': ''Dance like everybody's watching'', Kingston EMC, [http://www.emckingston.com/20120809/lifestyle/Dance+like+everybody%27s+watching] <br/><br />
'''Queen's University''': ''Dancing in the Lab'', The Queen's Journal, [http://queensjournal.ca/story/2012-09-11/news/dancing-lab/] <br/><br />
'''NRP UEA Norwich''': ''UEA Students Compete For Top Biology Prize'', UEA Press Release, [http://www.uea.ac.uk/mac/comm/media/press/2012/August/igem-competition-uea]<br><br />
'''NRP UEA Norwich''': ''Interview With Students From The UEA Talking About Synthetic Biology (Show 23)'', The Farming Show: STAR Radio, [http://www.star107.co.uk/farming-show.php]<br><br />
'''University of St Andrews''': "Streets of London: paved with platinum" [http://www.st-andrews.ac.uk/news/archive/2012/Title,89409,en.html] <br><br />
'''TU_Munich''': ''Technische Universität München: Brauhefe für leckeres und gesundes Bier'', biotechnologie.de, [http://www.biotechnologie.de/BIO/Navigation/DE/root,did=153818.html]<br><br />
'''TU_Munich''': ''TUMstudis: baker's yeast is synthetically converted'', TUMstudinews, [http://portal.mytum.de/ccc/newsletter/students/2012_03/02]<br><br />
'''TU_Munich''': ''Die Wunderhefe'', jetzt.de sueddeutsche zeitung, [http://jetzt.sueddeutsche.de/texte/anzeigen/544866/4/1#texttitel]<br><br />
'''TU_Munich''': ''Widerstand gegen die "Grüne Gentechnik" wächst weiter'', merkur, [http://www.merkur-online.de/lokales/freising/widerstand-gegen-gruene-gentechnik-waechst-weiter-2510648.html]<br><br />
'''TU_Munich''': ''iGEM: Fünf Teams lösen Ticket für Boston'', biotechnologie.de, [http://www.biotechnologie.de/BIO/Navigation/DE/root,did=154774.html]<br><br />
'''Tübingen''': ''Tübinger Studenten nehmen an internationalem Forscherwettbewerb teil'', Schwäbisches Tagblatt, [http://www.tagblatt.de/Home/nachrichten/hochschule_artikel,-Tuebinger-Studenten-nehmen-an-internationalem-Forscherwettbewerb-teil-_arid,184906.html]<br><br />
'''Bielefeld-Germany''': ''Studierende der Universität Bielefeld nehmen am iGEM-Wettbewerb teil'', Universität Bielefeld, [http://ekvv.uni-bielefeld.de/blog/pressemitteilungen/entry/östrogen_aus_trinkwasser_entfernen_nr]<br><br />
'''Bielefeld-Germany''': ''Östrogen aus Trinkwasser entfernen'', Press Release, [http://www.uni-protokolle.de/nachrichten/id/240229/][http://www.laborpraxis.vogel.de/forschung-und-entwicklung/analytik/articles/369125/][http://www.bio.nrw.de/nachrichten][http://idw-online.de/pages/de/news484982][http://www.eurekalert.org/pub_releases/2012-06/uob-ref062512.php]<br><br />
'''Bielefeld-Germany''': ''Turkey tail tree fungus could filter oestrogen from water'', Wired, [http://www.wired.co.uk/news/archive/2012-06/25/tree-fungus-oestrogen-filter]<br><br />
'''Bielefeld-Germany''': ''Biological filter to remove estrogens from waste water and drinking water'', News Medical Australia, [http://www.news-medical.net/news/20120626/Biological-filter-to-remove-estrogens-from-waste-water-and-drinking-water.aspx]<br><br />
'''Bielefeld-Germany''': ''Das Wasser wird weiblich'', Neue Westfälische, [https://2012.igem.org/Team:Bielefeld-Germany/Homeleft]<br><br />
'''Bielefeld-Germany''': ''Arzneimittel in Gewässern minimieren'', Neue Züricher Zeitung, [http://www.nzz.ch/wissen/wissenschaft/arzneimittel-in-gewaessern-minimieren-1.17368069]<br><br />
'''USP-UNESP-Brazil''': ''Brazilian Science Dreams Come Alive'', RocketHub Blog, [http://blog.rockethub.com/brazilian-science-dreams-come-alive]<br><br />
'''USP-UNESP-Brazil''': ''USP participa pela 1ª vez de campeonato de biologia sintética'', Jornal do Campus, [http://www.jornaldocampus.usp.br/index.php/2012/06/usp-participa-pela-1a-vez-de-campeonato-de-biologia-sintetica/]<br><br />
'''USP-UNESP-Brazil''': ''Cientistas da USP conseguem financiar projeto com vaquinha virtual'', Folha de S.Paulo, [http://www1.folha.uol.com.br/ciencia/1097202-cientistas-da-usp-conseguem-financiar-projeto-com-vaquinha-virtual.shtml]<br><br />
'''EPF-Lausanne''': ''Fabriquer des médicaments en allumant la lumière?'' (French), Flash EPFL, [http://actualites.epfl.ch/newspaper-edition?np_id=151] [https://static.igem.org/mediawiki/2012/4/42/Team-EPF-Lausanne_Flash_article_about_us.pdf]<br><br />
'''Lyon INSA iGEM''' : ''L’INSA de Lyon vise l’or à Amsterdam'' , En vue INSA Lyon, [http://envue.insa-lyon.fr/2012avril/art2a_0412.php]<br><br />
'''Trieste''': ''Human practices - SynBio for everyone (Italian)'', Il Tascapane, [http://tascapane.blogspot.se/2012/08/la-biologia-sintetica.html]<br><br />
'''Trieste''': ''Human practices - SynBio and medicine (Italian)'', Il Tascapane,[http://tascapane.blogspot.it/2012/09/la-biologia-sintetica-approda-in.html]<br><br />
'''Trieste''': '' Un'idea per proteggere l'intestino (Italian)'', ICGEB, [http://www.icgeb.org/notizie-in-italiano/items/squadra-igem-2012-un-idee-per-proteggere-lintestino.html]<br><br />
'''Lyon-INSA''':''M. concourt au titre mondial de biologie de synthèse'', Républicain Lorrain [https://docs.google.com/file/d/0BzhWvySCD_KzWmJTYjBYWHBCUnM/edit]<br><br />
'''Lyon-INSA''' : ''L'Insa de Lyon vise l'or à Amsterdam'', Insa de Lyon [http://www.insa-lyon.fr/fr/concours-igem-2012]<br><br />
'''UANL_Mty-Mexico''': ''Túnel de la Biología Sintética (Synthetic Biology Tunnel), Televisa'', Monterrey,[http://www.televisaregional.com/monterrey/video/169491536.html]<br/><br />
'''Wageningen_UR''': ''The Constructor: a web application optimizing cloning strategies based on modules from the registry of standard biological parts'', Journal of Biological Engineering,[http://www.jbioleng.org/content/6/1/14/abstract]<br/><br />
'''SDU-Denmark''': ''Fighting Obesity at the SDU in Odense'', SIJ, by Eliana van de Craats, [http://community.ejc.net/forum/topic/show?id=2345725%3ATopic%3A89026&xgs=1&xg_source=msg_share_topic]<br/><br />
'''Georgia_Tech''': ''<br />
Georgia Tech workshop well received: Lambert students embrace engineering'', ForsythNews.com, [http://www.forsythnews.com/m/section/3/article/14721/. ]<br/><br />
'''Cornell''': ''Using electroactive bacteria, students design toxin sensor'', Cornell Chronicle, [http://www.news.cornell.edu/stories/Oct12/oilSands.html]</div>JaraOhttp://2012.igem.org/IGEM_PublicityIGEM Publicity2012-10-24T15:23:22Z<p>JaraO: </p>
<hr />
<div>__NOTOC__<br />
<br />
<br />
<div style="color:#aaa; padding-bottom:20px;">(Members of the press, please see the [https://igem.org/Press_Kit | iGEM Press Kit])</div><br />
<br />
If you would like to share an article that was written about iGEM or your iGEM team, please link to it on this page. If you have multiple articles featuring your team, link to them all individually!<br />
<br />
Post your official team name, the title of your article, where it was featured, and provide a link to it. <br />
<br />
''Example'':<br> <br />
'''Team iGEM Headquarters''': ''Title of article'', Nature, [link]<br />
<br />
<br />
<span style="color:#1e90ff; font-size:150%">'''blogs </span><span style="color:#3cb371; font-size:150%">covering iGEM 2012'''</span><br><br />
'''Peking2012 iGEM''': ''Peking iGEM'', [http://page.renren.com/601444914] <br><br />
'''UANL Mexico''': ''Bio! iGEM Collection'', Blogspot, [http://biospot10.blogspot.de/2012/02/bio-about-igem.html]<br><br />
'''iGEM Valencia 2012''': ''Synth(ethic) Biology'', Wordpress, [http://igemvalencia2012.wordpress.com/]<br><br />
'''TU_Munich''': ''TUM iGEM 2012'', [http://tum-igem2012.blog.de/]<br><br />
<br />
<br><br />
<span style="color:#1e90ff; font-size:150%">'''video/radio</span><span style="color:#3cb371; font-size:150%"> about iGEM 2012'''</span><br><br />
'''TU_Munich''': LIVE radio interview, Kortex: TUM-Projektteam iGEM 2012 - Bier und synthetische Biologie, M94.5, soundcloud, [http://soundcloud.com/m945/kortex-tum-projektteam-igem]<br />
<br><br />
'''Peking2012 iGEM''': ''Human Practices ---"Sowing Tomorrow Synthetic Biologists"'', [http://youtu.be/cd7m5POAwRY] <br><br />
'''Peking2012 iGEM''': ''Human Practices --- Series Lectures --- Intro to Synthetic Biology'', [http://youtu.be/BpmCMeBqZuI]<br />
<br><br />
'''Peking2012 iGEM''': ''Human Practices --- Series Lectures --- Research guideline of Synthetic Biology'', [http://youtu.be/gfNFW6LQ1SQ]<br />
<br><br />
'''Peking2012 iGEM''': ''Human Practices --- Series Lectures --- iGEM HS-Division in a Nutshell'', [http://youtu.be/R3g0zVi0DgI]<br />
<br><br />
'''University College London''': ''#gemFM'', radioshow Mixcloud / Soundcloud, [https://2012.igem.org/Team:University_College_London/gemFM] <br><br />
'''Bielefeld-Germany''': ''LIVE radio interview'', Radio Hertz, [http://www.radiohertz.de/beta-site/2012/07/02/impuls-am-04-juli-2012/]<br><br />
'''SDU-Denmark''': ''Forsker i fritiden'', TV2 Fyn, [http://www.tv2fyn.dk/article/370281?autoplay=1&video_id=54463]<br><br />
'''University College London''':''London 'bio-hackers' catching the eye of professionals'', BBC News TV, [http://www.bbc.co.uk/news/uk-england-london-19964886]<br /><br />
<br><br />
<span style="color:#1e90ff; font-size:150%">'''news articles</span><span style="color:#3cb371; font-size:150%"> about iGEM 2012'''</span><br />
<br><br />
'''Peking2012 iGEM''': ''Lecture "Life as Machines --- Rational Design for Artificial Biological Systems"'', Beijing Teenager Science and Technology Club, [http://www.scitech-youth.org.cn/club_news/dt/4676.html] <br><br />
'''iGEM Leicester''', ''Google Campus hosts future gene-iuses'', University of Leicester press office, [http://www2.le.ac.uk/news/blog/2012/august/google-campus-hosts-future-gene-iuses]<br><br />
'''Lyon-INSA iGEM''': ''Ready, Set, Go for OSLI 2012 iGEM Teams'', Osli, [http://www.osli.ca/storybank/39/59/Ready-Set-Go-for-OSLI-2012-iGEM-Teams]<br />
'''TU_Munich''': ''TUM Brew'', [http://vimeo.com/51804324]<br><br />
<br />
====<font size=4><font color=dodgerblue>'''general'''</font></font>====<br />
<br />
'''Peking2012 iGEM''': ''Designing the Scientific Cradle for Quantitative Biologists'', ACS Synth. Biol., [http://pubs.acs.org/doi/abs/10.1021/sb3000386]<br />
<br><br />
'''iGEM Teams Germany''', ''iGEM: Elf Teams tüfteln für die Biokonstrukteurs-WM'', biotechnologie.de, [http://www.biotechnologie.de/BIO/Navigation/DE/root,did=153792.html]<br><br />
'''iGEM Teams France''', ''Site iGEM France'', [https://sites.google.com/site/igemfrance/]<br><br />
<br />
'''Buenos Aires 2012 iGEM Team''', Synthetic Communities, <br />
[http://blogs.scientificamerican.com/lab-rat/2012/09/09/igem-buenos-aires-synthetic-bacterial-communities/]<br><br />
'''SDU-Denmark''': ''iGEM – Verdens største konkurrence indenfor syntetisk biologi vender tilbage til SDU'', Sund & Hed, [http://sundoghed.dk/7038/igem-verdens-storste-konkurrence-indenfor-syntetisk-biologi-vender-tilbage-til-sdu/?fb_comment_id=fbc_365648506787388_4738817_366895283329377]<br><br />
<br />
'''Waterloo 2012 iGEM Team''', Educational Podcast on SynBio by Virtual Researcher On Call (Canadian Educational Resource Organization) [http://sns.vroc.ca/p.jsp?i=17]<br><br />
<br />
====<font size=4><font color=dodgerblue>'''team specific'''</font></font>====<br />
'''Arizona_State''': ''Students create low-cost biosensor to detect contaminated water in developing nations'',[https://asunews.asu.edu/20120906_waterbiosensor]<br/><br />
'''Potsdam-Bioware''': ''Hamsterzellen als Antikörperfabrik, biotechnologie.de'',[http://www.biotechnologie.de/BIO/Navigation/DE/root,did=153816.html]<br/><br />
'''Valencia (UPV,UCV)''' : ''Seminar at the Institute of marine science (CSIC, Barcelona)'',[http://igemvalencia2012.wordpress.com/2012/09/11/seminar-in-barcelona/] <br/><br />
'''Valencia (UPV,UCV)''' : ''Interview with D. Justo Aznar, an expert in Bioethics, Wordpress'',[http://igemvalencia2012.wordpress.com/2012/08/29/interview-with-justo-aznar-lucena-m-d/] <br/><br />
'''Valencia (UPV,UCV)''' : ''What do you know about Synthetic Biology?, Wordpress'',[http://igemvalencia2012.wordpress.com/2012/09/07/what-do-you-know-about-synthetic-biology/] <br/><br />
'''TU Delft''' : ''Our team gets featured in the national news paper'',[https://static.igem.org/mediawiki/igem.org/c/c7/ZomerrepoNRC7aug.pdf] <br/><br />
'''TU Delft''' : '' Team members reaching out to the general public at The Night of Art and Science in Groningen to spread awareness on Synthetic Biology '', [http://www.youtube.com/watch?v=j9t7-Qnho7o&feature=share] <br/><br />
'''TU Delft''' : ''Licht aan de bacterie - Our team member Sietske explains how cool and fascinating Synthetic Biology is for LowLabs!! '', [http://www.youtube.com/watch?v=4q2Ol89ekz4&feature=youtu.be] <br/><br />
'''Valencia (UPV,UCV)''': ''Biohacking: Do it yourself!'', Wordpress, [http://igemvalencia2012.wordpress.com/2012/08/21/biohacking-do-it-yourself/] <br/><br />
'''Valencia (UPV,UCV)''': ''Where will synthetic biology lead us?'', Wordpress, [http://igemvalencia2012.wordpress.com/2012/08/13/a-life-of-its-own-where-will-synthetic-biology-lead-us-by-michael-specter/] <br/><br />
'''Valencia (UPV,UCV)''': ''iGEM: Why of all this'', Wordpress, [http://igemvalencia2012.wordpress.com/2012/08/14/igem-the-why-of-all-this/] <br/><br />
'''University of Leicester''': ''Synthetic biology solution to polystyrene degradation'', Blogger, [http://uoleicesterigem2012.blogspot.co.uk/] <br/><br />
'''University College London''': ''Land Grab: Could Bioremediation Turn Pacific Garbage Patch Into Habitable Island?'', Good, [http://www.good.is/post/land-grab-could-bioremediation-turn-pacific-garbage-patch-into-habitable-island/] <br/><br />
'''University College London''': ''Synthetic Bacteria Could Turn Ocean Garbage into One Big Island'', Smithsonian, [http://blogs.smithsonianmag.com/smartnews/2012/07/synthetic-bacteria-could-turn-ocean-garbage-into-one-big-island/] <br/><br />
'''University College London''': ''Students synthesizing bacteria to create islands out of garbage'', DVICE, [http://dvice.com/archives/2012/07/students-synthe.php] <br/><br />
'''University College London''': ''Nanobots could turn 'Great Pacific Plastic Patch' into a floating island'', Wired, [http://www.wired.co.uk/news/archive/2012-07/18/nanobots-recycling-plastic] <br/><br />
'''University College London''': ''Synthetic Biology Speed Debate'', C-LAB, [http://c-lab.co.uk/events/synthetic-biology-speed-debate.html] <br/><br />
'''University College London''': ''From Pixels To DNA: The Next Frontier In Interaction Design'', Co.Design, [http://www.fastcodesign.com/1670825/from-pixels-to-dna-the-next-frontier-in-interaction-design#1]<br /><br />
'''University College London''': ''Biohacking, iGEM and the limits of citizen science'', Po Ve Sham, [http://povesham.wordpress.com/2012/10/05/biohacking-igem-and-the-limits-of-citizen-science/]<br /><br />
'''University College London''': ''Right or Risk? World's First Public BioBrick'', C-LAB, [http://c-lab.co.uk/events/right-or-risk-worlds-first-public-biobrick.html]<br /><br />
'''Queen's University''': ''Getting Biological over the Break'', Kingston This Week, [http://www.kingstonthisweek.com/2012/06/21/getting-biological-over-the-break] <br/><br />
'''Queen's University''': ''Dance like everybody's watching'', Kingston EMC, [http://www.emckingston.com/20120809/lifestyle/Dance+like+everybody%27s+watching] <br/><br />
'''Queen's University''': ''Dancing in the Lab'', The Queen's Journal, [http://queensjournal.ca/story/2012-09-11/news/dancing-lab/] <br/><br />
'''NRP UEA Norwich''': ''UEA Students Compete For Top Biology Prize'', UEA Press Release, [http://www.uea.ac.uk/mac/comm/media/press/2012/August/igem-competition-uea]<br><br />
'''NRP UEA Norwich''': ''Interview With Students From The UEA Talking About Synthetic Biology (Show 23)'', The Farming Show: STAR Radio, [http://www.star107.co.uk/farming-show.php]<br><br />
'''University of St Andrews''': "Streets of London: paved with platinum" [http://www.st-andrews.ac.uk/news/archive/2012/Title,89409,en.html] <br><br />
'''TU_Munich''': ''Technische Universität München: Brauhefe für leckeres und gesundes Bier'', biotechnologie.de, [http://www.biotechnologie.de/BIO/Navigation/DE/root,did=153818.html]<br><br />
'''TU_Munich''': ''TUMstudis: baker's yeast is synthetically converted'', TUMstudinews, [http://portal.mytum.de/ccc/newsletter/students/2012_03/02]<br><br />
'''TU_Munich''': ''Die Wunderhefe'', jetzt.de sueddeutsche zeitung, [http://jetzt.sueddeutsche.de/texte/anzeigen/544866/4/1#texttitel]<br><br />
'''TU_Munich''': ''Widerstand gegen die "Grüne Gentechnik" wächst weiter'', merkur, [http://www.merkur-online.de/lokales/freising/widerstand-gegen-gruene-gentechnik-waechst-weiter-2510648.html]<br><br />
'''TU_Munich''': ''iGEM: Fünf Teams lösen Ticket für Boston'', biotechnologie.de, [http://www.biotechnologie.de/BIO/Navigation/DE/root,did=154774.html]<br><br />
'''Tübingen''': ''Tübinger Studenten nehmen an internationalem Forscherwettbewerb teil'', Schwäbisches Tagblatt, [http://www.tagblatt.de/Home/nachrichten/hochschule_artikel,-Tuebinger-Studenten-nehmen-an-internationalem-Forscherwettbewerb-teil-_arid,184906.html]<br><br />
'''Bielefeld-Germany''': ''Studierende der Universität Bielefeld nehmen am iGEM-Wettbewerb teil'', Universität Bielefeld, [http://ekvv.uni-bielefeld.de/blog/pressemitteilungen/entry/östrogen_aus_trinkwasser_entfernen_nr]<br><br />
'''Bielefeld-Germany''': ''Östrogen aus Trinkwasser entfernen'', Press Release, [http://www.uni-protokolle.de/nachrichten/id/240229/][http://www.laborpraxis.vogel.de/forschung-und-entwicklung/analytik/articles/369125/][http://www.bio.nrw.de/nachrichten][http://idw-online.de/pages/de/news484982][http://www.eurekalert.org/pub_releases/2012-06/uob-ref062512.php]<br><br />
'''Bielefeld-Germany''': ''Turkey tail tree fungus could filter oestrogen from water'', Wired, [http://www.wired.co.uk/news/archive/2012-06/25/tree-fungus-oestrogen-filter]<br><br />
'''Bielefeld-Germany''': ''Biological filter to remove estrogens from waste water and drinking water'', News Medical Australia, [http://www.news-medical.net/news/20120626/Biological-filter-to-remove-estrogens-from-waste-water-and-drinking-water.aspx]<br><br />
'''Bielefeld-Germany''': ''Das Wasser wird weiblich'', Neue Westfälische, [https://2012.igem.org/Team:Bielefeld-Germany/Homeleft]<br><br />
'''Bielefeld-Germany''': ''Arzneimittel in Gewässern minimieren'', Neue Züricher Zeitung, [http://www.nzz.ch/wissen/wissenschaft/arzneimittel-in-gewaessern-minimieren-1.17368069]<br><br />
'''USP-UNESP-Brazil''': ''Brazilian Science Dreams Come Alive'', RocketHub Blog, [http://blog.rockethub.com/brazilian-science-dreams-come-alive]<br><br />
'''USP-UNESP-Brazil''': ''USP participa pela 1ª vez de campeonato de biologia sintética'', Jornal do Campus, [http://www.jornaldocampus.usp.br/index.php/2012/06/usp-participa-pela-1a-vez-de-campeonato-de-biologia-sintetica/]<br><br />
'''USP-UNESP-Brazil''': ''Cientistas da USP conseguem financiar projeto com vaquinha virtual'', Folha de S.Paulo, [http://www1.folha.uol.com.br/ciencia/1097202-cientistas-da-usp-conseguem-financiar-projeto-com-vaquinha-virtual.shtml]<br><br />
'''EPF-Lausanne''': ''Fabriquer des médicaments en allumant la lumière?'' (French), Flash EPFL, [http://actualites.epfl.ch/newspaper-edition?np_id=151] [https://static.igem.org/mediawiki/2012/4/42/Team-EPF-Lausanne_Flash_article_about_us.pdf]<br><br />
'''Lyon INSA iGEM''' : ''L’INSA de Lyon vise l’or à Amsterdam'' , En vue INSA Lyon, [http://envue.insa-lyon.fr/2012avril/art2a_0412.php]<br><br />
'''Trieste''': ''Human practices - SynBio for everyone (Italian)'', Il Tascapane, [http://tascapane.blogspot.se/2012/08/la-biologia-sintetica.html]<br><br />
'''Trieste''': ''Human practices - SynBio and medicine (Italian)'', Il Tascapane,[http://tascapane.blogspot.it/2012/09/la-biologia-sintetica-approda-in.html]<br><br />
'''Trieste''': '' Un'idea per proteggere l'intestino (Italian)'', ICGEB, [http://www.icgeb.org/notizie-in-italiano/items/squadra-igem-2012-un-idee-per-proteggere-lintestino.html]<br><br />
'''Lyon-INSA''':''M. concourt au titre mondial de biologie de synthèse'', Républicain Lorrain [https://docs.google.com/file/d/0BzhWvySCD_KzWmJTYjBYWHBCUnM/edit]<br><br />
'''Lyon-INSA''' : ''L'Insa de Lyon vise l'or à Amsterdam'', Insa de Lyon [http://www.insa-lyon.fr/fr/concours-igem-2012]<br><br />
'''UANL_Mty-Mexico''': ''Túnel de la Biología Sintética (Synthetic Biology Tunnel), Televisa'', Monterrey,[http://www.televisaregional.com/monterrey/video/169491536.html]<br/><br />
'''Wageningen_UR''': ''The Constructor: a web application optimizing cloning strategies based on modules from the registry of standard biological parts'', Journal of Biological Engineering,[http://www.jbioleng.org/content/6/1/14/abstract]<br/><br />
'''SDU-Denmark''': ''Fighting Obesity at the SDU in Odense'', SIJ, by Eliana van de Craats, [http://community.ejc.net/forum/topic/show?id=2345725%3ATopic%3A89026&xgs=1&xg_source=msg_share_topic]<br/><br />
'''Georgia_Tech''': ''<br />
Georgia Tech workshop well received: Lambert students embrace engineering'', ForsythNews.com, [http://www.forsythnews.com/m/section/3/article/14721/. ]<br/><br />
'''Cornell''': ''Using electroactive bacteria, students design toxin sensor'', Cornell Chronicle, [http://www.news.cornell.edu/stories/Oct12/oilSands.html]</div>JaraOhttp://2012.igem.org/IGEM_PublicityIGEM Publicity2012-10-24T15:21:09Z<p>JaraO: /* team specific */</p>
<hr />
<div>__NOTOC__<br />
<br />
<br />
<div style="color:#aaa; padding-bottom:20px;">(Members of the press, please see the [https://igem.org/Press_Kit | iGEM Press Kit])</div><br />
<br />
If you would like to share an article that was written about iGEM or your iGEM team, please link to it on this page. If you have multiple articles featuring your team, link to them all individually!<br />
<br />
Post your official team name, the title of your article, where it was featured, and provide a link to it. <br />
<br />
''Example'':<br> <br />
'''Team iGEM Headquarters''': ''Title of article'', Nature, [link]<br />
<br />
<br />
<span style="color:#1e90ff; font-size:150%">'''blogs </span><span style="color:#3cb371; font-size:150%">covering iGEM 2012'''</span><br><br />
'''Peking2012 iGEM''': ''Peking iGEM'', [http://page.renren.com/601444914] <br><br />
'''UANL Mexico''': ''Bio! iGEM Collection'', Blogspot, [http://biospot10.blogspot.de/2012/02/bio-about-igem.html]<br><br />
'''iGEM Valencia 2012''': ''Synth(ethic) Biology'', Wordpress, [http://igemvalencia2012.wordpress.com/]<br><br />
'''TU_Munich''': ''TUM iGEM 2012'', [http://tum-igem2012.blog.de/]<br><br />
<br />
<br><br />
<span style="color:#1e90ff; font-size:150%">'''video/radio</span><span style="color:#3cb371; font-size:150%"> about iGEM 2012'''</span><br><br />
'''TU_Munich''': LIVE radio interview, Kortex: TUM-Projektteam iGEM 2012 - Bier und synthetische Biologie, M94.5, soundcloud, [http://soundcloud.com/m945/kortex-tum-projektteam-igem]<br />
<br><br />
'''Peking2012 iGEM''': ''Human Practices ---"Sowing Tomorrow Synthetic Biologists"'', [http://youtu.be/cd7m5POAwRY] <br><br />
'''Peking2012 iGEM''': ''Human Practices --- Series Lectures --- Intro to Synthetic Biology'', [http://youtu.be/BpmCMeBqZuI]<br />
<br><br />
'''Peking2012 iGEM''': ''Human Practices --- Series Lectures --- Research guideline of Synthetic Biology'', [http://youtu.be/gfNFW6LQ1SQ]<br />
<br><br />
'''Peking2012 iGEM''': ''Human Practices --- Series Lectures --- iGEM HS-Division in a Nutshell'', [http://youtu.be/R3g0zVi0DgI]<br />
<br><br />
'''University College London''': ''#gemFM'', radioshow Mixcloud / Soundcloud, [https://2012.igem.org/Team:University_College_London/gemFM] <br><br />
'''Bielefeld-Germany''': ''LIVE radio interview'', Radio Hertz, [http://www.radiohertz.de/beta-site/2012/07/02/impuls-am-04-juli-2012/]<br><br />
'''SDU-Denmark''': ''Forsker i fritiden'', TV2 Fyn, [http://www.tv2fyn.dk/article/370281?autoplay=1&video_id=54463]<br><br />
'''University College London''':''London 'bio-hackers' catching the eye of professionals'', BBC News TV, [http://www.bbc.co.uk/news/uk-england-london-19964886]<br /><br />
<br><br />
<span style="color:#1e90ff; font-size:150%">'''news articles</span><span style="color:#3cb371; font-size:150%"> about iGEM 2012'''</span><br />
<br><br />
'''Peking2012 iGEM''': ''Lecture "Life as Machines --- Rational Design for Artificial Biological Systems"'', Beijing Teenager Science and Technology Club, [http://www.scitech-youth.org.cn/club_news/dt/4676.html] <br><br />
'''iGEM Leicester''', ''Google Campus hosts future gene-iuses'', University of Leicester press office, [http://www2.le.ac.uk/news/blog/2012/august/google-campus-hosts-future-gene-iuses]<br><br />
'''Lyon-INSA iGEM''': ''Ready, Set, Go for OSLI 2012 iGEM Teams'', Osli, [http://www.osli.ca/storybank/39/59/Ready-Set-Go-for-OSLI-2012-iGEM-Teams]<br />
<br />
====<font size=4><font color=dodgerblue>'''general'''</font></font>====<br />
<br />
'''Peking2012 iGEM''': ''Designing the Scientific Cradle for Quantitative Biologists'', ACS Synth. Biol., [http://pubs.acs.org/doi/abs/10.1021/sb3000386]<br />
<br><br />
'''iGEM Teams Germany''', ''iGEM: Elf Teams tüfteln für die Biokonstrukteurs-WM'', biotechnologie.de, [http://www.biotechnologie.de/BIO/Navigation/DE/root,did=153792.html]<br><br />
'''iGEM Teams France''', ''Site iGEM France'', [https://sites.google.com/site/igemfrance/]<br><br />
<br />
'''Buenos Aires 2012 iGEM Team''', Synthetic Communities, <br />
[http://blogs.scientificamerican.com/lab-rat/2012/09/09/igem-buenos-aires-synthetic-bacterial-communities/]<br><br />
'''SDU-Denmark''': ''iGEM – Verdens største konkurrence indenfor syntetisk biologi vender tilbage til SDU'', Sund & Hed, [http://sundoghed.dk/7038/igem-verdens-storste-konkurrence-indenfor-syntetisk-biologi-vender-tilbage-til-sdu/?fb_comment_id=fbc_365648506787388_4738817_366895283329377]<br><br />
<br />
'''Waterloo 2012 iGEM Team''', Educational Podcast on SynBio by Virtual Researcher On Call (Canadian Educational Resource Organization) [http://sns.vroc.ca/p.jsp?i=17]<br><br />
<br />
====<font size=4><font color=dodgerblue>'''team specific'''</font></font>====<br />
'''Arizona_State''': ''Students create low-cost biosensor to detect contaminated water in developing nations'',[https://asunews.asu.edu/20120906_waterbiosensor]<br/><br />
'''Potsdam-Bioware''': ''Hamsterzellen als Antikörperfabrik, biotechnologie.de'',[http://www.biotechnologie.de/BIO/Navigation/DE/root,did=153816.html]<br/><br />
'''Valencia (UPV,UCV)''' : ''Seminar at the Institute of marine science (CSIC, Barcelona)'',[http://igemvalencia2012.wordpress.com/2012/09/11/seminar-in-barcelona/] <br/><br />
'''Valencia (UPV,UCV)''' : ''Interview with D. Justo Aznar, an expert in Bioethics, Wordpress'',[http://igemvalencia2012.wordpress.com/2012/08/29/interview-with-justo-aznar-lucena-m-d/] <br/><br />
'''Valencia (UPV,UCV)''' : ''What do you know about Synthetic Biology?, Wordpress'',[http://igemvalencia2012.wordpress.com/2012/09/07/what-do-you-know-about-synthetic-biology/] <br/><br />
'''TU Delft''' : ''Our team gets featured in the national news paper'',[https://static.igem.org/mediawiki/igem.org/c/c7/ZomerrepoNRC7aug.pdf] <br/><br />
'''TU Delft''' : '' Team members reaching out to the general public at The Night of Art and Science in Groningen to spread awareness on Synthetic Biology '', [http://www.youtube.com/watch?v=j9t7-Qnho7o&feature=share] <br/><br />
'''TU Delft''' : ''Licht aan de bacterie - Our team member Sietske explains how cool and fascinating Synthetic Biology is for LowLabs!! '', [http://www.youtube.com/watch?v=4q2Ol89ekz4&feature=youtu.be] <br/><br />
'''Valencia (UPV,UCV)''': ''Biohacking: Do it yourself!'', Wordpress, [http://igemvalencia2012.wordpress.com/2012/08/21/biohacking-do-it-yourself/] <br/><br />
'''Valencia (UPV,UCV)''': ''Where will synthetic biology lead us?'', Wordpress, [http://igemvalencia2012.wordpress.com/2012/08/13/a-life-of-its-own-where-will-synthetic-biology-lead-us-by-michael-specter/] <br/><br />
'''Valencia (UPV,UCV)''': ''iGEM: Why of all this'', Wordpress, [http://igemvalencia2012.wordpress.com/2012/08/14/igem-the-why-of-all-this/] <br/><br />
'''University of Leicester''': ''Synthetic biology solution to polystyrene degradation'', Blogger, [http://uoleicesterigem2012.blogspot.co.uk/] <br/><br />
'''University College London''': ''Land Grab: Could Bioremediation Turn Pacific Garbage Patch Into Habitable Island?'', Good, [http://www.good.is/post/land-grab-could-bioremediation-turn-pacific-garbage-patch-into-habitable-island/] <br/><br />
'''University College London''': ''Synthetic Bacteria Could Turn Ocean Garbage into One Big Island'', Smithsonian, [http://blogs.smithsonianmag.com/smartnews/2012/07/synthetic-bacteria-could-turn-ocean-garbage-into-one-big-island/] <br/><br />
'''University College London''': ''Students synthesizing bacteria to create islands out of garbage'', DVICE, [http://dvice.com/archives/2012/07/students-synthe.php] <br/><br />
'''University College London''': ''Nanobots could turn 'Great Pacific Plastic Patch' into a floating island'', Wired, [http://www.wired.co.uk/news/archive/2012-07/18/nanobots-recycling-plastic] <br/><br />
'''University College London''': ''Synthetic Biology Speed Debate'', C-LAB, [http://c-lab.co.uk/events/synthetic-biology-speed-debate.html] <br/><br />
'''University College London''': ''From Pixels To DNA: The Next Frontier In Interaction Design'', Co.Design, [http://www.fastcodesign.com/1670825/from-pixels-to-dna-the-next-frontier-in-interaction-design#1]<br /><br />
'''University College London''': ''Biohacking, iGEM and the limits of citizen science'', Po Ve Sham, [http://povesham.wordpress.com/2012/10/05/biohacking-igem-and-the-limits-of-citizen-science/]<br /><br />
'''University College London''': ''Right or Risk? World's First Public BioBrick'', C-LAB, [http://c-lab.co.uk/events/right-or-risk-worlds-first-public-biobrick.html]<br /><br />
'''Queen's University''': ''Getting Biological over the Break'', Kingston This Week, [http://www.kingstonthisweek.com/2012/06/21/getting-biological-over-the-break] <br/><br />
'''Queen's University''': ''Dance like everybody's watching'', Kingston EMC, [http://www.emckingston.com/20120809/lifestyle/Dance+like+everybody%27s+watching] <br/><br />
'''Queen's University''': ''Dancing in the Lab'', The Queen's Journal, [http://queensjournal.ca/story/2012-09-11/news/dancing-lab/] <br/><br />
'''NRP UEA Norwich''': ''UEA Students Compete For Top Biology Prize'', UEA Press Release, [http://www.uea.ac.uk/mac/comm/media/press/2012/August/igem-competition-uea]<br><br />
'''NRP UEA Norwich''': ''Interview With Students From The UEA Talking About Synthetic Biology (Show 23)'', The Farming Show: STAR Radio, [http://www.star107.co.uk/farming-show.php]<br><br />
'''University of St Andrews''': "Streets of London: paved with platinum" [http://www.st-andrews.ac.uk/news/archive/2012/Title,89409,en.html] <br><br />
'''TU_Munich''': ''Technische Universität München: Brauhefe für leckeres und gesundes Bier'', biotechnologie.de, [http://www.biotechnologie.de/BIO/Navigation/DE/root,did=153818.html]<br><br />
'''TU_Munich''': ''TUMstudis: baker's yeast is synthetically converted'', TUMstudinews, [http://portal.mytum.de/ccc/newsletter/students/2012_03/02]<br><br />
'''TU_Munich''': ''Die Wunderhefe'', jetzt.de sueddeutsche zeitung, [http://jetzt.sueddeutsche.de/texte/anzeigen/544866/4/1#texttitel]<br><br />
'''TU_Munich''': ''Widerstand gegen die "Grüne Gentechnik" wächst weiter'', merkur, [http://www.merkur-online.de/lokales/freising/widerstand-gegen-gruene-gentechnik-waechst-weiter-2510648.html]<br><br />
'''TU_Munich''': ''iGEM: Fünf Teams lösen Ticket für Boston'', biotechnologie.de, [http://www.biotechnologie.de/BIO/Navigation/DE/root,did=154774.html]<br><br />
'''Tübingen''': ''Tübinger Studenten nehmen an internationalem Forscherwettbewerb teil'', Schwäbisches Tagblatt, [http://www.tagblatt.de/Home/nachrichten/hochschule_artikel,-Tuebinger-Studenten-nehmen-an-internationalem-Forscherwettbewerb-teil-_arid,184906.html]<br><br />
'''Bielefeld-Germany''': ''Studierende der Universität Bielefeld nehmen am iGEM-Wettbewerb teil'', Universität Bielefeld, [http://ekvv.uni-bielefeld.de/blog/pressemitteilungen/entry/östrogen_aus_trinkwasser_entfernen_nr]<br><br />
'''Bielefeld-Germany''': ''Östrogen aus Trinkwasser entfernen'', Press Release, [http://www.uni-protokolle.de/nachrichten/id/240229/][http://www.laborpraxis.vogel.de/forschung-und-entwicklung/analytik/articles/369125/][http://www.bio.nrw.de/nachrichten][http://idw-online.de/pages/de/news484982][http://www.eurekalert.org/pub_releases/2012-06/uob-ref062512.php]<br><br />
'''Bielefeld-Germany''': ''Turkey tail tree fungus could filter oestrogen from water'', Wired, [http://www.wired.co.uk/news/archive/2012-06/25/tree-fungus-oestrogen-filter]<br><br />
'''Bielefeld-Germany''': ''Biological filter to remove estrogens from waste water and drinking water'', News Medical Australia, [http://www.news-medical.net/news/20120626/Biological-filter-to-remove-estrogens-from-waste-water-and-drinking-water.aspx]<br><br />
'''Bielefeld-Germany''': ''Das Wasser wird weiblich'', Neue Westfälische, [https://2012.igem.org/Team:Bielefeld-Germany/Homeleft]<br><br />
'''Bielefeld-Germany''': ''Arzneimittel in Gewässern minimieren'', Neue Züricher Zeitung, [http://www.nzz.ch/wissen/wissenschaft/arzneimittel-in-gewaessern-minimieren-1.17368069]<br><br />
'''USP-UNESP-Brazil''': ''Brazilian Science Dreams Come Alive'', RocketHub Blog, [http://blog.rockethub.com/brazilian-science-dreams-come-alive]<br><br />
'''USP-UNESP-Brazil''': ''USP participa pela 1ª vez de campeonato de biologia sintética'', Jornal do Campus, [http://www.jornaldocampus.usp.br/index.php/2012/06/usp-participa-pela-1a-vez-de-campeonato-de-biologia-sintetica/]<br><br />
'''USP-UNESP-Brazil''': ''Cientistas da USP conseguem financiar projeto com vaquinha virtual'', Folha de S.Paulo, [http://www1.folha.uol.com.br/ciencia/1097202-cientistas-da-usp-conseguem-financiar-projeto-com-vaquinha-virtual.shtml]<br><br />
'''EPF-Lausanne''': ''Fabriquer des médicaments en allumant la lumière?'' (French), Flash EPFL, [http://actualites.epfl.ch/newspaper-edition?np_id=151] [https://static.igem.org/mediawiki/2012/4/42/Team-EPF-Lausanne_Flash_article_about_us.pdf]<br><br />
'''Lyon INSA iGEM''' : ''L’INSA de Lyon vise l’or à Amsterdam'' , En vue INSA Lyon, [http://envue.insa-lyon.fr/2012avril/art2a_0412.php]<br><br />
'''Trieste''': ''Human practices - SynBio for everyone (Italian)'', Il Tascapane, [http://tascapane.blogspot.se/2012/08/la-biologia-sintetica.html]<br><br />
'''Trieste''': ''Human practices - SynBio and medicine (Italian)'', Il Tascapane,[http://tascapane.blogspot.it/2012/09/la-biologia-sintetica-approda-in.html]<br><br />
'''Trieste''': '' Un'idea per proteggere l'intestino (Italian)'', ICGEB, [http://www.icgeb.org/notizie-in-italiano/items/squadra-igem-2012-un-idee-per-proteggere-lintestino.html]<br><br />
'''Lyon-INSA''':''M. concourt au titre mondial de biologie de synthèse'', Républicain Lorrain [https://docs.google.com/file/d/0BzhWvySCD_KzWmJTYjBYWHBCUnM/edit]<br><br />
'''Lyon-INSA''' : ''L'Insa de Lyon vise l'or à Amsterdam'', Insa de Lyon [http://www.insa-lyon.fr/fr/concours-igem-2012]<br><br />
'''UANL_Mty-Mexico''': ''Túnel de la Biología Sintética (Synthetic Biology Tunnel), Televisa'', Monterrey,[http://www.televisaregional.com/monterrey/video/169491536.html]<br/><br />
'''Wageningen_UR''': ''The Constructor: a web application optimizing cloning strategies based on modules from the registry of standard biological parts'', Journal of Biological Engineering,[http://www.jbioleng.org/content/6/1/14/abstract]<br/><br />
'''SDU-Denmark''': ''Fighting Obesity at the SDU in Odense'', SIJ, by Eliana van de Craats, [http://community.ejc.net/forum/topic/show?id=2345725%3ATopic%3A89026&xgs=1&xg_source=msg_share_topic]<br/><br />
'''Georgia_Tech''': ''<br />
Georgia Tech workshop well received: Lambert students embrace engineering'', ForsythNews.com, [http://www.forsythnews.com/m/section/3/article/14721/. ]<br/><br />
'''Cornell''': ''Using electroactive bacteria, students design toxin sensor'', Cornell Chronicle, [http://www.news.cornell.edu/stories/Oct12/oilSands.html]</div>JaraOhttp://2012.igem.org/Team:TU_Munich/Human_Practice/SurveyTeam:TU Munich/Human Practice/Survey2012-10-24T07:58:45Z<p>JaraO: /* Background */</p>
<hr />
<div>{{Team:TU_Munich/Header}}<br />
<br />
= Survey =<br />
<hr/><br />
===Background===<br />
The survey was carried out by the [http://portal.mytum.de/jungeakademie/index_html/document_view?/ TUM young academy], who planned to install a [http://www.genial-info.de/ website] to inform the public about genetic engineering. They made an online questionnaire and stood in Munich for one day, with a print out version.<br />
All in all''' 1183''' people participated, 979 online and 204 on the street, thereof 597 were male and 570 female. The problem is that, in this survey, the group of the age from 15-24 is drastically over represented with 70%, the reason could be that through the university project more students were reached.<br />
<br/><br />
This team allowed us to use their data for our project as well. As they had 33 questions we chose the ones directly relied to genetic engineering, the ones dealing with the webpage they wanted to create we left out.<br />
<br />
===Basic data===<br />
*Participants: 1183<br />
*Online participants: 979<br />
*Street participants: 204<br />
*Male: 597 <br />
*Female: 570<br />
*Age: mainly between 15-24 (70%)<br />
<br />
===Questions===<br />
====Evaluated questions====<br />
*How do you feel about genetic engineering in general?<br />
*How good do you think is your knowledge about genetic engineering?<br />
*Are you concerned about genetically modified food?<br />
*Do you think genetic engineering is reasonable in medicine?<br />
*Do you think genetic engineering is reasonable in farming?<br />
*Do you think genetic engineering is reasonable in food?<br />
*Do you think the commentatorship in Germany is neutral?<br />
*Would you eat a genetically modified apple if it was healthier?<br />
*Would you eat a genetically modified apple if it tasted better?<br />
<br />
====Answers====<br />
[[File:TUM12_Overall.png|thumb|left|400px|Overall impression about genetic engineering]] <br />
[[File:TUM12_Knowledge.png|thumb|right|400px|Knowledge about genetic engineering]]<br />
[[File:TUM12_Concerns about food.png|thumb|left|400px|Concerns about genetically modified food]]<br />
[[File:TUM12_Genetic engineering in farming.png|thumb|right|400px|Acceptance of genetic engineering in farming]]<br />
[[File:TUM12_Genetic engineering in medicine.png|thumb|left|400px|Acceptance of genetic engineering in medicine]]<br />
[[File:TUM12_Media.png|thumb|right|400px|Neutrality of the German media from the point of view of the public]]<br />
[[File:TUM12_Healthier apple.png|thumb|left|400px|Decision to eat an genetically modified apple if it was healthier]]<br />
[[File:TUM12_Tastier apple.png|thumb|right|400px|Decision to eat an genetically modified apple if it was tastier]]<br />
<br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br />
<br />
==Conclusion==<br />
In contrary to our first impression about half of the questioned people say that they have a "normal" or better amount of knowledge. In our former human practice events we often realised that the discussions are rather emotional than scientific. This difference could arise from the mostly younger people who participated. Because in Germany genetic engineering started to play a bigger role in schools and in the media, although as mentioned below, the media is not always neutral.<br />
The overall impression about genetically modified organism is not that bad, but it has to be differenced where genetic engineering is used. A lot of people agree that it is appropriate to use it in medicine (922).<br />
<br/><br />
Whereas in food and agriculture the acceptance is with 633 (food) and 573 (farming) people who say no, very low. As also mentioned on our [https://2012.igem.org/Team:TU_Munich/Human_Practice/Overview overview page] people do not accept GMO in their food. The skepsis in Germany is rather big.<br />
This is also depicted in the fact that people (533) will rather not eat a genetically modified apple even if it would taste the same. Eating a healthier apple is not that controversial but not many Germans would (404). So with around 50% of people who would not eat an healthier apple, when it is healthier because of genetic engineering, the problem lies in the acceptance. Even a positive result is not enough to convince people. But the question about medicine shows that the benefit is here that good that people accept genetic engineered therapeutics. So maybe the benefit of green biotechnology is not that present in the peoples minds. <br />
<br/><br/><br />
Nevertheless it is to be mentioned that a lot of people think that they are not informed impartial. This might lead to a rather negatic point of view, because a neutral reporting is essential to come up with his or her own mind. <br />
All in all it is to say that the red biotechnology is widely accepted in Germany, whereas green biotechnology has a hard stand. Although it is to say that the Germans think that the media is not neutral about GMO, 808 think so. Also they have the wish that plays a bigger role in politics (655).</div>JaraOhttp://2012.igem.org/Team:TU_Munich/Human_Practice/SurveyTeam:TU Munich/Human Practice/Survey2012-10-24T07:57:15Z<p>JaraO: /* Background */</p>
<hr />
<div>{{Team:TU_Munich/Header}}<br />
<br />
= Survey =<br />
<hr/><br />
===Background===<br />
The survey was carried out by the [http://portal.mytum.de/jungeakademie/index_html/document_view?/ TUM young academy], who planned to install a website to inform the public about genetic engineering. They made an online questionnaire and stood in Munich for one day, with a print out version.<br />
All in all''' 1183''' people participated, 979 online and 204 on the street, thereof 597 were male and 570 female. The problem is that, in this survey, the group of the age from 15-24 is drastically over represented with 70%, the reason could be that through the university project more students were reached.<br />
<br/><br />
This team allowed us to use their data for our project as well. As they had 33 questions we chose the ones directly relied to genetic engineering, the ones dealing with the webpage they wanted to create we left out.<br />
<br />
===Basic data===<br />
*Participants: 1183<br />
*Online participants: 979<br />
*Street participants: 204<br />
*Male: 597 <br />
*Female: 570<br />
*Age: mainly between 15-24 (70%)<br />
<br />
===Questions===<br />
====Evaluated questions====<br />
*How do you feel about genetic engineering in general?<br />
*How good do you think is your knowledge about genetic engineering?<br />
*Are you concerned about genetically modified food?<br />
*Do you think genetic engineering is reasonable in medicine?<br />
*Do you think genetic engineering is reasonable in farming?<br />
*Do you think genetic engineering is reasonable in food?<br />
*Do you think the commentatorship in Germany is neutral?<br />
*Would you eat a genetically modified apple if it was healthier?<br />
*Would you eat a genetically modified apple if it tasted better?<br />
<br />
====Answers====<br />
[[File:TUM12_Overall.png|thumb|left|400px|Overall impression about genetic engineering]] <br />
[[File:TUM12_Knowledge.png|thumb|right|400px|Knowledge about genetic engineering]]<br />
[[File:TUM12_Concerns about food.png|thumb|left|400px|Concerns about genetically modified food]]<br />
[[File:TUM12_Genetic engineering in farming.png|thumb|right|400px|Acceptance of genetic engineering in farming]]<br />
[[File:TUM12_Genetic engineering in medicine.png|thumb|left|400px|Acceptance of genetic engineering in medicine]]<br />
[[File:TUM12_Media.png|thumb|right|400px|Neutrality of the German media from the point of view of the public]]<br />
[[File:TUM12_Healthier apple.png|thumb|left|400px|Decision to eat an genetically modified apple if it was healthier]]<br />
[[File:TUM12_Tastier apple.png|thumb|right|400px|Decision to eat an genetically modified apple if it was tastier]]<br />
<br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br />
<br />
==Conclusion==<br />
In contrary to our first impression about half of the questioned people say that they have a "normal" or better amount of knowledge. In our former human practice events we often realised that the discussions are rather emotional than scientific. This difference could arise from the mostly younger people who participated. Because in Germany genetic engineering started to play a bigger role in schools and in the media, although as mentioned below, the media is not always neutral.<br />
The overall impression about genetically modified organism is not that bad, but it has to be differenced where genetic engineering is used. A lot of people agree that it is appropriate to use it in medicine (922).<br />
<br/><br />
Whereas in food and agriculture the acceptance is with 633 (food) and 573 (farming) people who say no, very low. As also mentioned on our [https://2012.igem.org/Team:TU_Munich/Human_Practice/Overview overview page] people do not accept GMO in their food. The skepsis in Germany is rather big.<br />
This is also depicted in the fact that people (533) will rather not eat a genetically modified apple even if it would taste the same. Eating a healthier apple is not that controversial but not many Germans would (404). So with around 50% of people who would not eat an healthier apple, when it is healthier because of genetic engineering, the problem lies in the acceptance. Even a positive result is not enough to convince people. But the question about medicine shows that the benefit is here that good that people accept genetic engineered therapeutics. So maybe the benefit of green biotechnology is not that present in the peoples minds. <br />
<br/><br/><br />
Nevertheless it is to be mentioned that a lot of people think that they are not informed impartial. This might lead to a rather negatic point of view, because a neutral reporting is essential to come up with his or her own mind. <br />
All in all it is to say that the red biotechnology is widely accepted in Germany, whereas green biotechnology has a hard stand. Although it is to say that the Germans think that the media is not neutral about GMO, 808 think so. Also they have the wish that plays a bigger role in politics (655).</div>JaraOhttp://2012.igem.org/Team:TU_Munich/Human_Practice/SurveyTeam:TU Munich/Human Practice/Survey2012-10-23T20:56:35Z<p>JaraO: /* Survey */</p>
<hr />
<div>{{Team:TU_Munich/Header}}<br />
<br />
= Survey =<br />
<hr/><br />
===Background===<br />
The survey was carried out by the [http://www.genial-info.de/ TUM young academy], who planned to install a website to inform the public about genetic engineering. They made an online questionnaire and stood in Munich for one day, with a print out version.<br />
All in all''' 1183''' people participated, 979 online and 204 on the street, thereof 597 were male and 570 female. The problem is that, in this survey, the group of the age from 15-24 is drastically over represented with 70%, the reason could be that through the university project more students were reached.<br />
<br/><br />
This team allowed us to use their data for our project as well. As they had 33 questions we chose the ones directly relied to genetic engineering, the ones dealing with the webpage they wanted to create we left out.<br />
<br />
===Basic data===<br />
*Participants: 1183<br />
*Online participants: 979<br />
*Street participants: 204<br />
*Male: 597 <br />
*Female: 570<br />
*Age: mainly between 15-24 (70%)<br />
<br />
===Questions===<br />
====Evaluated questions====<br />
*How do you feel about genetic engineering in general?<br />
*How good do you think is your knowledge about genetic engineering?<br />
*Are you concerned about genetically modified food?<br />
*Do you think genetic engineering is reasonable in medicine?<br />
*Do you think genetic engineering is reasonable in farming?<br />
*Do you think genetic engineering is reasonable in food?<br />
*Do you think the commentatorship in Germany is neutral?<br />
*Would you eat a genetically modified apple if it was healthier?<br />
*Would you eat a genetically modified apple if it tasted better?<br />
<br />
====Answers====<br />
[[File:TUM12_Overall.png|thumb|left|400px|Overall impression about genetic engineering]] <br />
[[File:TUM12_Knowledge.png|thumb|right|400px|Knowledge about genetic engineering]]<br />
[[File:TUM12_Concerns about food.png|thumb|left|400px|Concerns about genetically modified food]]<br />
[[File:TUM12_Genetic engineering in farming.png|thumb|right|400px|Acceptance of genetic engineering in farming]]<br />
[[File:TUM12_Genetic engineering in medicine.png|thumb|left|400px|Acceptance of genetic engineering in medicine]]<br />
[[File:TUM12_Media.png|thumb|right|400px|Neutrality of the German media from the point of view of the public]]<br />
[[File:TUM12_Healthier apple.png|thumb|left|400px|Decision to eat an genetically modified apple if it was healthier]]<br />
[[File:TUM12_Tastier apple.png|thumb|right|400px|Decision to eat an genetically modified apple if it was tastier]]<br />
<br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br />
<br />
==Conclusion==<br />
In contrary to our first impression about half of the questioned people say that they have a "normal" or better amount of knowledge. In our former human practice events we often realised that the discussions are rather emotional than scientific. This difference could arise from the mostly younger people who participated. Because in Germany genetic engineering started to play a bigger role in schools and in the media, although as mentioned below, the media is not always neutral.<br />
The overall impression about genetically modified organism is not that bad, but it has to be differenced where genetic engineering is used. A lot of people agree that it is appropriate to use it in medicine (922).<br />
<br/><br />
Whereas in food and agriculture the acceptance is with 633 (food) and 573 (farming) people who say no, very low. As also mentioned on our [https://2012.igem.org/Team:TU_Munich/Human_Practice/Overview overview page] people do not accept GMO in their food. The skepsis in Germany is rather big.<br />
This is also depicted in the fact that people (533) will rather not eat a genetically modified apple even if it would taste the same. Eating a healthier apple is not that controversial but not many Germans would (404). So with around 50% of people who would not eat an healthier apple, when it is healthier because of genetic engineering, the problem lies in the acceptance. Even a positive result is not enough to convince people. But the question about medicine shows that the benefit is here that good that people accept genetic engineered therapeutics. So maybe the benefit of green biotechnology is not that present in the peoples minds. <br />
<br/><br/><br />
Nevertheless it is to be mentioned that a lot of people think that they are not informed impartial. This might lead to a rather negatic point of view, because a neutral reporting is essential to come up with his or her own mind. <br />
All in all it is to say that the red biotechnology is widely accepted in Germany, whereas green biotechnology has a hard stand. Although it is to say that the Germans think that the media is not neutral about GMO, 808 think so. Also they have the wish that plays a bigger role in politics (655).</div>JaraOhttp://2012.igem.org/Team:TU_Munich/Project/Genome_IntegrationTeam:TU Munich/Project/Genome Integration2012-10-23T20:50:47Z<p>JaraO: /* Background and principles */</p>
<hr />
<div>{{Team:TU_Munich/Header}}<br />
<br />
{{Team:TU_Munich/ExCol}}<br />
<br />
= Genome Integration =<br />
----<br />
[[File:Martin_einzel_TUM12.jpg|200px|thumb||Responsible: Martin Schappert]]<br />
As we cannot obey the letter of the German purity law (there is a zero tolerance policy concerning transgenic ingredients), we try our best to meet the spirit. Thus, it is '''unacceptable for us to work with antibiotics''' to keep up the selective environment. Since we cannot work with '''auxotrophies in beer''' either, we have to make sure the yeasts do not lose the BioBricks. The most promising way to accomplish a long lasting presence of our constructs is to achieve '''genome integration'''.<br />
<br />
By means of genome integration we do not have to induce a selective stress in the brewing process and yet can be sure that the yeasts express our constructs properly - under [[Team:TU_Munich/Project/Constitutive_Promoter|constitutive promoters]] like '''TEF1 or TEF2''' as well as [[Team:TU_Munich/Project/Ethanol_Inducible_Promoter|ethanol-inducible promoter]] or the [[Team:TU_Munich/Project/Light_Switchable_Promoter|light-switchable promoter]].<br />
== Background and principles ==<br />
----<br />
<div><br />
To accomplish genome integration we could resort to a preexisting BioBrick [http://partsregistry.org/Part:BBa_K300001]. <br />
<div style="float:right"><br />
'''BBa K300001 (Pavia '10)'''<br />
[[File:integration_vector_of_iGEM10_UNIPV-Pavia_-TUM12.png|left|x200px]] <br />
[[File:Glossary_K300001_iGEM_UNIPV-Pavia_-TUM12.png|right|x200px]]<br />
</div><br />
'''Summary of principle:'''<br />
<br />
* "The part of interest, contained in the yeast integrative vector, will be inserted in the genomic region comprised between the two target sequences by homologous recombination, thus disrupting the original genomic DNA between them."[http://partsregistry.org/wiki/index.php/Part:BBa_K300986]<br />
* The recombinant region includes the divergent GAL1/GAL10 promoter and several base pairs of the GAL1 and GAL10 ORFs.<br />
* With SbfI linearized plasmids show a 10- to 50-fold higher integration efficiency than non-linearized ones [https://www.urmc.rochester.edu/labs/Sherman-Lab/publications/pdfs/Saccharomyces-Cerevisiae-Yeast-Intro.pdf]<br />
* As the plasmid itself contains no yeast ORI, colonies proliferating on G418 plates must have integrated the resistance gene.<br />
</div><br />
<br />
== Idea ==<br />
----<br />
<br />
For proof of principle, we want to insert a functional mOrange cassette (BBa K300007), cut the integrative vector with SbfI in order to linearize it and transfect it into yeasts. After we've shown the general functionality, we'll work on and integrate one gene after another until all of our BioBricks are integrated. In order to do that, we have to engineer new homologous regions to enable multiple transgenic integrations.<br />
<br />
<br><br />
[[File:Integrational_vector_method_TUM12.jpg|right|406px]]<br />
<br />
<br />
To show, that the stable integration worked, we picked 36 clones from the plates with yeasts transformed with the linearized integration vector plasmid (insert: BBa K300007[http://partsregistry.org/Part:BBa_K300007]).<br />
These 36 clones were put into non-selective YPD medium and passaged 4 times. After 48 hours, the last culture was used to inoculate an overnight culture. The OD of all candidates was measured. <br />
We had 5 negatives, probably due to mistakes in pipetting. The rest of the cultures were diluted to OD 0.4 and 100 ml of it were used to plate on selective (meaning G418+) petri dishes.<br />
<br />
== Results ==<br />
----<br />
<br />
Of the 31 remaining plates, 21 were empty. The cfu of the positive plates were: [[File:Genome_integration_cfu_TUM12.png|right|350px]]<br />
<br />
* 3<br />
* 8<br />
* 12<br />
* 49<br />
* 68<br />
* 282<br />
* >1000<br />
* >10 000<br />
* >10 000<br />
* >10 000<br />
<br />
The plan is to PCR at least two of the clones to gain knowledge why there's been such drastic differences in between the different clones regarding the in the integration efficiency and endurance.<br />
<br />
As of the mOrange cassette (BBa K300007[http://partsregistry.org/Part:BBa_K300007]), we couldn't show any fluorescence which suggests a corrupt insert.</div>JaraOhttp://2012.igem.org/Team:TU_Munich/Project/OverviewTeam:TU Munich/Project/Overview2012-10-23T20:45:06Z<p>JaraO: /* Brewing our SynBio Beer */</p>
<hr />
<div>{{Team:TU_Munich/Header}}<br />
<br />
= Overview =<br />
<hr/><br />
<br />
== Vision ==<br />
----<br />
We, TU Munich’s 2012 iGEM team, strive to catalyze the diffusion process of knowledge about genetic engineering and synthetic biology among the general public. Using the example of iGEM’s first and finest SynBio Beer we involve, interest and inspire people to reconsider preconceived ideas and encourage them to openly engage in a broad discussion weighing pros and cons of genetic engineering in foodstuff. We sketch a future where new technology can be applied in a meaningful way to complement traditional foods or beverages.<br />
<br />
==Biosynthesis pathways==<br />
----<br />
<br />
<br />
<div class="bezel mfull"><br />
===Limonene===<br />
Limonene is a cyclic terpene and a major constituent of several citrus oils. D-Limonene has been used as a component of flavorings and fragrances. It is formed from geranyl pyrophosphate by limonene synthase.<br />
<br />
We successfully demonstrated the production of the flavoring substance limonene by expressing limonene synthase in ''S. cerevisiae'', which naturally synthesizes the educt geranyl pyrophosphate.<br />
<hr><br />
[[file:TUM12_experiment_overwiew_limonene.png|500px|thumb|right| Main results from our limonene subproject: reaction mechanism (A), constructed BioBricks (B) and proof of principle for the in vivo production of limonene]]<br />
<center>'''Experimental results:'''</center><br><br />
(+)-Limonene synthase 1 [[http://partsregistry.org/Part:BBa_K801065 BBa_K801065]] and (+)-limonene synthase 1 with yeast consensus sequence [[http://partsregistry.org/Part:BBa_K801060 BBa_K801060]] were successfully cloned into our new yeast expression vector pTUM100. Expression of recombinant limonene synthase in ''Saccharomyces cerevisiae'' was proven by western blotting. Subsequently the protein was purified using SA-chromatography and size exclusion chromatography. The functionality of the enzyme was verified by ''in vivo'' and ''in vitro'' detection of limonene via GC-MS. <br />
<br />
Furthermore, we established gene constructs of the limonene synthase coding sequence with different yeast specific promoters and terminators [[http://partsregistry.org/Part:BBa_K801062 BBa_K801062]], [[http://partsregistry.org/Part:BBa_K801063 BBa_K801063]], [[http://partsregistry.org/Part:BBa_K801064 BBa_K801064]]. <br />
<br />
Last but not least, we have brewed iGEM's first SynBio beer containing limonene.<br />
<br />
<center>'''Conclusion and outlook:'''</center><br><br />
<br />
We have achieved functional expression of ''Citrus limon'' limonene synthase and production of limonene in yeast. The last remaining step to a SynBio beer would be the integration of our gene constructs [[http://partsregistry.org/Part:BBa_K801062 BBa_K801062]], [[http://partsregistry.org/Part:BBa_K801063 BBa_K801063]], [[http://partsregistry.org/Part:BBa_K801064 BBa_K801064]] into the yeast genome.<br />
<br />
<div class="noborder" style="float:left"><br />
<html><a href="https://2012.igem.org/Team:TU_Munich/Project/Limonene"><img width="200px" src="https://static.igem.org/mediawiki/2012/3/33/TUM12_readmore.png"/></a></html><br />
</div><br />
</div><br />
<br />
<div class="bezel mfull"><br />
<br />
===Thaumatin===<br />
Thaumatin is a natural α+β-protein which is synthesized by the katamfe plant (''Thaumatococcus daniellii''). It is said to be 2,000 to 100,000 times sweeter than sucrose on molar basis, but the sweetness builds up slow and lasts long. It has been approved as a sweetener by the European Union (E957).<br />
<br />
Our aim is to have ''S. cerevisiae'' secrete functional thaumatin by expressing preprothaumatin – a principle which has been proven by [[http://www.ncbi.nlm.nih.gov/pubmed/6327079 Edens et al., 1984]].<br />
<hr><br />
[[file:TUM12_experiment_overwiew_Thaumati.png|500px|thumb|right| Main results from the Thaumatin subproject: Structure of Thaumatin (A), constructed BioBricks (B), profile of an ion exchange chromatography (IEC) used to detect our recombinant Thaumatin and a SDS-PAGE gel showing IEC elution fractions containing Thaumatin]]<br />
<center>'''Experimental results:'''</center><br><br />
The BioBrick for preprothaumatin [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801080 BBa_K801080] as well as an expression cassette [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801080 K801080] were successfully cloned, expressed in yeast, purified using an ion exchange chromatography (see figure C) and detected in the SDS-PAGE. Therefore, the expression of thaumatin in yeast could be demonstrated and functionality of the BioBrick is confirmed.<br />
<br />
<center>'''Conclusion and outlook:'''</center><br><br />
A proof of principle for the expression of thaumatin was achieved. Further goals are the increase of the expression of thaumatin and the investigation of the secretion.<br />
<br />
<div class="noborder" style="float:left"><br />
<html><a href="https://2012.igem.org/Team:TU_Munich/Project/Thaumatin"><img width="200px" src="https://static.igem.org/mediawiki/2012/3/33/TUM12_readmore.png"/></a></html><br />
</div><br />
</div><br />
<br />
<div class="bezel mfull"><br />
<br />
===Caffeine===<br />
Caffeine is a purine-alkaloid and its biosynthesis is known from coffee plants and tea plants. The molecule acts as a competitive antagonist of adenosine receptors and, therefore, increases indirectly neurotransmitter concentrations resulting in warding off drowsiness and restoring alertness. <br />
<br />
The idea is to perform a heterologous gene expression of the three enzymes 7-methylxanthosine synthase (CaXMT1), N-methyl nucleosidase (CaMXMT1) and caffeine synthase (CaDXMT1) required for caffeine biosynthesis in ''Saccharomyces cerevisiae''. <br />
<br />
<br />
<hr><br />
[[file:TUM12_experiment_overwiew_caffeine2.png|500px|thumb|right| Figure showing a schematic overview of the reaction in (A), a western blot against the Strep-tag II for GFP, [[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801070 BBa_K801070]] and [[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801071 BBa_K801071]] in (B) and finally the same western blot development for [[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801072 BBa_K801072]]]]<br />
<center>'''Experimental results:'''</center><br><br />
* Successful cloning of the three enzymes [http://partsregistry.org/Part:BBa_K801070 7-methylxanthosine synthase (CaXMT1)], [http://partsregistry.org/Part:BBa_K801071 N-methyl nucleosidase (CaMXMT1)] and the [http://partsregistry.org/Part:BBa_K801072 caffeine synthase (CaDXMT1)] into the shuttle vector pTUM104 and pSB1C3 each. <br />
* Successful assembly of the BioBricks to form expression cassettes consisting of promoter, gene and terminator: [http://partsregistry.org/Part:BBa_K801073 pTEF2-CaXMT1-tADH1], [http://partsregistry.org/Part:BBa_K801074 pTEF1-CaMXMT1-tADH1] and [http://partsregistry.org/Part:BBa_K801075 pTEF2-CaDXMT1-tADH1]) into pSB1C3.<br />
* Successful assembly of the expression cassettes of the three relevant enzymes forming a composite part of 6.4 kb capable of caffeine production in yeast ([http://partsregistry.org/Part:BBa_K801077 Caffeine Synthesis Pathway]) into pSB1C3.<br />
* Successful expression of CaXMT1, CaMXMT1 and CaDXMT1 in ''Saccharomyces cerevisiae'' INVSc1 in selective Sc minimal induction medium lacking uracil with 2 % galactose.<br />
<br />
<br />
<center>'''Conclusion and outlook:'''</center><br><br />
The homologue expression of the three required enzymes for caffeine synthesis in ''Saccharomyces cerevisiae'' INVSc1 transformed with pTUM102_CaXMT1, pTUM102_CaMXMT1 and pTUM102_CaDXMT1 was successful. Further testing of caffeine production using crude extracts from lysed transformed yeast cells and working on genome integration of the expression cassette containing all three enzymes flanked by promoter and terminator are in progress.<br />
<br />
<div class="noborder" style="float:left"><br />
<html><a href="https://2012.igem.org/Team:TU_Munich/Project/Caffeine"><img width="200px" src="https://static.igem.org/mediawiki/2012/3/33/TUM12_readmore.png"/></a></html><br />
</div><br />
</div><br />
<br />
<div class="bezel mfull"><br />
<br />
===Xanthohumol===<br />
Xanthohumol is known as a putative cancer chemopreventive agent due to its antioxidant activities [[http://www.ncbi.nlm.nih.gov/pubmed/10995285 Miranda et al., 2000]]. Our goal is a heterologous gene expression of all enzymes required for xanthohumol biosynthesis in ''S. cerevisiae''.<br />
<br />
The pathway for the production of this plant secondary metabolite is composed of five steps, starting with the conversion of phenylalanine and followed by four further enzymatic reactions.<br />
<hr><br />
[[file:TUM12_experiment_overwiew_xanto2.png|500px|thumb|right| Main results from the Xanthohumol subproject: Successful reconstruction of the metabolic pathway for Xanthohumol]]<br />
<center>'''Experimental results:'''</center><br><br />
The whole biosynthetic pathway for the production of xanthohumol was converted into BioBricks. Except for APT each of the enzymes were cloned in two versions one having the proposed consensus sequence for more efficient expression in yeast chassis and another for usage of these BioBricks in other chassis. All BioBricks were sequenced. Sequences can be found in the registry of standard biological parts:<br />
[http://partsregistry.org/Part:BBa_K801090 BBa_K801090], [http://partsregistry.org/Part:BBa_K801091 BBa_K801091], [http://partsregistry.org/Part:BBa_K801092 BBa_K801092], [http://partsregistry.org/Part:BBa_K801093 BBa_K801093], [http://partsregistry.org/Part:BBa_K801094 BBa_K801094], [http://partsregistry.org/Part:BBa_K801095 BBa_K801095], [http://partsregistry.org/Part:BBa_K801096 BBa_K801096], [http://partsregistry.org/Part:BBa_K801097 BBa_K801097] and [http://partsregistry.org/Part:BBa_K801098 BBa_K801098] <br />
<br />
<br />
<center>'''Conclusion and outlook:'''</center><br><br />
<br />
The construction of the xanthohumol pathway was achieved, whereas the expression and characterization have to be postponed after the European Jamboree or might be an interesting task for next year's iGEM teams.<br />
<br />
<div class="noborder" style="float:left"><br />
<html><a href="https://2012.igem.org/Team:TU_Munich/Project/Xanthohumol"><img width="200px" src="https://static.igem.org/mediawiki/2012/3/33/TUM12_readmore.png"/></a></html><br />
</div><br />
</div><br />
<br />
==Vector Design==<br />
----<br />
<div class="bezel mfull"><br />
===pTUM100===<br />
Designing an expression vector for yeast which is compatible to the iGEM cloning principles and standards was the main aim of this subproject. Based on the commercially available pYES2 vector we created vectors containing inducible and constitutive promoters in order to establish efficient possibilities to clone and express our enzymes.<br />
<hr><br />
[[file:TUM12_experiment_overwiew_vector.png|500px|thumb|right| Explanations on the figure:<br />
<br />
Figure A shows the new multiple cloning site (MCS) containing the RFC 10/25 restriction sides and the DNA sequence coding for the ''Strep''-tag II.<br />
<br />
Figure B gives an overview of all important functional elements located on the vector backbone. Upstream to the new MCS lies a T7 promoter primer binding site allowing easy forward sequencing of integrated gene constructs using the standard T7 primer. The URA 3 gene is a prototrophy marker used for the selection of transfected cells.<br />
<br />
Figure C to E present the successfully designed BioBricks: pTUM100 simply contains the new MCS, the transcription terminator and further elements required for cloning and transfection. pTUM102 to pTUM104 contain in addition the constitutive promoters pTef1, pTef2 and ADH. On pTUM104 is the galactose inducible promoter pGAL1 located. ]]<br />
<br />
<center>'''Experimental results:'''</center><br><br />
Using the pYES vector from Invitrogen we first deleted five forbidden restriction sites in the vector backbone via side directed mutagenesis. Furthermore, the original multiple cloning site was replaced by a multiple cloning site compatible with the RFC 10/25 cloning standards. To allow easy extraction and purification of proteins for ''in vitro'' applications the new multiple cloning site allows to express proteins with a ''Strep''-tag II. <br />
Exclusion of the galactose inducible promoter provided a powerful basis vector for the integration of user-defined promoters. This way the pTUM100 vector gives a valuable contribution to our and to further protein expression and promoter characterization experiments in ''Saccharomyces cerevisiae''.<br />
Moreover, we used the pTUM100 to integrate the three constitutive promoters Tef1, Tef2 and ADH which come all with different promoter intensities.<br />
<br />
<center>'''Outlook and conclusion:'''</center><br><br />
<br />
The galactose inducible expression system was a great aid for the majority of all subprojects. Especially the opportunity to purify and detect (via Western blot) proteins using the ''Strep''-tag II did facilitate our laboratory practice and accelerated our work progress.<br />
To cover even more demands we are planning to design a second vector template containing a His-tag.<br />
<br />
All BioBricks were sequenced. Sequences can be found in the registry of standard biological parts under the following entries:<br />
<br />
[http://partsregistry.org/Part:BBa_K801000 pTUM100 BBa_K801000], [http://partsregistry.org/Part:BBa_K801001 pTUM101 BBa_K801001], [http://partsregistry.org/Part:BBa_K801002 pTUM102 BBa_K801002], [[http://partsregistry.org/Part:BBa_K801003 pTUM103 BBa_K801003] and [[http://partsregistry.org/Part:BBa_K801004 pTUM104 BBa_K801004]] <br />
<br />
<br />
<div class="noborder" style="float:left"><br />
<html><a href="https://2012.igem.org/Team:TU_Munich/Project/Vector_Design"><img width="200px" src="https://static.igem.org/mediawiki/2012/3/33/TUM12_readmore.png"/></a></html><br />
</div><br />
</div><br />
<br />
==Regulation of Genexpression==<br />
----<br />
By developing inducible promoters and placing them upstream of our biosynthetic pathways we create the possibility to make ''S. cerevisiae'' dynamically respond to concentration changes in its medium as well as to external stimuli. <br />
<br />
An optimal inducing substance needs to be inexpensive, nontoxic and fully controllable in its application. Only substances with these characteristics allow to precisely regulate a system temporally, spatially and quantitatively. <br />
<br />
<br />
<br />
<div class="bezel mfull"><br />
===Ethanol-inducible promoter===<br />
The KlADH4-promoter from the yeast ''Kluyveromyces lactis'' regulates the expression of a mitochondrial alcohol dehydrogenase in an ethanol-dependent way. An alcohol-inducible promoter would be incredibly useful for anyone planning to brew a beer with a transgenic yeast - it would allow for the induction of the target genes after the main fermentation has finished and this way, the metabolic burden for the yeast cells could be lowered. All the transcription factors known to be involved in the regulation of the KlADH4-promoter in ''K. lactis'' also occur in ''S. cerevisiae'' [[http://www.ncbi.nlm.nih.gov/pubmed/10724480 Mazzoni et al., 2000]]. This is why we are confident that this promoter maintains its unique characteristics when transformed into ''S. cerevisiae''.<hr><br />
[[file:TUM12_experiment_overwiew_alcohol.png|400px|thumb|right| '''Top''': Emission spectrum, excitation spectrum and visible fluorescence of a yeast culture expressing eGFP under the control of the KlADH4-promoter at 1.72 Vol.-% ethanol. Significant amounts of eGFP detectable '''Center''': Emission spectrum and excitation spectrum of a yeast culture carrying a plasmid in which eGFP-expression is controlled by the KlADH4-promoter cultivated with glycerol as only carbon source. Ethanol concentration: 0.21 Vol.-%. No eGFP detectable. '''Bottom''': Fluorescence per cell and ethanol concentration plotted against time after induction.]]<br />
<center>'''Experimental results:'''</center><br><br />
At this time our results concerning the KlADH4-promoter (originally from the yeast ''Kluyveromyces lactis'') suggest that this promoter is ethanol inducible in ''S. cerevisiae''. Further experiments are still being done to abolish residual ambiguities. The lowest ethanol concentration at which eGFP-expression was detected is 0.9 Vol.-%.<br />
<br />
<center>'''Conclusion and outlook:'''</center><br><br />
<br />
Because ''S. cerevisiae'' is such a good brewer, it was difficult to produce a stringent negative control in our characterization experiments. However, we finally figured out some experiments that allowed us to keep the ethanol concentration below 0.5 % v/v, which is a concentration at which induction is observed in ''K. lactis''. We are working hard on providing additional data, but we are confident that we will be able to provide clear evidence that this promoter is ethanol-inducible not only in ''K. lactis'', but also in ''S. cerevisiae''.<br />
<br />
<div class="noborder" style="float:left"><br />
<html><a href="https://2012.igem.org/Team:TU_Munich/Project/Ethanol_Inducible_Promoter"><img width="200px" src="https://static.igem.org/mediawiki/2012/3/33/TUM12_readmore.png"/></a></html><br />
</div><br />
</div><br />
<br />
<div class="bezel mfull"><br />
<br />
===Light-switchable promoter===<br />
The idea behind a light-switchable system is to create a gene expression system which can be induced and deactivated by light of a certain wavelength.<br />
<br />
This system is extremely attractive, as induction does not require the addition of a specific substance. This makes induction '''cheap, fast, precise''' and also compatible with the Bavarian purity law.<br />
<hr><br />
[[Image:TUM12_light.jpg|thumb|right|300px|Principle of light-dependent switching of gene-expression.]]<br />
<center>'''Experimental results:'''</center><br><br />
All fusion proteins for the two types of a light-switchable promoter system has been finished ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801039 BBa_K801039], [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801040 BBa_K801040] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801041 BBa_K801041]), also gene expression batteries coding for all components of each type of our light-switchable promoter system has been done ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801042 BBa_K801042] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801043 BBa_K801043]). Since lacking of a second functional yeast vector carrying another auxotrophy marker than URA3 of the pTUM plasmids, which is already reserved for the biosynthesis enzymes, proteins and also reporters, we were not able to clone the whole gene expression battery, into a yeast vector, in order to co-transfect the yeast with one plasmid with the reporter construct and the second plasmid coding for all the devices needed in a light-switchable promoter system.<br />
<br />
<center>'''Conclusion and outlook:'''</center><br><br />
<br />
To get gene expression casette for both of the light-switchable promoter systems ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801042 BBa_K801042] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801043 BBa_K801043]) into a yeast plasmid, we want to use pSB6A0 ([http://partsregistry.org/Part:BBa_K268000 BBa_K268000]) carrying a TRP1 auxotrophy marker. But we've discovered some discrepancies in this part, e.&nbsp;g. there is still at least one forbidden restriction site (XbaI or PstI, please see experience site of the part). So we have to correct the part by quickchange mutagenesis.<br />
<br />
After successful cloning and transfection of the gene expression batteries including their reporters, we will proceed with the characterization of the part.<br />
<br />
In parallel, we will implement also the whole biosynthesis pathway for the phycocyanobilin synthesis, downstream of our both batteries for a light-switchable promoter system ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801042 BBa_K801042] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801043 BBa_K801043]); after that, we don't have to add phycocyanobilin in our medium anymore!<br />
<br />
If there is still time in the end, we will clone our system including the biosynthetic genes for PCB synthesis in the yeast integrative vector ([http://partsregistry.org/Part:BBa_K300001 BBa_K300001]) and will start a genome integration in order to create a new yeast strain with a fully functional light-switchable promoter system.<br />
<br />
<div class="noborder" style="float:left"><br />
<html><a href="https://2012.igem.org/Team:TU_Munich/Project/Light_Switchable_Promoter"><img width="200px" src="https://static.igem.org/mediawiki/2012/3/33/TUM12_readmore.png"/></a></html><br />
</div><br />
</div><br />
<br />
==Genome Integration==<br />
----<br />
<div class="bezel mfull"><br />
As we cannot obey the letter of the German purity law (there is a zero tolerance policy concerning transgenic ingredients), we try our best to meet the spirit. Thus, it is <b>unacceptable for us to work with antibiotics</b> to keep up the selective environment. Since we <b>cannot work with auxotrophies</b> in beer either, we have to make sure the yeasts do not lose the BioBricks. The most promising way to accomplish a long lasting presence of our constructs is to achieve <b>genome integration</b>.<br />
<hr><br />
[[file:TUM12_experiment_overwiew_genome.png|500px|thumb|right| Plasmid backbone used for integration of our expression cassettes]]<br />
<center>'''Experimental results:'''</center><br><br />
First experiments to characterize the function of the yeast integration system were performed and the used selection marker was maintained in the yeast culture, although the selection pressure was switched off. This indicates that first integrations were achieved. <br />
<br />
<center>'''Conclusion and outlook:'''</center><br><br />
Maintaining the plasmids harboring our expression cassettes in the yeast cells during the brewing process is best possible using genome integration. This becomes increasingly interesting, when a yeast strain with different expression cassettes is to be created. Because this is intended for the next step of our project the integration of our expression cassettes becomes increasingly important.<br />
<br />
<div class="noborder" style="float:left"><br />
<html><a href="https://2012.igem.org/Team:TU_Munich/Project/Genome_Integration"><img width="200px" src="https://static.igem.org/mediawiki/2012/3/33/TUM12_readmore.png"/></a></html><br />
</div><br />
</div><br />
<br />
==Brewing our SynBio Beer==<br />
----<br />
<div class="bezel mfull"><br />
Contrary to popular opinion the chief ingredient of beer is not '''YPD''' but '''gyle''', a carefully prepared mixture of malt, hop and water. Although the name of the yeast strain commonly used in the lab, '''S. cerevisiae''', suggests that it is used in the beer brewing process, the yeast strains generally employed in brewing process have '''strongly adapted to gyle''', as they are reutilized in every succeeding brewing cycle.<br />
Hence some investigation on how our yeast '''performs''' in gyle was necessary.<br />
<br><hr><br />
[[file:TUM12_experiment_overwiew_Brewing.png|200px|thumb|right| Picture of the first SynBio Beer brewed during the iGEM competition in 2012]]<br />
<center>'''Experimental results:'''</center><br><br />
Our experiments show that the growth of several different yeast strains is '''not impaired in gyle'''!<br />
<br />
Expression assays proved the necessity of [[Team:TU_Munich/Project/Genome_Integration|'''genome integration''']] for a proper '''SynBio Beer'''.<br />
<br />
<br />
<center>'''Conclusion and outlook:'''</center><br><br />
As soon as cloning of integration vectors containing expression cassettes for our biosynthetic pathways are finished we will repeat the brewing experiments.<br />
<br />
<div class="noborder" style="float:left"><br />
<html><a href="https://2012.igem.org/Team:TU_Munich/Project/Brewing"><img width="200px" src="https://static.igem.org/mediawiki/2012/3/33/TUM12_readmore.png"/></a></html><br />
</div><br />
</div><br />
<br />
== References ==<br />
----<br />
* [[http://www.ncbi.nlm.nih.gov/pubmed/6327079 Edens et al., 1984]] Edens, L., Bom, I., Ledeboer, A. M., Maat, J., Toonen, M. Y., Visser, C., and Verrips, C. T. (1984). Synthesis and processing of the plant protein thaumatin in yeast. ''Cell'', 37(2):629–33.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/10995285 Miranda et al., 2000]] Miranda, C. L., Stevens, J. F., Ivanov, V., McCall, M., Frei, B., Deinzer, M. L., and Buhler, D. R. (2000). Antioxidant and prooxidant actions of prenylated and nonprenylated chalcones and flavanones in vitro. ''J Agric Food Chem'', 48(9):3876–84.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/10724480 Mazzoni et al., 2000]] Mazzoni, C., Santori, F., Saliola, M., and Falcone, C. (2000). Molecular analysis of uas(e), a cis element containing stress response elements responsible for ethanol induction of the kladh4 gene of ''kluyveromyces lactis''. ''Res Microbiol'', 151(1):19–28.</div>JaraOhttp://2012.igem.org/Team:TU_Munich/Project/OverviewTeam:TU Munich/Project/Overview2012-10-23T20:42:21Z<p>JaraO: /* Light-switchable promoter */</p>
<hr />
<div>{{Team:TU_Munich/Header}}<br />
<br />
= Overview =<br />
<hr/><br />
<br />
== Vision ==<br />
----<br />
We, TU Munich’s 2012 iGEM team, strive to catalyze the diffusion process of knowledge about genetic engineering and synthetic biology among the general public. Using the example of iGEM’s first and finest SynBio Beer we involve, interest and inspire people to reconsider preconceived ideas and encourage them to openly engage in a broad discussion weighing pros and cons of genetic engineering in foodstuff. We sketch a future where new technology can be applied in a meaningful way to complement traditional foods or beverages.<br />
<br />
==Biosynthesis pathways==<br />
----<br />
<br />
<br />
<div class="bezel mfull"><br />
===Limonene===<br />
Limonene is a cyclic terpene and a major constituent of several citrus oils. D-Limonene has been used as a component of flavorings and fragrances. It is formed from geranyl pyrophosphate by limonene synthase.<br />
<br />
We successfully demonstrated the production of the flavoring substance limonene by expressing limonene synthase in ''S. cerevisiae'', which naturally synthesizes the educt geranyl pyrophosphate.<br />
<hr><br />
[[file:TUM12_experiment_overwiew_limonene.png|500px|thumb|right| Main results from our limonene subproject: reaction mechanism (A), constructed BioBricks (B) and proof of principle for the in vivo production of limonene]]<br />
<center>'''Experimental results:'''</center><br><br />
(+)-Limonene synthase 1 [[http://partsregistry.org/Part:BBa_K801065 BBa_K801065]] and (+)-limonene synthase 1 with yeast consensus sequence [[http://partsregistry.org/Part:BBa_K801060 BBa_K801060]] were successfully cloned into our new yeast expression vector pTUM100. Expression of recombinant limonene synthase in ''Saccharomyces cerevisiae'' was proven by western blotting. Subsequently the protein was purified using SA-chromatography and size exclusion chromatography. The functionality of the enzyme was verified by ''in vivo'' and ''in vitro'' detection of limonene via GC-MS. <br />
<br />
Furthermore, we established gene constructs of the limonene synthase coding sequence with different yeast specific promoters and terminators [[http://partsregistry.org/Part:BBa_K801062 BBa_K801062]], [[http://partsregistry.org/Part:BBa_K801063 BBa_K801063]], [[http://partsregistry.org/Part:BBa_K801064 BBa_K801064]]. <br />
<br />
Last but not least, we have brewed iGEM's first SynBio beer containing limonene.<br />
<br />
<center>'''Conclusion and outlook:'''</center><br><br />
<br />
We have achieved functional expression of ''Citrus limon'' limonene synthase and production of limonene in yeast. The last remaining step to a SynBio beer would be the integration of our gene constructs [[http://partsregistry.org/Part:BBa_K801062 BBa_K801062]], [[http://partsregistry.org/Part:BBa_K801063 BBa_K801063]], [[http://partsregistry.org/Part:BBa_K801064 BBa_K801064]] into the yeast genome.<br />
<br />
<div class="noborder" style="float:left"><br />
<html><a href="https://2012.igem.org/Team:TU_Munich/Project/Limonene"><img width="200px" src="https://static.igem.org/mediawiki/2012/3/33/TUM12_readmore.png"/></a></html><br />
</div><br />
</div><br />
<br />
<div class="bezel mfull"><br />
<br />
===Thaumatin===<br />
Thaumatin is a natural α+β-protein which is synthesized by the katamfe plant (''Thaumatococcus daniellii''). It is said to be 2,000 to 100,000 times sweeter than sucrose on molar basis, but the sweetness builds up slow and lasts long. It has been approved as a sweetener by the European Union (E957).<br />
<br />
Our aim is to have ''S. cerevisiae'' secrete functional thaumatin by expressing preprothaumatin – a principle which has been proven by [[http://www.ncbi.nlm.nih.gov/pubmed/6327079 Edens et al., 1984]].<br />
<hr><br />
[[file:TUM12_experiment_overwiew_Thaumati.png|500px|thumb|right| Main results from the Thaumatin subproject: Structure of Thaumatin (A), constructed BioBricks (B), profile of an ion exchange chromatography (IEC) used to detect our recombinant Thaumatin and a SDS-PAGE gel showing IEC elution fractions containing Thaumatin]]<br />
<center>'''Experimental results:'''</center><br><br />
The BioBrick for preprothaumatin [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801080 BBa_K801080] as well as an expression cassette [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801080 K801080] were successfully cloned, expressed in yeast, purified using an ion exchange chromatography (see figure C) and detected in the SDS-PAGE. Therefore, the expression of thaumatin in yeast could be demonstrated and functionality of the BioBrick is confirmed.<br />
<br />
<center>'''Conclusion and outlook:'''</center><br><br />
A proof of principle for the expression of thaumatin was achieved. Further goals are the increase of the expression of thaumatin and the investigation of the secretion.<br />
<br />
<div class="noborder" style="float:left"><br />
<html><a href="https://2012.igem.org/Team:TU_Munich/Project/Thaumatin"><img width="200px" src="https://static.igem.org/mediawiki/2012/3/33/TUM12_readmore.png"/></a></html><br />
</div><br />
</div><br />
<br />
<div class="bezel mfull"><br />
<br />
===Caffeine===<br />
Caffeine is a purine-alkaloid and its biosynthesis is known from coffee plants and tea plants. The molecule acts as a competitive antagonist of adenosine receptors and, therefore, increases indirectly neurotransmitter concentrations resulting in warding off drowsiness and restoring alertness. <br />
<br />
The idea is to perform a heterologous gene expression of the three enzymes 7-methylxanthosine synthase (CaXMT1), N-methyl nucleosidase (CaMXMT1) and caffeine synthase (CaDXMT1) required for caffeine biosynthesis in ''Saccharomyces cerevisiae''. <br />
<br />
<br />
<hr><br />
[[file:TUM12_experiment_overwiew_caffeine2.png|500px|thumb|right| Figure showing a schematic overview of the reaction in (A), a western blot against the Strep-tag II for GFP, [[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801070 BBa_K801070]] and [[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801071 BBa_K801071]] in (B) and finally the same western blot development for [[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801072 BBa_K801072]]]]<br />
<center>'''Experimental results:'''</center><br><br />
* Successful cloning of the three enzymes [http://partsregistry.org/Part:BBa_K801070 7-methylxanthosine synthase (CaXMT1)], [http://partsregistry.org/Part:BBa_K801071 N-methyl nucleosidase (CaMXMT1)] and the [http://partsregistry.org/Part:BBa_K801072 caffeine synthase (CaDXMT1)] into the shuttle vector pTUM104 and pSB1C3 each. <br />
* Successful assembly of the BioBricks to form expression cassettes consisting of promoter, gene and terminator: [http://partsregistry.org/Part:BBa_K801073 pTEF2-CaXMT1-tADH1], [http://partsregistry.org/Part:BBa_K801074 pTEF1-CaMXMT1-tADH1] and [http://partsregistry.org/Part:BBa_K801075 pTEF2-CaDXMT1-tADH1]) into pSB1C3.<br />
* Successful assembly of the expression cassettes of the three relevant enzymes forming a composite part of 6.4 kb capable of caffeine production in yeast ([http://partsregistry.org/Part:BBa_K801077 Caffeine Synthesis Pathway]) into pSB1C3.<br />
* Successful expression of CaXMT1, CaMXMT1 and CaDXMT1 in ''Saccharomyces cerevisiae'' INVSc1 in selective Sc minimal induction medium lacking uracil with 2 % galactose.<br />
<br />
<br />
<center>'''Conclusion and outlook:'''</center><br><br />
The homologue expression of the three required enzymes for caffeine synthesis in ''Saccharomyces cerevisiae'' INVSc1 transformed with pTUM102_CaXMT1, pTUM102_CaMXMT1 and pTUM102_CaDXMT1 was successful. Further testing of caffeine production using crude extracts from lysed transformed yeast cells and working on genome integration of the expression cassette containing all three enzymes flanked by promoter and terminator are in progress.<br />
<br />
<div class="noborder" style="float:left"><br />
<html><a href="https://2012.igem.org/Team:TU_Munich/Project/Caffeine"><img width="200px" src="https://static.igem.org/mediawiki/2012/3/33/TUM12_readmore.png"/></a></html><br />
</div><br />
</div><br />
<br />
<div class="bezel mfull"><br />
<br />
===Xanthohumol===<br />
Xanthohumol is known as a putative cancer chemopreventive agent due to its antioxidant activities [[http://www.ncbi.nlm.nih.gov/pubmed/10995285 Miranda et al., 2000]]. Our goal is a heterologous gene expression of all enzymes required for xanthohumol biosynthesis in ''S. cerevisiae''.<br />
<br />
The pathway for the production of this plant secondary metabolite is composed of five steps, starting with the conversion of phenylalanine and followed by four further enzymatic reactions.<br />
<hr><br />
[[file:TUM12_experiment_overwiew_xanto2.png|500px|thumb|right| Main results from the Xanthohumol subproject: Successful reconstruction of the metabolic pathway for Xanthohumol]]<br />
<center>'''Experimental results:'''</center><br><br />
The whole biosynthetic pathway for the production of xanthohumol was converted into BioBricks. Except for APT each of the enzymes were cloned in two versions one having the proposed consensus sequence for more efficient expression in yeast chassis and another for usage of these BioBricks in other chassis. All BioBricks were sequenced. Sequences can be found in the registry of standard biological parts:<br />
[http://partsregistry.org/Part:BBa_K801090 BBa_K801090], [http://partsregistry.org/Part:BBa_K801091 BBa_K801091], [http://partsregistry.org/Part:BBa_K801092 BBa_K801092], [http://partsregistry.org/Part:BBa_K801093 BBa_K801093], [http://partsregistry.org/Part:BBa_K801094 BBa_K801094], [http://partsregistry.org/Part:BBa_K801095 BBa_K801095], [http://partsregistry.org/Part:BBa_K801096 BBa_K801096], [http://partsregistry.org/Part:BBa_K801097 BBa_K801097] and [http://partsregistry.org/Part:BBa_K801098 BBa_K801098] <br />
<br />
<br />
<center>'''Conclusion and outlook:'''</center><br><br />
<br />
The construction of the xanthohumol pathway was achieved, whereas the expression and characterization have to be postponed after the European Jamboree or might be an interesting task for next year's iGEM teams.<br />
<br />
<div class="noborder" style="float:left"><br />
<html><a href="https://2012.igem.org/Team:TU_Munich/Project/Xanthohumol"><img width="200px" src="https://static.igem.org/mediawiki/2012/3/33/TUM12_readmore.png"/></a></html><br />
</div><br />
</div><br />
<br />
==Vector Design==<br />
----<br />
<div class="bezel mfull"><br />
===pTUM100===<br />
Designing an expression vector for yeast which is compatible to the iGEM cloning principles and standards was the main aim of this subproject. Based on the commercially available pYES2 vector we created vectors containing inducible and constitutive promoters in order to establish efficient possibilities to clone and express our enzymes.<br />
<hr><br />
[[file:TUM12_experiment_overwiew_vector.png|500px|thumb|right| Explanations on the figure:<br />
<br />
Figure A shows the new multiple cloning site (MCS) containing the RFC 10/25 restriction sides and the DNA sequence coding for the ''Strep''-tag II.<br />
<br />
Figure B gives an overview of all important functional elements located on the vector backbone. Upstream to the new MCS lies a T7 promoter primer binding site allowing easy forward sequencing of integrated gene constructs using the standard T7 primer. The URA 3 gene is a prototrophy marker used for the selection of transfected cells.<br />
<br />
Figure C to E present the successfully designed BioBricks: pTUM100 simply contains the new MCS, the transcription terminator and further elements required for cloning and transfection. pTUM102 to pTUM104 contain in addition the constitutive promoters pTef1, pTef2 and ADH. On pTUM104 is the galactose inducible promoter pGAL1 located. ]]<br />
<br />
<center>'''Experimental results:'''</center><br><br />
Using the pYES vector from Invitrogen we first deleted five forbidden restriction sites in the vector backbone via side directed mutagenesis. Furthermore, the original multiple cloning site was replaced by a multiple cloning site compatible with the RFC 10/25 cloning standards. To allow easy extraction and purification of proteins for ''in vitro'' applications the new multiple cloning site allows to express proteins with a ''Strep''-tag II. <br />
Exclusion of the galactose inducible promoter provided a powerful basis vector for the integration of user-defined promoters. This way the pTUM100 vector gives a valuable contribution to our and to further protein expression and promoter characterization experiments in ''Saccharomyces cerevisiae''.<br />
Moreover, we used the pTUM100 to integrate the three constitutive promoters Tef1, Tef2 and ADH which come all with different promoter intensities.<br />
<br />
<center>'''Outlook and conclusion:'''</center><br><br />
<br />
The galactose inducible expression system was a great aid for the majority of all subprojects. Especially the opportunity to purify and detect (via Western blot) proteins using the ''Strep''-tag II did facilitate our laboratory practice and accelerated our work progress.<br />
To cover even more demands we are planning to design a second vector template containing a His-tag.<br />
<br />
All BioBricks were sequenced. Sequences can be found in the registry of standard biological parts under the following entries:<br />
<br />
[http://partsregistry.org/Part:BBa_K801000 pTUM100 BBa_K801000], [http://partsregistry.org/Part:BBa_K801001 pTUM101 BBa_K801001], [http://partsregistry.org/Part:BBa_K801002 pTUM102 BBa_K801002], [[http://partsregistry.org/Part:BBa_K801003 pTUM103 BBa_K801003] and [[http://partsregistry.org/Part:BBa_K801004 pTUM104 BBa_K801004]] <br />
<br />
<br />
<div class="noborder" style="float:left"><br />
<html><a href="https://2012.igem.org/Team:TU_Munich/Project/Vector_Design"><img width="200px" src="https://static.igem.org/mediawiki/2012/3/33/TUM12_readmore.png"/></a></html><br />
</div><br />
</div><br />
<br />
==Regulation of Genexpression==<br />
----<br />
By developing inducible promoters and placing them upstream of our biosynthetic pathways we create the possibility to make ''S. cerevisiae'' dynamically respond to concentration changes in its medium as well as to external stimuli. <br />
<br />
An optimal inducing substance needs to be inexpensive, nontoxic and fully controllable in its application. Only substances with these characteristics allow to precisely regulate a system temporally, spatially and quantitatively. <br />
<br />
<br />
<br />
<div class="bezel mfull"><br />
===Ethanol-inducible promoter===<br />
The KlADH4-promoter from the yeast ''Kluyveromyces lactis'' regulates the expression of a mitochondrial alcohol dehydrogenase in an ethanol-dependent way. An alcohol-inducible promoter would be incredibly useful for anyone planning to brew a beer with a transgenic yeast - it would allow for the induction of the target genes after the main fermentation has finished and this way, the metabolic burden for the yeast cells could be lowered. All the transcription factors known to be involved in the regulation of the KlADH4-promoter in ''K. lactis'' also occur in ''S. cerevisiae'' [[http://www.ncbi.nlm.nih.gov/pubmed/10724480 Mazzoni et al., 2000]]. This is why we are confident that this promoter maintains its unique characteristics when transformed into ''S. cerevisiae''.<hr><br />
[[file:TUM12_experiment_overwiew_alcohol.png|400px|thumb|right| '''Top''': Emission spectrum, excitation spectrum and visible fluorescence of a yeast culture expressing eGFP under the control of the KlADH4-promoter at 1.72 Vol.-% ethanol. Significant amounts of eGFP detectable '''Center''': Emission spectrum and excitation spectrum of a yeast culture carrying a plasmid in which eGFP-expression is controlled by the KlADH4-promoter cultivated with glycerol as only carbon source. Ethanol concentration: 0.21 Vol.-%. No eGFP detectable. '''Bottom''': Fluorescence per cell and ethanol concentration plotted against time after induction.]]<br />
<center>'''Experimental results:'''</center><br><br />
At this time our results concerning the KlADH4-promoter (originally from the yeast ''Kluyveromyces lactis'') suggest that this promoter is ethanol inducible in ''S. cerevisiae''. Further experiments are still being done to abolish residual ambiguities. The lowest ethanol concentration at which eGFP-expression was detected is 0.9 Vol.-%.<br />
<br />
<center>'''Conclusion and outlook:'''</center><br><br />
<br />
Because ''S. cerevisiae'' is such a good brewer, it was difficult to produce a stringent negative control in our characterization experiments. However, we finally figured out some experiments that allowed us to keep the ethanol concentration below 0.5 % v/v, which is a concentration at which induction is observed in ''K. lactis''. We are working hard on providing additional data, but we are confident that we will be able to provide clear evidence that this promoter is ethanol-inducible not only in ''K. lactis'', but also in ''S. cerevisiae''.<br />
<br />
<div class="noborder" style="float:left"><br />
<html><a href="https://2012.igem.org/Team:TU_Munich/Project/Ethanol_Inducible_Promoter"><img width="200px" src="https://static.igem.org/mediawiki/2012/3/33/TUM12_readmore.png"/></a></html><br />
</div><br />
</div><br />
<br />
<div class="bezel mfull"><br />
<br />
===Light-switchable promoter===<br />
The idea behind a light-switchable system is to create a gene expression system which can be induced and deactivated by light of a certain wavelength.<br />
<br />
This system is extremely attractive, as induction does not require the addition of a specific substance. This makes induction '''cheap, fast, precise''' and also compatible with the Bavarian purity law.<br />
<hr><br />
[[Image:TUM12_light.jpg|thumb|right|300px|Principle of light-dependent switching of gene-expression.]]<br />
<center>'''Experimental results:'''</center><br><br />
All fusion proteins for the two types of a light-switchable promoter system has been finished ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801039 BBa_K801039], [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801040 BBa_K801040] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801041 BBa_K801041]), also gene expression batteries coding for all components of each type of our light-switchable promoter system has been done ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801042 BBa_K801042] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801043 BBa_K801043]). Since lacking of a second functional yeast vector carrying another auxotrophy marker than URA3 of the pTUM plasmids, which is already reserved for the biosynthesis enzymes, proteins and also reporters, we were not able to clone the whole gene expression battery, into a yeast vector, in order to co-transfect the yeast with one plasmid with the reporter construct and the second plasmid coding for all the devices needed in a light-switchable promoter system.<br />
<br />
<center>'''Conclusion and outlook:'''</center><br><br />
<br />
To get gene expression casette for both of the light-switchable promoter systems ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801042 BBa_K801042] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801043 BBa_K801043]) into a yeast plasmid, we want to use pSB6A0 ([http://partsregistry.org/Part:BBa_K268000 BBa_K268000]) carrying a TRP1 auxotrophy marker. But we've discovered some discrepancies in this part, e.&nbsp;g. there is still at least one forbidden restriction site (XbaI or PstI, please see experience site of the part). So we have to correct the part by quickchange mutagenesis.<br />
<br />
After successful cloning and transfection of the gene expression batteries including their reporters, we will proceed with the characterization of the part.<br />
<br />
In parallel, we will implement also the whole biosynthesis pathway for the phycocyanobilin synthesis, downstream of our both batteries for a light-switchable promoter system ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801042 BBa_K801042] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801043 BBa_K801043]); after that, we don't have to add phycocyanobilin in our medium anymore!<br />
<br />
If there is still time in the end, we will clone our system including the biosynthetic genes for PCB synthesis in the yeast integrative vector ([http://partsregistry.org/Part:BBa_K300001 BBa_K300001]) and will start a genome integration in order to create a new yeast strain with a fully functional light-switchable promoter system.<br />
<br />
<div class="noborder" style="float:left"><br />
<html><a href="https://2012.igem.org/Team:TU_Munich/Project/Light_Switchable_Promoter"><img width="200px" src="https://static.igem.org/mediawiki/2012/3/33/TUM12_readmore.png"/></a></html><br />
</div><br />
</div><br />
<br />
==Genome Integration==<br />
----<br />
<div class="bezel mfull"><br />
As we cannot obey the letter of the German purity law (there is a zero tolerance policy concerning transgenic ingredients), we try our best to meet the spirit. Thus, it is <b>unacceptable for us to work with antibiotics</b> to keep up the selective environment. Since we <b>cannot work with auxotrophies</b> in beer either, we have to make sure the yeasts do not lose the BioBricks. The most promising way to accomplish a long lasting presence of our constructs is to achieve <b>genome integration</b>.<br />
<hr><br />
[[file:TUM12_experiment_overwiew_genome.png|500px|thumb|right| Plasmid backbone used for integration of our expression cassettes]]<br />
<center>'''Experimental results:'''</center><br><br />
First experiments to characterize the function of the yeast integration system were performed and the used selection marker was maintained in the yeast culture, although the selection pressure was switched off. This indicates that first integrations were achieved. <br />
<br />
<center>'''Conclusion and outlook:'''</center><br><br />
Maintaining the plasmids harboring our expression cassettes in the yeast cells during the brewing process is best possible using genome integration. This becomes increasingly interesting, when a yeast strain with different expression cassettes is to be created. Because this is intended for the next step of our project the integration of our expression cassettes becomes increasingly important.<br />
<br />
<div class="noborder" style="float:left"><br />
<html><a href="https://2012.igem.org/Team:TU_Munich/Project/Genome_Integration"><img width="200px" src="https://static.igem.org/mediawiki/2012/3/33/TUM12_readmore.png"/></a></html><br />
</div><br />
</div><br />
<br />
==Brewing our SynBio Beer==<br />
----<br />
<div class="bezel mfull"><br />
Contrary to popular opinion the chief ingredient of beer is not '''YPD''' but '''gyle''', a carefully prepared mixture of malt, hop and water. Although the name of the yeast strain commonly used in the lab, '''S. cerevisiae''', suggests that it is used in the beer brewing process, the yeast strains generally employed in brewing process have '''strongly adapted to gyle''', as they are reutilized in every succeeding brewing cycle.<br />
Hence some investigation on how our yeast '''performs''' in gyle was necessary.<br />
<br><hr><br />
[[file:TUM12_experiment_overwiew_Brewing.png|200px|thumb|right| Picture of the first SynBio Beer brewed during the iGEM competition in 2012]]<br />
<center>'''Experimental results:'''</center><br><br />
Our experiments show that the growth of several different yeast strains is '''not impaired in gyle'''!<br />
<br />
Expression assays proved the necessity of [[Team:TU_Munich/Project/Genome_Integration|'''genome integration''']] for a proper '''SynBio Beer'''.<br />
<br />
<center>'''Conclusion and outlook:'''</center><br><br />
As soon as cloning of integration vectors containing expression cassettes for our biosynthetic pathways are finished we will repeat the brewing experiments.<br />
<br />
<div class="noborder" style="float:left"><br />
<html><a href="https://2012.igem.org/Team:TU_Munich/Project/Brewing"><img width="200px" src="https://static.igem.org/mediawiki/2012/3/33/TUM12_readmore.png"/></a></html><br />
</div><br />
</div><br />
<br />
== References ==<br />
----<br />
* [[http://www.ncbi.nlm.nih.gov/pubmed/6327079 Edens et al., 1984]] Edens, L., Bom, I., Ledeboer, A. M., Maat, J., Toonen, M. Y., Visser, C., and Verrips, C. T. (1984). Synthesis and processing of the plant protein thaumatin in yeast. ''Cell'', 37(2):629–33.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/10995285 Miranda et al., 2000]] Miranda, C. L., Stevens, J. F., Ivanov, V., McCall, M., Frei, B., Deinzer, M. L., and Buhler, D. R. (2000). Antioxidant and prooxidant actions of prenylated and nonprenylated chalcones and flavanones in vitro. ''J Agric Food Chem'', 48(9):3876–84.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/10724480 Mazzoni et al., 2000]] Mazzoni, C., Santori, F., Saliola, M., and Falcone, C. (2000). Molecular analysis of uas(e), a cis element containing stress response elements responsible for ethanol induction of the kladh4 gene of ''kluyveromyces lactis''. ''Res Microbiol'', 151(1):19–28.</div>JaraOhttp://2012.igem.org/Team:TU_Munich/Project/OverviewTeam:TU Munich/Project/Overview2012-10-23T20:41:39Z<p>JaraO: /* Light-switchable promoter */</p>
<hr />
<div>{{Team:TU_Munich/Header}}<br />
<br />
= Overview =<br />
<hr/><br />
<br />
== Vision ==<br />
----<br />
We, TU Munich’s 2012 iGEM team, strive to catalyze the diffusion process of knowledge about genetic engineering and synthetic biology among the general public. Using the example of iGEM’s first and finest SynBio Beer we involve, interest and inspire people to reconsider preconceived ideas and encourage them to openly engage in a broad discussion weighing pros and cons of genetic engineering in foodstuff. We sketch a future where new technology can be applied in a meaningful way to complement traditional foods or beverages.<br />
<br />
==Biosynthesis pathways==<br />
----<br />
<br />
<br />
<div class="bezel mfull"><br />
===Limonene===<br />
Limonene is a cyclic terpene and a major constituent of several citrus oils. D-Limonene has been used as a component of flavorings and fragrances. It is formed from geranyl pyrophosphate by limonene synthase.<br />
<br />
We successfully demonstrated the production of the flavoring substance limonene by expressing limonene synthase in ''S. cerevisiae'', which naturally synthesizes the educt geranyl pyrophosphate.<br />
<hr><br />
[[file:TUM12_experiment_overwiew_limonene.png|500px|thumb|right| Main results from our limonene subproject: reaction mechanism (A), constructed BioBricks (B) and proof of principle for the in vivo production of limonene]]<br />
<center>'''Experimental results:'''</center><br><br />
(+)-Limonene synthase 1 [[http://partsregistry.org/Part:BBa_K801065 BBa_K801065]] and (+)-limonene synthase 1 with yeast consensus sequence [[http://partsregistry.org/Part:BBa_K801060 BBa_K801060]] were successfully cloned into our new yeast expression vector pTUM100. Expression of recombinant limonene synthase in ''Saccharomyces cerevisiae'' was proven by western blotting. Subsequently the protein was purified using SA-chromatography and size exclusion chromatography. The functionality of the enzyme was verified by ''in vivo'' and ''in vitro'' detection of limonene via GC-MS. <br />
<br />
Furthermore, we established gene constructs of the limonene synthase coding sequence with different yeast specific promoters and terminators [[http://partsregistry.org/Part:BBa_K801062 BBa_K801062]], [[http://partsregistry.org/Part:BBa_K801063 BBa_K801063]], [[http://partsregistry.org/Part:BBa_K801064 BBa_K801064]]. <br />
<br />
Last but not least, we have brewed iGEM's first SynBio beer containing limonene.<br />
<br />
<center>'''Conclusion and outlook:'''</center><br><br />
<br />
We have achieved functional expression of ''Citrus limon'' limonene synthase and production of limonene in yeast. The last remaining step to a SynBio beer would be the integration of our gene constructs [[http://partsregistry.org/Part:BBa_K801062 BBa_K801062]], [[http://partsregistry.org/Part:BBa_K801063 BBa_K801063]], [[http://partsregistry.org/Part:BBa_K801064 BBa_K801064]] into the yeast genome.<br />
<br />
<div class="noborder" style="float:left"><br />
<html><a href="https://2012.igem.org/Team:TU_Munich/Project/Limonene"><img width="200px" src="https://static.igem.org/mediawiki/2012/3/33/TUM12_readmore.png"/></a></html><br />
</div><br />
</div><br />
<br />
<div class="bezel mfull"><br />
<br />
===Thaumatin===<br />
Thaumatin is a natural α+β-protein which is synthesized by the katamfe plant (''Thaumatococcus daniellii''). It is said to be 2,000 to 100,000 times sweeter than sucrose on molar basis, but the sweetness builds up slow and lasts long. It has been approved as a sweetener by the European Union (E957).<br />
<br />
Our aim is to have ''S. cerevisiae'' secrete functional thaumatin by expressing preprothaumatin – a principle which has been proven by [[http://www.ncbi.nlm.nih.gov/pubmed/6327079 Edens et al., 1984]].<br />
<hr><br />
[[file:TUM12_experiment_overwiew_Thaumati.png|500px|thumb|right| Main results from the Thaumatin subproject: Structure of Thaumatin (A), constructed BioBricks (B), profile of an ion exchange chromatography (IEC) used to detect our recombinant Thaumatin and a SDS-PAGE gel showing IEC elution fractions containing Thaumatin]]<br />
<center>'''Experimental results:'''</center><br><br />
The BioBrick for preprothaumatin [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801080 BBa_K801080] as well as an expression cassette [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801080 K801080] were successfully cloned, expressed in yeast, purified using an ion exchange chromatography (see figure C) and detected in the SDS-PAGE. Therefore, the expression of thaumatin in yeast could be demonstrated and functionality of the BioBrick is confirmed.<br />
<br />
<center>'''Conclusion and outlook:'''</center><br><br />
A proof of principle for the expression of thaumatin was achieved. Further goals are the increase of the expression of thaumatin and the investigation of the secretion.<br />
<br />
<div class="noborder" style="float:left"><br />
<html><a href="https://2012.igem.org/Team:TU_Munich/Project/Thaumatin"><img width="200px" src="https://static.igem.org/mediawiki/2012/3/33/TUM12_readmore.png"/></a></html><br />
</div><br />
</div><br />
<br />
<div class="bezel mfull"><br />
<br />
===Caffeine===<br />
Caffeine is a purine-alkaloid and its biosynthesis is known from coffee plants and tea plants. The molecule acts as a competitive antagonist of adenosine receptors and, therefore, increases indirectly neurotransmitter concentrations resulting in warding off drowsiness and restoring alertness. <br />
<br />
The idea is to perform a heterologous gene expression of the three enzymes 7-methylxanthosine synthase (CaXMT1), N-methyl nucleosidase (CaMXMT1) and caffeine synthase (CaDXMT1) required for caffeine biosynthesis in ''Saccharomyces cerevisiae''. <br />
<br />
<br />
<hr><br />
[[file:TUM12_experiment_overwiew_caffeine2.png|500px|thumb|right| Figure showing a schematic overview of the reaction in (A), a western blot against the Strep-tag II for GFP, [[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801070 BBa_K801070]] and [[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801071 BBa_K801071]] in (B) and finally the same western blot development for [[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801072 BBa_K801072]]]]<br />
<center>'''Experimental results:'''</center><br><br />
* Successful cloning of the three enzymes [http://partsregistry.org/Part:BBa_K801070 7-methylxanthosine synthase (CaXMT1)], [http://partsregistry.org/Part:BBa_K801071 N-methyl nucleosidase (CaMXMT1)] and the [http://partsregistry.org/Part:BBa_K801072 caffeine synthase (CaDXMT1)] into the shuttle vector pTUM104 and pSB1C3 each. <br />
* Successful assembly of the BioBricks to form expression cassettes consisting of promoter, gene and terminator: [http://partsregistry.org/Part:BBa_K801073 pTEF2-CaXMT1-tADH1], [http://partsregistry.org/Part:BBa_K801074 pTEF1-CaMXMT1-tADH1] and [http://partsregistry.org/Part:BBa_K801075 pTEF2-CaDXMT1-tADH1]) into pSB1C3.<br />
* Successful assembly of the expression cassettes of the three relevant enzymes forming a composite part of 6.4 kb capable of caffeine production in yeast ([http://partsregistry.org/Part:BBa_K801077 Caffeine Synthesis Pathway]) into pSB1C3.<br />
* Successful expression of CaXMT1, CaMXMT1 and CaDXMT1 in ''Saccharomyces cerevisiae'' INVSc1 in selective Sc minimal induction medium lacking uracil with 2 % galactose.<br />
<br />
<br />
<center>'''Conclusion and outlook:'''</center><br><br />
The homologue expression of the three required enzymes for caffeine synthesis in ''Saccharomyces cerevisiae'' INVSc1 transformed with pTUM102_CaXMT1, pTUM102_CaMXMT1 and pTUM102_CaDXMT1 was successful. Further testing of caffeine production using crude extracts from lysed transformed yeast cells and working on genome integration of the expression cassette containing all three enzymes flanked by promoter and terminator are in progress.<br />
<br />
<div class="noborder" style="float:left"><br />
<html><a href="https://2012.igem.org/Team:TU_Munich/Project/Caffeine"><img width="200px" src="https://static.igem.org/mediawiki/2012/3/33/TUM12_readmore.png"/></a></html><br />
</div><br />
</div><br />
<br />
<div class="bezel mfull"><br />
<br />
===Xanthohumol===<br />
Xanthohumol is known as a putative cancer chemopreventive agent due to its antioxidant activities [[http://www.ncbi.nlm.nih.gov/pubmed/10995285 Miranda et al., 2000]]. Our goal is a heterologous gene expression of all enzymes required for xanthohumol biosynthesis in ''S. cerevisiae''.<br />
<br />
The pathway for the production of this plant secondary metabolite is composed of five steps, starting with the conversion of phenylalanine and followed by four further enzymatic reactions.<br />
<hr><br />
[[file:TUM12_experiment_overwiew_xanto2.png|500px|thumb|right| Main results from the Xanthohumol subproject: Successful reconstruction of the metabolic pathway for Xanthohumol]]<br />
<center>'''Experimental results:'''</center><br><br />
The whole biosynthetic pathway for the production of xanthohumol was converted into BioBricks. Except for APT each of the enzymes were cloned in two versions one having the proposed consensus sequence for more efficient expression in yeast chassis and another for usage of these BioBricks in other chassis. All BioBricks were sequenced. Sequences can be found in the registry of standard biological parts:<br />
[http://partsregistry.org/Part:BBa_K801090 BBa_K801090], [http://partsregistry.org/Part:BBa_K801091 BBa_K801091], [http://partsregistry.org/Part:BBa_K801092 BBa_K801092], [http://partsregistry.org/Part:BBa_K801093 BBa_K801093], [http://partsregistry.org/Part:BBa_K801094 BBa_K801094], [http://partsregistry.org/Part:BBa_K801095 BBa_K801095], [http://partsregistry.org/Part:BBa_K801096 BBa_K801096], [http://partsregistry.org/Part:BBa_K801097 BBa_K801097] and [http://partsregistry.org/Part:BBa_K801098 BBa_K801098] <br />
<br />
<br />
<center>'''Conclusion and outlook:'''</center><br><br />
<br />
The construction of the xanthohumol pathway was achieved, whereas the expression and characterization have to be postponed after the European Jamboree or might be an interesting task for next year's iGEM teams.<br />
<br />
<div class="noborder" style="float:left"><br />
<html><a href="https://2012.igem.org/Team:TU_Munich/Project/Xanthohumol"><img width="200px" src="https://static.igem.org/mediawiki/2012/3/33/TUM12_readmore.png"/></a></html><br />
</div><br />
</div><br />
<br />
==Vector Design==<br />
----<br />
<div class="bezel mfull"><br />
===pTUM100===<br />
Designing an expression vector for yeast which is compatible to the iGEM cloning principles and standards was the main aim of this subproject. Based on the commercially available pYES2 vector we created vectors containing inducible and constitutive promoters in order to establish efficient possibilities to clone and express our enzymes.<br />
<hr><br />
[[file:TUM12_experiment_overwiew_vector.png|500px|thumb|right| Explanations on the figure:<br />
<br />
Figure A shows the new multiple cloning site (MCS) containing the RFC 10/25 restriction sides and the DNA sequence coding for the ''Strep''-tag II.<br />
<br />
Figure B gives an overview of all important functional elements located on the vector backbone. Upstream to the new MCS lies a T7 promoter primer binding site allowing easy forward sequencing of integrated gene constructs using the standard T7 primer. The URA 3 gene is a prototrophy marker used for the selection of transfected cells.<br />
<br />
Figure C to E present the successfully designed BioBricks: pTUM100 simply contains the new MCS, the transcription terminator and further elements required for cloning and transfection. pTUM102 to pTUM104 contain in addition the constitutive promoters pTef1, pTef2 and ADH. On pTUM104 is the galactose inducible promoter pGAL1 located. ]]<br />
<br />
<center>'''Experimental results:'''</center><br><br />
Using the pYES vector from Invitrogen we first deleted five forbidden restriction sites in the vector backbone via side directed mutagenesis. Furthermore, the original multiple cloning site was replaced by a multiple cloning site compatible with the RFC 10/25 cloning standards. To allow easy extraction and purification of proteins for ''in vitro'' applications the new multiple cloning site allows to express proteins with a ''Strep''-tag II. <br />
Exclusion of the galactose inducible promoter provided a powerful basis vector for the integration of user-defined promoters. This way the pTUM100 vector gives a valuable contribution to our and to further protein expression and promoter characterization experiments in ''Saccharomyces cerevisiae''.<br />
Moreover, we used the pTUM100 to integrate the three constitutive promoters Tef1, Tef2 and ADH which come all with different promoter intensities.<br />
<br />
<center>'''Outlook and conclusion:'''</center><br><br />
<br />
The galactose inducible expression system was a great aid for the majority of all subprojects. Especially the opportunity to purify and detect (via Western blot) proteins using the ''Strep''-tag II did facilitate our laboratory practice and accelerated our work progress.<br />
To cover even more demands we are planning to design a second vector template containing a His-tag.<br />
<br />
All BioBricks were sequenced. Sequences can be found in the registry of standard biological parts under the following entries:<br />
<br />
[http://partsregistry.org/Part:BBa_K801000 pTUM100 BBa_K801000], [http://partsregistry.org/Part:BBa_K801001 pTUM101 BBa_K801001], [http://partsregistry.org/Part:BBa_K801002 pTUM102 BBa_K801002], [[http://partsregistry.org/Part:BBa_K801003 pTUM103 BBa_K801003] and [[http://partsregistry.org/Part:BBa_K801004 pTUM104 BBa_K801004]] <br />
<br />
<br />
<div class="noborder" style="float:left"><br />
<html><a href="https://2012.igem.org/Team:TU_Munich/Project/Vector_Design"><img width="200px" src="https://static.igem.org/mediawiki/2012/3/33/TUM12_readmore.png"/></a></html><br />
</div><br />
</div><br />
<br />
==Regulation of Genexpression==<br />
----<br />
By developing inducible promoters and placing them upstream of our biosynthetic pathways we create the possibility to make ''S. cerevisiae'' dynamically respond to concentration changes in its medium as well as to external stimuli. <br />
<br />
An optimal inducing substance needs to be inexpensive, nontoxic and fully controllable in its application. Only substances with these characteristics allow to precisely regulate a system temporally, spatially and quantitatively. <br />
<br />
<br />
<br />
<div class="bezel mfull"><br />
===Ethanol-inducible promoter===<br />
The KlADH4-promoter from the yeast ''Kluyveromyces lactis'' regulates the expression of a mitochondrial alcohol dehydrogenase in an ethanol-dependent way. An alcohol-inducible promoter would be incredibly useful for anyone planning to brew a beer with a transgenic yeast - it would allow for the induction of the target genes after the main fermentation has finished and this way, the metabolic burden for the yeast cells could be lowered. All the transcription factors known to be involved in the regulation of the KlADH4-promoter in ''K. lactis'' also occur in ''S. cerevisiae'' [[http://www.ncbi.nlm.nih.gov/pubmed/10724480 Mazzoni et al., 2000]]. This is why we are confident that this promoter maintains its unique characteristics when transformed into ''S. cerevisiae''.<hr><br />
[[file:TUM12_experiment_overwiew_alcohol.png|400px|thumb|right| '''Top''': Emission spectrum, excitation spectrum and visible fluorescence of a yeast culture expressing eGFP under the control of the KlADH4-promoter at 1.72 Vol.-% ethanol. Significant amounts of eGFP detectable '''Center''': Emission spectrum and excitation spectrum of a yeast culture carrying a plasmid in which eGFP-expression is controlled by the KlADH4-promoter cultivated with glycerol as only carbon source. Ethanol concentration: 0.21 Vol.-%. No eGFP detectable. '''Bottom''': Fluorescence per cell and ethanol concentration plotted against time after induction.]]<br />
<center>'''Experimental results:'''</center><br><br />
At this time our results concerning the KlADH4-promoter (originally from the yeast ''Kluyveromyces lactis'') suggest that this promoter is ethanol inducible in ''S. cerevisiae''. Further experiments are still being done to abolish residual ambiguities. The lowest ethanol concentration at which eGFP-expression was detected is 0.9 Vol.-%.<br />
<br />
<center>'''Conclusion and outlook:'''</center><br><br />
<br />
Because ''S. cerevisiae'' is such a good brewer, it was difficult to produce a stringent negative control in our characterization experiments. However, we finally figured out some experiments that allowed us to keep the ethanol concentration below 0.5 % v/v, which is a concentration at which induction is observed in ''K. lactis''. We are working hard on providing additional data, but we are confident that we will be able to provide clear evidence that this promoter is ethanol-inducible not only in ''K. lactis'', but also in ''S. cerevisiae''.<br />
<br />
<div class="noborder" style="float:left"><br />
<html><a href="https://2012.igem.org/Team:TU_Munich/Project/Ethanol_Inducible_Promoter"><img width="200px" src="https://static.igem.org/mediawiki/2012/3/33/TUM12_readmore.png"/></a></html><br />
</div><br />
</div><br />
<br />
<div class="bezel mfull"><br />
<br />
===Light-switchable promoter===<br />
The idea behind a light-switchable system is to create a gene expression system which can be induced and deactivated by light of a certain wavelength.<br />
<br />
This system is extremely attractive, as induction does not require the addition of a specific substance. This makes induction '''cheap, fast, precise''' and also compatible with the Bavarian purity law.<br />
<hr><br />
[[Image:TUM12_light.jpg|thumb|right|300px|Principle of light-dependent switching of gene-expression.]]<br />
<center>'''Experimental results:'''</center><br><br />
All fusion proteins for the two types of a light-switchable promoter system has been finished ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801039 BBa_K801039], [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801040 BBa_K801040] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801041 BBa_K801041]), also gene expression batteries coding for all components of each type of our light-switchable promoter system has been done ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801042 BBa_K801042] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801043 BBa_K801043]). Since lacking of a second functional yeast vector carrying another auxotrophy marker than URA3 of the pTUM plasmids, which is already reserved for the biosynthesis enzymes, proteins and also reporters, we were not able to clone the whole gene expression battery, into a yeast vector, in order to co-transfect the yeast with one plasmid with the reporter construct and the second plasmid coding for all the devices needed in a light-switchable promoter system.<br />
<br />
<center>'''Conclusion and outlook:'''</center><br><br />
<br />
To get gene expression casette for both of the light-switchable promoter systems ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801042 BBa_K801042] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801043 BBa_K801043]) into a yeast plasmid, we want to use pSB6A0 ([http://partsregistry.org/Part:BBa_K268000 BBa_K268000]) carrying a TRP1 auxotrophy marker. But we've discovered some discrepancies in this part, e.&nbsp;g. there is still at least one forbidden restriction site (XbaI or PstI, please see experience site of the part). So we have to correct the part by quickchange mutagenesis.<br />
<br />
After successful cloning and transfection of the gene expression batteries including their reporters, we will proceed with the characterization of the part.<br />
<br />
In parallel, we will implement also the whole biosynthesis pathway for the phycocyanobilin synthesis, donwstream of our both batteries for a light-switchable promoter system ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801042 BBa_K801042] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801043 BBa_K801043]); after that, we don't have to add phycocyanobilin in our medium anymore!<br />
<br />
If there is still time in the end, we will clone our system including the biosynthetic genes for PCB synthesis in the yeast integrative vector ([http://partsregistry.org/Part:BBa_K300001 BBa_K300001]) and will start a genome integration in order to create a new yeast strain with a fully functional light-switchable promoter system.<br />
<br />
<div class="noborder" style="float:left"><br />
<html><a href="https://2012.igem.org/Team:TU_Munich/Project/Light_Switchable_Promoter"><img width="200px" src="https://static.igem.org/mediawiki/2012/3/33/TUM12_readmore.png"/></a></html><br />
</div><br />
</div><br />
<br />
==Genome Integration==<br />
----<br />
<div class="bezel mfull"><br />
As we cannot obey the letter of the German purity law (there is a zero tolerance policy concerning transgenic ingredients), we try our best to meet the spirit. Thus, it is <b>unacceptable for us to work with antibiotics</b> to keep up the selective environment. Since we <b>cannot work with auxotrophies</b> in beer either, we have to make sure the yeasts do not lose the BioBricks. The most promising way to accomplish a long lasting presence of our constructs is to achieve <b>genome integration</b>.<br />
<hr><br />
[[file:TUM12_experiment_overwiew_genome.png|500px|thumb|right| Plasmid backbone used for integration of our expression cassettes]]<br />
<center>'''Experimental results:'''</center><br><br />
First experiments to characterize the function of the yeast integration system were performed and the used selection marker was maintained in the yeast culture, although the selection pressure was switched off. This indicates that first integrations were achieved. <br />
<br />
<center>'''Conclusion and outlook:'''</center><br><br />
Maintaining the plasmids harboring our expression cassettes in the yeast cells during the brewing process is best possible using genome integration. This becomes increasingly interesting, when a yeast strain with different expression cassettes is to be created. Because this is intended for the next step of our project the integration of our expression cassettes becomes increasingly important.<br />
<br />
<div class="noborder" style="float:left"><br />
<html><a href="https://2012.igem.org/Team:TU_Munich/Project/Genome_Integration"><img width="200px" src="https://static.igem.org/mediawiki/2012/3/33/TUM12_readmore.png"/></a></html><br />
</div><br />
</div><br />
<br />
==Brewing our SynBio Beer==<br />
----<br />
<div class="bezel mfull"><br />
Contrary to popular opinion the chief ingredient of beer is not '''YPD''' but '''gyle''', a carefully prepared mixture of malt, hop and water. Although the name of the yeast strain commonly used in the lab, '''S. cerevisiae''', suggests that it is used in the beer brewing process, the yeast strains generally employed in brewing process have '''strongly adapted to gyle''', as they are reutilized in every succeeding brewing cycle.<br />
Hence some investigation on how our yeast '''performs''' in gyle was necessary.<br />
<br><hr><br />
[[file:TUM12_experiment_overwiew_Brewing.png|200px|thumb|right| Picture of the first SynBio Beer brewed during the iGEM competition in 2012]]<br />
<center>'''Experimental results:'''</center><br><br />
Our experiments show that the growth of several different yeast strains is '''not impaired in gyle'''!<br />
<br />
Expression assays proved the necessity of [[Team:TU_Munich/Project/Genome_Integration|'''genome integration''']] for a proper '''SynBio Beer'''.<br />
<br />
<center>'''Conclusion and outlook:'''</center><br><br />
As soon as cloning of integration vectors containing expression cassettes for our biosynthetic pathways are finished we will repeat the brewing experiments.<br />
<br />
<div class="noborder" style="float:left"><br />
<html><a href="https://2012.igem.org/Team:TU_Munich/Project/Brewing"><img width="200px" src="https://static.igem.org/mediawiki/2012/3/33/TUM12_readmore.png"/></a></html><br />
</div><br />
</div><br />
<br />
== References ==<br />
----<br />
* [[http://www.ncbi.nlm.nih.gov/pubmed/6327079 Edens et al., 1984]] Edens, L., Bom, I., Ledeboer, A. M., Maat, J., Toonen, M. Y., Visser, C., and Verrips, C. T. (1984). Synthesis and processing of the plant protein thaumatin in yeast. ''Cell'', 37(2):629–33.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/10995285 Miranda et al., 2000]] Miranda, C. L., Stevens, J. F., Ivanov, V., McCall, M., Frei, B., Deinzer, M. L., and Buhler, D. R. (2000). Antioxidant and prooxidant actions of prenylated and nonprenylated chalcones and flavanones in vitro. ''J Agric Food Chem'', 48(9):3876–84.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/10724480 Mazzoni et al., 2000]] Mazzoni, C., Santori, F., Saliola, M., and Falcone, C. (2000). Molecular analysis of uas(e), a cis element containing stress response elements responsible for ethanol induction of the kladh4 gene of ''kluyveromyces lactis''. ''Res Microbiol'', 151(1):19–28.</div>JaraOhttp://2012.igem.org/Team:TU_Munich/Project/OverviewTeam:TU Munich/Project/Overview2012-10-23T20:40:37Z<p>JaraO: /* Light-switchable promoter */</p>
<hr />
<div>{{Team:TU_Munich/Header}}<br />
<br />
= Overview =<br />
<hr/><br />
<br />
== Vision ==<br />
----<br />
We, TU Munich’s 2012 iGEM team, strive to catalyze the diffusion process of knowledge about genetic engineering and synthetic biology among the general public. Using the example of iGEM’s first and finest SynBio Beer we involve, interest and inspire people to reconsider preconceived ideas and encourage them to openly engage in a broad discussion weighing pros and cons of genetic engineering in foodstuff. We sketch a future where new technology can be applied in a meaningful way to complement traditional foods or beverages.<br />
<br />
==Biosynthesis pathways==<br />
----<br />
<br />
<br />
<div class="bezel mfull"><br />
===Limonene===<br />
Limonene is a cyclic terpene and a major constituent of several citrus oils. D-Limonene has been used as a component of flavorings and fragrances. It is formed from geranyl pyrophosphate by limonene synthase.<br />
<br />
We successfully demonstrated the production of the flavoring substance limonene by expressing limonene synthase in ''S. cerevisiae'', which naturally synthesizes the educt geranyl pyrophosphate.<br />
<hr><br />
[[file:TUM12_experiment_overwiew_limonene.png|500px|thumb|right| Main results from our limonene subproject: reaction mechanism (A), constructed BioBricks (B) and proof of principle for the in vivo production of limonene]]<br />
<center>'''Experimental results:'''</center><br><br />
(+)-Limonene synthase 1 [[http://partsregistry.org/Part:BBa_K801065 BBa_K801065]] and (+)-limonene synthase 1 with yeast consensus sequence [[http://partsregistry.org/Part:BBa_K801060 BBa_K801060]] were successfully cloned into our new yeast expression vector pTUM100. Expression of recombinant limonene synthase in ''Saccharomyces cerevisiae'' was proven by western blotting. Subsequently the protein was purified using SA-chromatography and size exclusion chromatography. The functionality of the enzyme was verified by ''in vivo'' and ''in vitro'' detection of limonene via GC-MS. <br />
<br />
Furthermore, we established gene constructs of the limonene synthase coding sequence with different yeast specific promoters and terminators [[http://partsregistry.org/Part:BBa_K801062 BBa_K801062]], [[http://partsregistry.org/Part:BBa_K801063 BBa_K801063]], [[http://partsregistry.org/Part:BBa_K801064 BBa_K801064]]. <br />
<br />
Last but not least, we have brewed iGEM's first SynBio beer containing limonene.<br />
<br />
<center>'''Conclusion and outlook:'''</center><br><br />
<br />
We have achieved functional expression of ''Citrus limon'' limonene synthase and production of limonene in yeast. The last remaining step to a SynBio beer would be the integration of our gene constructs [[http://partsregistry.org/Part:BBa_K801062 BBa_K801062]], [[http://partsregistry.org/Part:BBa_K801063 BBa_K801063]], [[http://partsregistry.org/Part:BBa_K801064 BBa_K801064]] into the yeast genome.<br />
<br />
<div class="noborder" style="float:left"><br />
<html><a href="https://2012.igem.org/Team:TU_Munich/Project/Limonene"><img width="200px" src="https://static.igem.org/mediawiki/2012/3/33/TUM12_readmore.png"/></a></html><br />
</div><br />
</div><br />
<br />
<div class="bezel mfull"><br />
<br />
===Thaumatin===<br />
Thaumatin is a natural α+β-protein which is synthesized by the katamfe plant (''Thaumatococcus daniellii''). It is said to be 2,000 to 100,000 times sweeter than sucrose on molar basis, but the sweetness builds up slow and lasts long. It has been approved as a sweetener by the European Union (E957).<br />
<br />
Our aim is to have ''S. cerevisiae'' secrete functional thaumatin by expressing preprothaumatin – a principle which has been proven by [[http://www.ncbi.nlm.nih.gov/pubmed/6327079 Edens et al., 1984]].<br />
<hr><br />
[[file:TUM12_experiment_overwiew_Thaumati.png|500px|thumb|right| Main results from the Thaumatin subproject: Structure of Thaumatin (A), constructed BioBricks (B), profile of an ion exchange chromatography (IEC) used to detect our recombinant Thaumatin and a SDS-PAGE gel showing IEC elution fractions containing Thaumatin]]<br />
<center>'''Experimental results:'''</center><br><br />
The BioBrick for preprothaumatin [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801080 BBa_K801080] as well as an expression cassette [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801080 K801080] were successfully cloned, expressed in yeast, purified using an ion exchange chromatography (see figure C) and detected in the SDS-PAGE. Therefore, the expression of thaumatin in yeast could be demonstrated and functionality of the BioBrick is confirmed.<br />
<br />
<center>'''Conclusion and outlook:'''</center><br><br />
A proof of principle for the expression of thaumatin was achieved. Further goals are the increase of the expression of thaumatin and the investigation of the secretion.<br />
<br />
<div class="noborder" style="float:left"><br />
<html><a href="https://2012.igem.org/Team:TU_Munich/Project/Thaumatin"><img width="200px" src="https://static.igem.org/mediawiki/2012/3/33/TUM12_readmore.png"/></a></html><br />
</div><br />
</div><br />
<br />
<div class="bezel mfull"><br />
<br />
===Caffeine===<br />
Caffeine is a purine-alkaloid and its biosynthesis is known from coffee plants and tea plants. The molecule acts as a competitive antagonist of adenosine receptors and, therefore, increases indirectly neurotransmitter concentrations resulting in warding off drowsiness and restoring alertness. <br />
<br />
The idea is to perform a heterologous gene expression of the three enzymes 7-methylxanthosine synthase (CaXMT1), N-methyl nucleosidase (CaMXMT1) and caffeine synthase (CaDXMT1) required for caffeine biosynthesis in ''Saccharomyces cerevisiae''. <br />
<br />
<br />
<hr><br />
[[file:TUM12_experiment_overwiew_caffeine2.png|500px|thumb|right| Figure showing a schematic overview of the reaction in (A), a western blot against the Strep-tag II for GFP, [[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801070 BBa_K801070]] and [[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801071 BBa_K801071]] in (B) and finally the same western blot development for [[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801072 BBa_K801072]]]]<br />
<center>'''Experimental results:'''</center><br><br />
* Successful cloning of the three enzymes [http://partsregistry.org/Part:BBa_K801070 7-methylxanthosine synthase (CaXMT1)], [http://partsregistry.org/Part:BBa_K801071 N-methyl nucleosidase (CaMXMT1)] and the [http://partsregistry.org/Part:BBa_K801072 caffeine synthase (CaDXMT1)] into the shuttle vector pTUM104 and pSB1C3 each. <br />
* Successful assembly of the BioBricks to form expression cassettes consisting of promoter, gene and terminator: [http://partsregistry.org/Part:BBa_K801073 pTEF2-CaXMT1-tADH1], [http://partsregistry.org/Part:BBa_K801074 pTEF1-CaMXMT1-tADH1] and [http://partsregistry.org/Part:BBa_K801075 pTEF2-CaDXMT1-tADH1]) into pSB1C3.<br />
* Successful assembly of the expression cassettes of the three relevant enzymes forming a composite part of 6.4 kb capable of caffeine production in yeast ([http://partsregistry.org/Part:BBa_K801077 Caffeine Synthesis Pathway]) into pSB1C3.<br />
* Successful expression of CaXMT1, CaMXMT1 and CaDXMT1 in ''Saccharomyces cerevisiae'' INVSc1 in selective Sc minimal induction medium lacking uracil with 2 % galactose.<br />
<br />
<br />
<center>'''Conclusion and outlook:'''</center><br><br />
The homologue expression of the three required enzymes for caffeine synthesis in ''Saccharomyces cerevisiae'' INVSc1 transformed with pTUM102_CaXMT1, pTUM102_CaMXMT1 and pTUM102_CaDXMT1 was successful. Further testing of caffeine production using crude extracts from lysed transformed yeast cells and working on genome integration of the expression cassette containing all three enzymes flanked by promoter and terminator are in progress.<br />
<br />
<div class="noborder" style="float:left"><br />
<html><a href="https://2012.igem.org/Team:TU_Munich/Project/Caffeine"><img width="200px" src="https://static.igem.org/mediawiki/2012/3/33/TUM12_readmore.png"/></a></html><br />
</div><br />
</div><br />
<br />
<div class="bezel mfull"><br />
<br />
===Xanthohumol===<br />
Xanthohumol is known as a putative cancer chemopreventive agent due to its antioxidant activities [[http://www.ncbi.nlm.nih.gov/pubmed/10995285 Miranda et al., 2000]]. Our goal is a heterologous gene expression of all enzymes required for xanthohumol biosynthesis in ''S. cerevisiae''.<br />
<br />
The pathway for the production of this plant secondary metabolite is composed of five steps, starting with the conversion of phenylalanine and followed by four further enzymatic reactions.<br />
<hr><br />
[[file:TUM12_experiment_overwiew_xanto2.png|500px|thumb|right| Main results from the Xanthohumol subproject: Successful reconstruction of the metabolic pathway for Xanthohumol]]<br />
<center>'''Experimental results:'''</center><br><br />
The whole biosynthetic pathway for the production of xanthohumol was converted into BioBricks. Except for APT each of the enzymes were cloned in two versions one having the proposed consensus sequence for more efficient expression in yeast chassis and another for usage of these BioBricks in other chassis. All BioBricks were sequenced. Sequences can be found in the registry of standard biological parts:<br />
[http://partsregistry.org/Part:BBa_K801090 BBa_K801090], [http://partsregistry.org/Part:BBa_K801091 BBa_K801091], [http://partsregistry.org/Part:BBa_K801092 BBa_K801092], [http://partsregistry.org/Part:BBa_K801093 BBa_K801093], [http://partsregistry.org/Part:BBa_K801094 BBa_K801094], [http://partsregistry.org/Part:BBa_K801095 BBa_K801095], [http://partsregistry.org/Part:BBa_K801096 BBa_K801096], [http://partsregistry.org/Part:BBa_K801097 BBa_K801097] and [http://partsregistry.org/Part:BBa_K801098 BBa_K801098] <br />
<br />
<br />
<center>'''Conclusion and outlook:'''</center><br><br />
<br />
The construction of the xanthohumol pathway was achieved, whereas the expression and characterization have to be postponed after the European Jamboree or might be an interesting task for next year's iGEM teams.<br />
<br />
<div class="noborder" style="float:left"><br />
<html><a href="https://2012.igem.org/Team:TU_Munich/Project/Xanthohumol"><img width="200px" src="https://static.igem.org/mediawiki/2012/3/33/TUM12_readmore.png"/></a></html><br />
</div><br />
</div><br />
<br />
==Vector Design==<br />
----<br />
<div class="bezel mfull"><br />
===pTUM100===<br />
Designing an expression vector for yeast which is compatible to the iGEM cloning principles and standards was the main aim of this subproject. Based on the commercially available pYES2 vector we created vectors containing inducible and constitutive promoters in order to establish efficient possibilities to clone and express our enzymes.<br />
<hr><br />
[[file:TUM12_experiment_overwiew_vector.png|500px|thumb|right| Explanations on the figure:<br />
<br />
Figure A shows the new multiple cloning site (MCS) containing the RFC 10/25 restriction sides and the DNA sequence coding for the ''Strep''-tag II.<br />
<br />
Figure B gives an overview of all important functional elements located on the vector backbone. Upstream to the new MCS lies a T7 promoter primer binding site allowing easy forward sequencing of integrated gene constructs using the standard T7 primer. The URA 3 gene is a prototrophy marker used for the selection of transfected cells.<br />
<br />
Figure C to E present the successfully designed BioBricks: pTUM100 simply contains the new MCS, the transcription terminator and further elements required for cloning and transfection. pTUM102 to pTUM104 contain in addition the constitutive promoters pTef1, pTef2 and ADH. On pTUM104 is the galactose inducible promoter pGAL1 located. ]]<br />
<br />
<center>'''Experimental results:'''</center><br><br />
Using the pYES vector from Invitrogen we first deleted five forbidden restriction sites in the vector backbone via side directed mutagenesis. Furthermore, the original multiple cloning site was replaced by a multiple cloning site compatible with the RFC 10/25 cloning standards. To allow easy extraction and purification of proteins for ''in vitro'' applications the new multiple cloning site allows to express proteins with a ''Strep''-tag II. <br />
Exclusion of the galactose inducible promoter provided a powerful basis vector for the integration of user-defined promoters. This way the pTUM100 vector gives a valuable contribution to our and to further protein expression and promoter characterization experiments in ''Saccharomyces cerevisiae''.<br />
Moreover, we used the pTUM100 to integrate the three constitutive promoters Tef1, Tef2 and ADH which come all with different promoter intensities.<br />
<br />
<center>'''Outlook and conclusion:'''</center><br><br />
<br />
The galactose inducible expression system was a great aid for the majority of all subprojects. Especially the opportunity to purify and detect (via Western blot) proteins using the ''Strep''-tag II did facilitate our laboratory practice and accelerated our work progress.<br />
To cover even more demands we are planning to design a second vector template containing a His-tag.<br />
<br />
All BioBricks were sequenced. Sequences can be found in the registry of standard biological parts under the following entries:<br />
<br />
[http://partsregistry.org/Part:BBa_K801000 pTUM100 BBa_K801000], [http://partsregistry.org/Part:BBa_K801001 pTUM101 BBa_K801001], [http://partsregistry.org/Part:BBa_K801002 pTUM102 BBa_K801002], [[http://partsregistry.org/Part:BBa_K801003 pTUM103 BBa_K801003] and [[http://partsregistry.org/Part:BBa_K801004 pTUM104 BBa_K801004]] <br />
<br />
<br />
<div class="noborder" style="float:left"><br />
<html><a href="https://2012.igem.org/Team:TU_Munich/Project/Vector_Design"><img width="200px" src="https://static.igem.org/mediawiki/2012/3/33/TUM12_readmore.png"/></a></html><br />
</div><br />
</div><br />
<br />
==Regulation of Genexpression==<br />
----<br />
By developing inducible promoters and placing them upstream of our biosynthetic pathways we create the possibility to make ''S. cerevisiae'' dynamically respond to concentration changes in its medium as well as to external stimuli. <br />
<br />
An optimal inducing substance needs to be inexpensive, nontoxic and fully controllable in its application. Only substances with these characteristics allow to precisely regulate a system temporally, spatially and quantitatively. <br />
<br />
<br />
<br />
<div class="bezel mfull"><br />
===Ethanol-inducible promoter===<br />
The KlADH4-promoter from the yeast ''Kluyveromyces lactis'' regulates the expression of a mitochondrial alcohol dehydrogenase in an ethanol-dependent way. An alcohol-inducible promoter would be incredibly useful for anyone planning to brew a beer with a transgenic yeast - it would allow for the induction of the target genes after the main fermentation has finished and this way, the metabolic burden for the yeast cells could be lowered. All the transcription factors known to be involved in the regulation of the KlADH4-promoter in ''K. lactis'' also occur in ''S. cerevisiae'' [[http://www.ncbi.nlm.nih.gov/pubmed/10724480 Mazzoni et al., 2000]]. This is why we are confident that this promoter maintains its unique characteristics when transformed into ''S. cerevisiae''.<hr><br />
[[file:TUM12_experiment_overwiew_alcohol.png|400px|thumb|right| '''Top''': Emission spectrum, excitation spectrum and visible fluorescence of a yeast culture expressing eGFP under the control of the KlADH4-promoter at 1.72 Vol.-% ethanol. Significant amounts of eGFP detectable '''Center''': Emission spectrum and excitation spectrum of a yeast culture carrying a plasmid in which eGFP-expression is controlled by the KlADH4-promoter cultivated with glycerol as only carbon source. Ethanol concentration: 0.21 Vol.-%. No eGFP detectable. '''Bottom''': Fluorescence per cell and ethanol concentration plotted against time after induction.]]<br />
<center>'''Experimental results:'''</center><br><br />
At this time our results concerning the KlADH4-promoter (originally from the yeast ''Kluyveromyces lactis'') suggest that this promoter is ethanol inducible in ''S. cerevisiae''. Further experiments are still being done to abolish residual ambiguities. The lowest ethanol concentration at which eGFP-expression was detected is 0.9 Vol.-%.<br />
<br />
<center>'''Conclusion and outlook:'''</center><br><br />
<br />
Because ''S. cerevisiae'' is such a good brewer, it was difficult to produce a stringent negative control in our characterization experiments. However, we finally figured out some experiments that allowed us to keep the ethanol concentration below 0.5 % v/v, which is a concentration at which induction is observed in ''K. lactis''. We are working hard on providing additional data, but we are confident that we will be able to provide clear evidence that this promoter is ethanol-inducible not only in ''K. lactis'', but also in ''S. cerevisiae''.<br />
<br />
<div class="noborder" style="float:left"><br />
<html><a href="https://2012.igem.org/Team:TU_Munich/Project/Ethanol_Inducible_Promoter"><img width="200px" src="https://static.igem.org/mediawiki/2012/3/33/TUM12_readmore.png"/></a></html><br />
</div><br />
</div><br />
<br />
<div class="bezel mfull"><br />
<br />
===Light-switchable promoter===<br />
The idea behind a light-switchable system is to create a gene expression system which can be induced and deactivated by light of a certain wavelength.<br />
<br />
This system is extremely attractive, as induction does not require the addition of a specific substance. This makes induction '''cheap, fast, precise''' and also compatible with the Bavarian purity law.<br />
<hr><br />
[[Image:TUM12_light.jpg|thumb|right|300px|Principle of light-dependent switching of gene-expression.]]<br />
<center>'''Experimental results:'''</center><br><br />
All fusion proteins for the two types of a light-switchable promoter system has been finished ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801039 BBa_K801039], [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801040 BBa_K801040] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801041 BBa_K801041]), also gene expression batteries coding for all components of each type of our light-switchable promoter system has been done ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801042 BBa_K801042] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801043 BBa_K801043]). Since lacking of a second functional yeast vector carrying another auxotrophy marker than URA3 of the pTUM plasmids, which is already reserved for the biosynthesis enzymes, proteins and also reporters, we were not able to clone the whole gene expression battery, into a yeast vector, in order to co-transfect the yeast with one plasmid with the reporter construct and the second plasmid coding for all the devices needed in a light-switchable promoter system.<br />
<br />
<center>'''Conclusion and outlook:'''</center><br><br />
<br />
To get gene expression casette for both of the light-switchable promoter systems ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801042 BBa_K801042] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801043 BBa_K801043]) into a yeast plasmid, we want to use pSB6A0 ([http://partsregistry.org/Part:BBa_K268000 BBa_K268000]) carrying a TRP1 auxotrophy marker. But we've discovered some discrepancies in this part, e.&nbsp;g. there is still at least one forbidden restriction site (XbaI or PstI, please see experience site of the part). So we have to correct the part by quickchange mutagenesis.<br />
<br />
After successful cloning and transfection of the gene expression batteries including there reporters, we will proceed with the characterization of the part.<br />
<br />
In parallel, we will implement also the whole biosynthesis pathway for the phycocyanobilin synthesis, donwstream of our both batteries for a light-switchable promoter system ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801042 BBa_K801042] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801043 BBa_K801043]); after that, we don't have to add phycocyanobilin in our medium anymore!<br />
<br />
If there is still time in the end, we will clone our system including the biosynthetic genes for PCB synthesis in the yeast integrative vector ([http://partsregistry.org/Part:BBa_K300001 BBa_K300001]) and will start a genome integration in order to create a new yeast strain with a fully functional light-switchable promoter system.<br />
<br />
<div class="noborder" style="float:left"><br />
<html><a href="https://2012.igem.org/Team:TU_Munich/Project/Light_Switchable_Promoter"><img width="200px" src="https://static.igem.org/mediawiki/2012/3/33/TUM12_readmore.png"/></a></html><br />
</div><br />
</div><br />
<br />
==Genome Integration==<br />
----<br />
<div class="bezel mfull"><br />
As we cannot obey the letter of the German purity law (there is a zero tolerance policy concerning transgenic ingredients), we try our best to meet the spirit. Thus, it is <b>unacceptable for us to work with antibiotics</b> to keep up the selective environment. Since we <b>cannot work with auxotrophies</b> in beer either, we have to make sure the yeasts do not lose the BioBricks. The most promising way to accomplish a long lasting presence of our constructs is to achieve <b>genome integration</b>.<br />
<hr><br />
[[file:TUM12_experiment_overwiew_genome.png|500px|thumb|right| Plasmid backbone used for integration of our expression cassettes]]<br />
<center>'''Experimental results:'''</center><br><br />
First experiments to characterize the function of the yeast integration system were performed and the used selection marker was maintained in the yeast culture, although the selection pressure was switched off. This indicates that first integrations were achieved. <br />
<br />
<center>'''Conclusion and outlook:'''</center><br><br />
Maintaining the plasmids harboring our expression cassettes in the yeast cells during the brewing process is best possible using genome integration. This becomes increasingly interesting, when a yeast strain with different expression cassettes is to be created. Because this is intended for the next step of our project the integration of our expression cassettes becomes increasingly important.<br />
<br />
<div class="noborder" style="float:left"><br />
<html><a href="https://2012.igem.org/Team:TU_Munich/Project/Genome_Integration"><img width="200px" src="https://static.igem.org/mediawiki/2012/3/33/TUM12_readmore.png"/></a></html><br />
</div><br />
</div><br />
<br />
==Brewing our SynBio Beer==<br />
----<br />
<div class="bezel mfull"><br />
Contrary to popular opinion the chief ingredient of beer is not '''YPD''' but '''gyle''', a carefully prepared mixture of malt, hop and water. Although the name of the yeast strain commonly used in the lab, '''S. cerevisiae''', suggests that it is used in the beer brewing process, the yeast strains generally employed in brewing process have '''strongly adapted to gyle''', as they are reutilized in every succeeding brewing cycle.<br />
Hence some investigation on how our yeast '''performs''' in gyle was necessary.<br />
<br><hr><br />
[[file:TUM12_experiment_overwiew_Brewing.png|200px|thumb|right| Picture of the first SynBio Beer brewed during the iGEM competition in 2012]]<br />
<center>'''Experimental results:'''</center><br><br />
Our experiments show that the growth of several different yeast strains is '''not impaired in gyle'''!<br />
<br />
Expression assays proved the necessity of [[Team:TU_Munich/Project/Genome_Integration|'''genome integration''']] for a proper '''SynBio Beer'''.<br />
<br />
<center>'''Conclusion and outlook:'''</center><br><br />
As soon as cloning of integration vectors containing expression cassettes for our biosynthetic pathways are finished we will repeat the brewing experiments.<br />
<br />
<div class="noborder" style="float:left"><br />
<html><a href="https://2012.igem.org/Team:TU_Munich/Project/Brewing"><img width="200px" src="https://static.igem.org/mediawiki/2012/3/33/TUM12_readmore.png"/></a></html><br />
</div><br />
</div><br />
<br />
== References ==<br />
----<br />
* [[http://www.ncbi.nlm.nih.gov/pubmed/6327079 Edens et al., 1984]] Edens, L., Bom, I., Ledeboer, A. M., Maat, J., Toonen, M. Y., Visser, C., and Verrips, C. T. (1984). Synthesis and processing of the plant protein thaumatin in yeast. ''Cell'', 37(2):629–33.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/10995285 Miranda et al., 2000]] Miranda, C. L., Stevens, J. F., Ivanov, V., McCall, M., Frei, B., Deinzer, M. L., and Buhler, D. R. (2000). Antioxidant and prooxidant actions of prenylated and nonprenylated chalcones and flavanones in vitro. ''J Agric Food Chem'', 48(9):3876–84.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/10724480 Mazzoni et al., 2000]] Mazzoni, C., Santori, F., Saliola, M., and Falcone, C. (2000). Molecular analysis of uas(e), a cis element containing stress response elements responsible for ethanol induction of the kladh4 gene of ''kluyveromyces lactis''. ''Res Microbiol'', 151(1):19–28.</div>JaraOhttp://2012.igem.org/Team:TU_Munich/Project/OverviewTeam:TU Munich/Project/Overview2012-10-23T20:38:15Z<p>JaraO: /* Ethanol-inducible promoter */</p>
<hr />
<div>{{Team:TU_Munich/Header}}<br />
<br />
= Overview =<br />
<hr/><br />
<br />
== Vision ==<br />
----<br />
We, TU Munich’s 2012 iGEM team, strive to catalyze the diffusion process of knowledge about genetic engineering and synthetic biology among the general public. Using the example of iGEM’s first and finest SynBio Beer we involve, interest and inspire people to reconsider preconceived ideas and encourage them to openly engage in a broad discussion weighing pros and cons of genetic engineering in foodstuff. We sketch a future where new technology can be applied in a meaningful way to complement traditional foods or beverages.<br />
<br />
==Biosynthesis pathways==<br />
----<br />
<br />
<br />
<div class="bezel mfull"><br />
===Limonene===<br />
Limonene is a cyclic terpene and a major constituent of several citrus oils. D-Limonene has been used as a component of flavorings and fragrances. It is formed from geranyl pyrophosphate by limonene synthase.<br />
<br />
We successfully demonstrated the production of the flavoring substance limonene by expressing limonene synthase in ''S. cerevisiae'', which naturally synthesizes the educt geranyl pyrophosphate.<br />
<hr><br />
[[file:TUM12_experiment_overwiew_limonene.png|500px|thumb|right| Main results from our limonene subproject: reaction mechanism (A), constructed BioBricks (B) and proof of principle for the in vivo production of limonene]]<br />
<center>'''Experimental results:'''</center><br><br />
(+)-Limonene synthase 1 [[http://partsregistry.org/Part:BBa_K801065 BBa_K801065]] and (+)-limonene synthase 1 with yeast consensus sequence [[http://partsregistry.org/Part:BBa_K801060 BBa_K801060]] were successfully cloned into our new yeast expression vector pTUM100. Expression of recombinant limonene synthase in ''Saccharomyces cerevisiae'' was proven by western blotting. Subsequently the protein was purified using SA-chromatography and size exclusion chromatography. The functionality of the enzyme was verified by ''in vivo'' and ''in vitro'' detection of limonene via GC-MS. <br />
<br />
Furthermore, we established gene constructs of the limonene synthase coding sequence with different yeast specific promoters and terminators [[http://partsregistry.org/Part:BBa_K801062 BBa_K801062]], [[http://partsregistry.org/Part:BBa_K801063 BBa_K801063]], [[http://partsregistry.org/Part:BBa_K801064 BBa_K801064]]. <br />
<br />
Last but not least, we have brewed iGEM's first SynBio beer containing limonene.<br />
<br />
<center>'''Conclusion and outlook:'''</center><br><br />
<br />
We have achieved functional expression of ''Citrus limon'' limonene synthase and production of limonene in yeast. The last remaining step to a SynBio beer would be the integration of our gene constructs [[http://partsregistry.org/Part:BBa_K801062 BBa_K801062]], [[http://partsregistry.org/Part:BBa_K801063 BBa_K801063]], [[http://partsregistry.org/Part:BBa_K801064 BBa_K801064]] into the yeast genome.<br />
<br />
<div class="noborder" style="float:left"><br />
<html><a href="https://2012.igem.org/Team:TU_Munich/Project/Limonene"><img width="200px" src="https://static.igem.org/mediawiki/2012/3/33/TUM12_readmore.png"/></a></html><br />
</div><br />
</div><br />
<br />
<div class="bezel mfull"><br />
<br />
===Thaumatin===<br />
Thaumatin is a natural α+β-protein which is synthesized by the katamfe plant (''Thaumatococcus daniellii''). It is said to be 2,000 to 100,000 times sweeter than sucrose on molar basis, but the sweetness builds up slow and lasts long. It has been approved as a sweetener by the European Union (E957).<br />
<br />
Our aim is to have ''S. cerevisiae'' secrete functional thaumatin by expressing preprothaumatin – a principle which has been proven by [[http://www.ncbi.nlm.nih.gov/pubmed/6327079 Edens et al., 1984]].<br />
<hr><br />
[[file:TUM12_experiment_overwiew_Thaumati.png|500px|thumb|right| Main results from the Thaumatin subproject: Structure of Thaumatin (A), constructed BioBricks (B), profile of an ion exchange chromatography (IEC) used to detect our recombinant Thaumatin and a SDS-PAGE gel showing IEC elution fractions containing Thaumatin]]<br />
<center>'''Experimental results:'''</center><br><br />
The BioBrick for preprothaumatin [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801080 BBa_K801080] as well as an expression cassette [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801080 K801080] were successfully cloned, expressed in yeast, purified using an ion exchange chromatography (see figure C) and detected in the SDS-PAGE. Therefore, the expression of thaumatin in yeast could be demonstrated and functionality of the BioBrick is confirmed.<br />
<br />
<center>'''Conclusion and outlook:'''</center><br><br />
A proof of principle for the expression of thaumatin was achieved. Further goals are the increase of the expression of thaumatin and the investigation of the secretion.<br />
<br />
<div class="noborder" style="float:left"><br />
<html><a href="https://2012.igem.org/Team:TU_Munich/Project/Thaumatin"><img width="200px" src="https://static.igem.org/mediawiki/2012/3/33/TUM12_readmore.png"/></a></html><br />
</div><br />
</div><br />
<br />
<div class="bezel mfull"><br />
<br />
===Caffeine===<br />
Caffeine is a purine-alkaloid and its biosynthesis is known from coffee plants and tea plants. The molecule acts as a competitive antagonist of adenosine receptors and, therefore, increases indirectly neurotransmitter concentrations resulting in warding off drowsiness and restoring alertness. <br />
<br />
The idea is to perform a heterologous gene expression of the three enzymes 7-methylxanthosine synthase (CaXMT1), N-methyl nucleosidase (CaMXMT1) and caffeine synthase (CaDXMT1) required for caffeine biosynthesis in ''Saccharomyces cerevisiae''. <br />
<br />
<br />
<hr><br />
[[file:TUM12_experiment_overwiew_caffeine2.png|500px|thumb|right| Figure showing a schematic overview of the reaction in (A), a western blot against the Strep-tag II for GFP, [[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801070 BBa_K801070]] and [[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801071 BBa_K801071]] in (B) and finally the same western blot development for [[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801072 BBa_K801072]]]]<br />
<center>'''Experimental results:'''</center><br><br />
* Successful cloning of the three enzymes [http://partsregistry.org/Part:BBa_K801070 7-methylxanthosine synthase (CaXMT1)], [http://partsregistry.org/Part:BBa_K801071 N-methyl nucleosidase (CaMXMT1)] and the [http://partsregistry.org/Part:BBa_K801072 caffeine synthase (CaDXMT1)] into the shuttle vector pTUM104 and pSB1C3 each. <br />
* Successful assembly of the BioBricks to form expression cassettes consisting of promoter, gene and terminator: [http://partsregistry.org/Part:BBa_K801073 pTEF2-CaXMT1-tADH1], [http://partsregistry.org/Part:BBa_K801074 pTEF1-CaMXMT1-tADH1] and [http://partsregistry.org/Part:BBa_K801075 pTEF2-CaDXMT1-tADH1]) into pSB1C3.<br />
* Successful assembly of the expression cassettes of the three relevant enzymes forming a composite part of 6.4 kb capable of caffeine production in yeast ([http://partsregistry.org/Part:BBa_K801077 Caffeine Synthesis Pathway]) into pSB1C3.<br />
* Successful expression of CaXMT1, CaMXMT1 and CaDXMT1 in ''Saccharomyces cerevisiae'' INVSc1 in selective Sc minimal induction medium lacking uracil with 2 % galactose.<br />
<br />
<br />
<center>'''Conclusion and outlook:'''</center><br><br />
The homologue expression of the three required enzymes for caffeine synthesis in ''Saccharomyces cerevisiae'' INVSc1 transformed with pTUM102_CaXMT1, pTUM102_CaMXMT1 and pTUM102_CaDXMT1 was successful. Further testing of caffeine production using crude extracts from lysed transformed yeast cells and working on genome integration of the expression cassette containing all three enzymes flanked by promoter and terminator are in progress.<br />
<br />
<div class="noborder" style="float:left"><br />
<html><a href="https://2012.igem.org/Team:TU_Munich/Project/Caffeine"><img width="200px" src="https://static.igem.org/mediawiki/2012/3/33/TUM12_readmore.png"/></a></html><br />
</div><br />
</div><br />
<br />
<div class="bezel mfull"><br />
<br />
===Xanthohumol===<br />
Xanthohumol is known as a putative cancer chemopreventive agent due to its antioxidant activities [[http://www.ncbi.nlm.nih.gov/pubmed/10995285 Miranda et al., 2000]]. Our goal is a heterologous gene expression of all enzymes required for xanthohumol biosynthesis in ''S. cerevisiae''.<br />
<br />
The pathway for the production of this plant secondary metabolite is composed of five steps, starting with the conversion of phenylalanine and followed by four further enzymatic reactions.<br />
<hr><br />
[[file:TUM12_experiment_overwiew_xanto2.png|500px|thumb|right| Main results from the Xanthohumol subproject: Successful reconstruction of the metabolic pathway for Xanthohumol]]<br />
<center>'''Experimental results:'''</center><br><br />
The whole biosynthetic pathway for the production of xanthohumol was converted into BioBricks. Except for APT each of the enzymes were cloned in two versions one having the proposed consensus sequence for more efficient expression in yeast chassis and another for usage of these BioBricks in other chassis. All BioBricks were sequenced. Sequences can be found in the registry of standard biological parts:<br />
[http://partsregistry.org/Part:BBa_K801090 BBa_K801090], [http://partsregistry.org/Part:BBa_K801091 BBa_K801091], [http://partsregistry.org/Part:BBa_K801092 BBa_K801092], [http://partsregistry.org/Part:BBa_K801093 BBa_K801093], [http://partsregistry.org/Part:BBa_K801094 BBa_K801094], [http://partsregistry.org/Part:BBa_K801095 BBa_K801095], [http://partsregistry.org/Part:BBa_K801096 BBa_K801096], [http://partsregistry.org/Part:BBa_K801097 BBa_K801097] and [http://partsregistry.org/Part:BBa_K801098 BBa_K801098] <br />
<br />
<br />
<center>'''Conclusion and outlook:'''</center><br><br />
<br />
The construction of the xanthohumol pathway was achieved, whereas the expression and characterization have to be postponed after the European Jamboree or might be an interesting task for next year's iGEM teams.<br />
<br />
<div class="noborder" style="float:left"><br />
<html><a href="https://2012.igem.org/Team:TU_Munich/Project/Xanthohumol"><img width="200px" src="https://static.igem.org/mediawiki/2012/3/33/TUM12_readmore.png"/></a></html><br />
</div><br />
</div><br />
<br />
==Vector Design==<br />
----<br />
<div class="bezel mfull"><br />
===pTUM100===<br />
Designing an expression vector for yeast which is compatible to the iGEM cloning principles and standards was the main aim of this subproject. Based on the commercially available pYES2 vector we created vectors containing inducible and constitutive promoters in order to establish efficient possibilities to clone and express our enzymes.<br />
<hr><br />
[[file:TUM12_experiment_overwiew_vector.png|500px|thumb|right| Explanations on the figure:<br />
<br />
Figure A shows the new multiple cloning site (MCS) containing the RFC 10/25 restriction sides and the DNA sequence coding for the ''Strep''-tag II.<br />
<br />
Figure B gives an overview of all important functional elements located on the vector backbone. Upstream to the new MCS lies a T7 promoter primer binding site allowing easy forward sequencing of integrated gene constructs using the standard T7 primer. The URA 3 gene is a prototrophy marker used for the selection of transfected cells.<br />
<br />
Figure C to E present the successfully designed BioBricks: pTUM100 simply contains the new MCS, the transcription terminator and further elements required for cloning and transfection. pTUM102 to pTUM104 contain in addition the constitutive promoters pTef1, pTef2 and ADH. On pTUM104 is the galactose inducible promoter pGAL1 located. ]]<br />
<br />
<center>'''Experimental results:'''</center><br><br />
Using the pYES vector from Invitrogen we first deleted five forbidden restriction sites in the vector backbone via side directed mutagenesis. Furthermore, the original multiple cloning site was replaced by a multiple cloning site compatible with the RFC 10/25 cloning standards. To allow easy extraction and purification of proteins for ''in vitro'' applications the new multiple cloning site allows to express proteins with a ''Strep''-tag II. <br />
Exclusion of the galactose inducible promoter provided a powerful basis vector for the integration of user-defined promoters. This way the pTUM100 vector gives a valuable contribution to our and to further protein expression and promoter characterization experiments in ''Saccharomyces cerevisiae''.<br />
Moreover, we used the pTUM100 to integrate the three constitutive promoters Tef1, Tef2 and ADH which come all with different promoter intensities.<br />
<br />
<center>'''Outlook and conclusion:'''</center><br><br />
<br />
The galactose inducible expression system was a great aid for the majority of all subprojects. Especially the opportunity to purify and detect (via Western blot) proteins using the ''Strep''-tag II did facilitate our laboratory practice and accelerated our work progress.<br />
To cover even more demands we are planning to design a second vector template containing a His-tag.<br />
<br />
All BioBricks were sequenced. Sequences can be found in the registry of standard biological parts under the following entries:<br />
<br />
[http://partsregistry.org/Part:BBa_K801000 pTUM100 BBa_K801000], [http://partsregistry.org/Part:BBa_K801001 pTUM101 BBa_K801001], [http://partsregistry.org/Part:BBa_K801002 pTUM102 BBa_K801002], [[http://partsregistry.org/Part:BBa_K801003 pTUM103 BBa_K801003] and [[http://partsregistry.org/Part:BBa_K801004 pTUM104 BBa_K801004]] <br />
<br />
<br />
<div class="noborder" style="float:left"><br />
<html><a href="https://2012.igem.org/Team:TU_Munich/Project/Vector_Design"><img width="200px" src="https://static.igem.org/mediawiki/2012/3/33/TUM12_readmore.png"/></a></html><br />
</div><br />
</div><br />
<br />
==Regulation of Genexpression==<br />
----<br />
By developing inducible promoters and placing them upstream of our biosynthetic pathways we create the possibility to make ''S. cerevisiae'' dynamically respond to concentration changes in its medium as well as to external stimuli. <br />
<br />
An optimal inducing substance needs to be inexpensive, nontoxic and fully controllable in its application. Only substances with these characteristics allow to precisely regulate a system temporally, spatially and quantitatively. <br />
<br />
<br />
<br />
<div class="bezel mfull"><br />
===Ethanol-inducible promoter===<br />
The KlADH4-promoter from the yeast ''Kluyveromyces lactis'' regulates the expression of a mitochondrial alcohol dehydrogenase in an ethanol-dependent way. An alcohol-inducible promoter would be incredibly useful for anyone planning to brew a beer with a transgenic yeast - it would allow for the induction of the target genes after the main fermentation has finished and this way, the metabolic burden for the yeast cells could be lowered. All the transcription factors known to be involved in the regulation of the KlADH4-promoter in ''K. lactis'' also occur in ''S. cerevisiae'' [[http://www.ncbi.nlm.nih.gov/pubmed/10724480 Mazzoni et al., 2000]]. This is why we are confident that this promoter maintains its unique characteristics when transformed into ''S. cerevisiae''.<hr><br />
[[file:TUM12_experiment_overwiew_alcohol.png|400px|thumb|right| '''Top''': Emission spectrum, excitation spectrum and visible fluorescence of a yeast culture expressing eGFP under the control of the KlADH4-promoter at 1.72 Vol.-% ethanol. Significant amounts of eGFP detectable '''Center''': Emission spectrum and excitation spectrum of a yeast culture carrying a plasmid in which eGFP-expression is controlled by the KlADH4-promoter cultivated with glycerol as only carbon source. Ethanol concentration: 0.21 Vol.-%. No eGFP detectable. '''Bottom''': Fluorescence per cell and ethanol concentration plotted against time after induction.]]<br />
<center>'''Experimental results:'''</center><br><br />
At this time our results concerning the KlADH4-promoter (originally from the yeast ''Kluyveromyces lactis'') suggest that this promoter is ethanol inducible in ''S. cerevisiae''. Further experiments are still being done to abolish residual ambiguities. The lowest ethanol concentration at which eGFP-expression was detected is 0.9 Vol.-%.<br />
<br />
<center>'''Conclusion and outlook:'''</center><br><br />
<br />
Because ''S. cerevisiae'' is such a good brewer, it was difficult to produce a stringent negative control in our characterization experiments. However, we finally figured out some experiments that allowed us to keep the ethanol concentration below 0.5 % v/v, which is a concentration at which induction is observed in ''K. lactis''. We are working hard on providing additional data, but we are confident that we will be able to provide clear evidence that this promoter is ethanol-inducible not only in ''K. lactis'', but also in ''S. cerevisiae''.<br />
<br />
<div class="noborder" style="float:left"><br />
<html><a href="https://2012.igem.org/Team:TU_Munich/Project/Ethanol_Inducible_Promoter"><img width="200px" src="https://static.igem.org/mediawiki/2012/3/33/TUM12_readmore.png"/></a></html><br />
</div><br />
</div><br />
<br />
<div class="bezel mfull"><br />
<br />
===Light-switchable promoter===<br />
The idea behind a light-switchable system is to create a gene expression system which can be induced and deactivated by light of a certain wavelength.<br />
<br />
This system is extremely attractive, as induction does not require the addition of a specific substance. This makes induction '''cheap, fast, precise''' and also compatible with the Bavarian purity law.<br />
<hr><br />
[[Image:TUM12_light.jpg|thumb|right|300px|Principle of light-dependent switching of gene-expression.]]<br />
<center>'''Experimental results:'''</center><br><br />
All fusion proteins for the two types of a light-switchable promoter system has been finished ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801039 BBa_K801039], [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801040 BBa_K801040] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801041 BBa_K801041]), also gene expression batteries coding for all components of each type of our light-switchable promoter system has been done ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801042 BBa_K801042] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801043 BBa_K801043]). Since lacking of a second functional yeast vector carrying another auxotrophy marker than URA3 of the pTUM plasmids, which is already reserved for the biosynthesis enzymes, proteins and also reporters, we were not able to clone the whole gene expression battery, into a yeast vector, in order to co-transfect the yeast with one plasmid with the reporter construct and the second plasmid coding for all the devices needed in a light-switchable promoter system.<br />
<br />
<center>'''Conclusion and outlook:'''</center><br><br />
<br />
To get gene expression casette for both of the light-switchable promoter systems ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801042 BBa_K801042] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801043 BBa_K801043]) into an yeast plasmid, we want to use pSB6A0 ([http://partsregistry.org/Part:BBa_K268000 BBa_K268000]) carrying a TRP1 auxotrophy marker. But we've discovered some discrepancies in this part, e.&nbsp;g. there is still at least one forbidden restriction site (XbaI or PstI, please see experience site of the part). So we have to correct the part by quickchange mutagenesis.<br />
<br />
After successful cloning and transfection of the gene expression batteries including there reporters, we will proceed with the characterization of the part.<br />
<br />
In parallel, we will implement also the whole biosynthesis pathway for the phycocyanobilin synthesis, donwstream of our both batteries for a light-switchable promoter system ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801042 BBa_K801042] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801043 BBa_K801043]); after that, we don't have to add phycocyanobilin in our medium anymore!<br />
<br />
If there is still time in the end, we will clone our system including the biosynthetic genes for PCB synthesis in the yeast integrative vector ([http://partsregistry.org/Part:BBa_K300001 BBa_K300001]) and will start a genome integration in order to create a new yeast strain with a fully functional light-switchable promoter system.<br />
<br />
<div class="noborder" style="float:left"><br />
<html><a href="https://2012.igem.org/Team:TU_Munich/Project/Light_Switchable_Promoter"><img width="200px" src="https://static.igem.org/mediawiki/2012/3/33/TUM12_readmore.png"/></a></html><br />
</div><br />
</div><br />
<br />
==Genome Integration==<br />
----<br />
<div class="bezel mfull"><br />
As we cannot obey the letter of the German purity law (there is a zero tolerance policy concerning transgenic ingredients), we try our best to meet the spirit. Thus, it is <b>unacceptable for us to work with antibiotics</b> to keep up the selective environment. Since we <b>cannot work with auxotrophies</b> in beer either, we have to make sure the yeasts do not lose the BioBricks. The most promising way to accomplish a long lasting presence of our constructs is to achieve <b>genome integration</b>.<br />
<hr><br />
[[file:TUM12_experiment_overwiew_genome.png|500px|thumb|right| Plasmid backbone used for integration of our expression cassettes]]<br />
<center>'''Experimental results:'''</center><br><br />
First experiments to characterize the function of the yeast integration system were performed and the used selection marker was maintained in the yeast culture, although the selection pressure was switched off. This indicates that first integrations were achieved. <br />
<br />
<center>'''Conclusion and outlook:'''</center><br><br />
Maintaining the plasmids harboring our expression cassettes in the yeast cells during the brewing process is best possible using genome integration. This becomes increasingly interesting, when a yeast strain with different expression cassettes is to be created. Because this is intended for the next step of our project the integration of our expression cassettes becomes increasingly important.<br />
<br />
<div class="noborder" style="float:left"><br />
<html><a href="https://2012.igem.org/Team:TU_Munich/Project/Genome_Integration"><img width="200px" src="https://static.igem.org/mediawiki/2012/3/33/TUM12_readmore.png"/></a></html><br />
</div><br />
</div><br />
<br />
==Brewing our SynBio Beer==<br />
----<br />
<div class="bezel mfull"><br />
Contrary to popular opinion the chief ingredient of beer is not '''YPD''' but '''gyle''', a carefully prepared mixture of malt, hop and water. Although the name of the yeast strain commonly used in the lab, '''S. cerevisiae''', suggests that it is used in the beer brewing process, the yeast strains generally employed in brewing process have '''strongly adapted to gyle''', as they are reutilized in every succeeding brewing cycle.<br />
Hence some investigation on how our yeast '''performs''' in gyle was necessary.<br />
<br><hr><br />
[[file:TUM12_experiment_overwiew_Brewing.png|200px|thumb|right| Picture of the first SynBio Beer brewed during the iGEM competition in 2012]]<br />
<center>'''Experimental results:'''</center><br><br />
Our experiments show that the growth of several different yeast strains is '''not impaired in gyle'''!<br />
<br />
Expression assays proved the necessity of [[Team:TU_Munich/Project/Genome_Integration|'''genome integration''']] for a proper '''SynBio Beer'''.<br />
<br />
<center>'''Conclusion and outlook:'''</center><br><br />
As soon as cloning of integration vectors containing expression cassettes for our biosynthetic pathways are finished we will repeat the brewing experiments.<br />
<br />
<div class="noborder" style="float:left"><br />
<html><a href="https://2012.igem.org/Team:TU_Munich/Project/Brewing"><img width="200px" src="https://static.igem.org/mediawiki/2012/3/33/TUM12_readmore.png"/></a></html><br />
</div><br />
</div><br />
<br />
== References ==<br />
----<br />
* [[http://www.ncbi.nlm.nih.gov/pubmed/6327079 Edens et al., 1984]] Edens, L., Bom, I., Ledeboer, A. M., Maat, J., Toonen, M. Y., Visser, C., and Verrips, C. T. (1984). Synthesis and processing of the plant protein thaumatin in yeast. ''Cell'', 37(2):629–33.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/10995285 Miranda et al., 2000]] Miranda, C. L., Stevens, J. F., Ivanov, V., McCall, M., Frei, B., Deinzer, M. L., and Buhler, D. R. (2000). Antioxidant and prooxidant actions of prenylated and nonprenylated chalcones and flavanones in vitro. ''J Agric Food Chem'', 48(9):3876–84.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/10724480 Mazzoni et al., 2000]] Mazzoni, C., Santori, F., Saliola, M., and Falcone, C. (2000). Molecular analysis of uas(e), a cis element containing stress response elements responsible for ethanol induction of the kladh4 gene of ''kluyveromyces lactis''. ''Res Microbiol'', 151(1):19–28.</div>JaraOhttp://2012.igem.org/Team:TU_Munich/Project/OverviewTeam:TU Munich/Project/Overview2012-10-23T20:31:52Z<p>JaraO: /* pTUM100 */</p>
<hr />
<div>{{Team:TU_Munich/Header}}<br />
<br />
= Overview =<br />
<hr/><br />
<br />
== Vision ==<br />
----<br />
We, TU Munich’s 2012 iGEM team, strive to catalyze the diffusion process of knowledge about genetic engineering and synthetic biology among the general public. Using the example of iGEM’s first and finest SynBio Beer we involve, interest and inspire people to reconsider preconceived ideas and encourage them to openly engage in a broad discussion weighing pros and cons of genetic engineering in foodstuff. We sketch a future where new technology can be applied in a meaningful way to complement traditional foods or beverages.<br />
<br />
==Biosynthesis pathways==<br />
----<br />
<br />
<br />
<div class="bezel mfull"><br />
===Limonene===<br />
Limonene is a cyclic terpene and a major constituent of several citrus oils. D-Limonene has been used as a component of flavorings and fragrances. It is formed from geranyl pyrophosphate by limonene synthase.<br />
<br />
We successfully demonstrated the production of the flavoring substance limonene by expressing limonene synthase in ''S. cerevisiae'', which naturally synthesizes the educt geranyl pyrophosphate.<br />
<hr><br />
[[file:TUM12_experiment_overwiew_limonene.png|500px|thumb|right| Main results from our limonene subproject: reaction mechanism (A), constructed BioBricks (B) and proof of principle for the in vivo production of limonene]]<br />
<center>'''Experimental results:'''</center><br><br />
(+)-Limonene synthase 1 [[http://partsregistry.org/Part:BBa_K801065 BBa_K801065]] and (+)-limonene synthase 1 with yeast consensus sequence [[http://partsregistry.org/Part:BBa_K801060 BBa_K801060]] were successfully cloned into our new yeast expression vector pTUM100. Expression of recombinant limonene synthase in ''Saccharomyces cerevisiae'' was proven by western blotting. Subsequently the protein was purified using SA-chromatography and size exclusion chromatography. The functionality of the enzyme was verified by ''in vivo'' and ''in vitro'' detection of limonene via GC-MS. <br />
<br />
Furthermore, we established gene constructs of the limonene synthase coding sequence with different yeast specific promoters and terminators [[http://partsregistry.org/Part:BBa_K801062 BBa_K801062]], [[http://partsregistry.org/Part:BBa_K801063 BBa_K801063]], [[http://partsregistry.org/Part:BBa_K801064 BBa_K801064]]. <br />
<br />
Last but not least, we have brewed iGEM's first SynBio beer containing limonene.<br />
<br />
<center>'''Conclusion and outlook:'''</center><br><br />
<br />
We have achieved functional expression of ''Citrus limon'' limonene synthase and production of limonene in yeast. The last remaining step to a SynBio beer would be the integration of our gene constructs [[http://partsregistry.org/Part:BBa_K801062 BBa_K801062]], [[http://partsregistry.org/Part:BBa_K801063 BBa_K801063]], [[http://partsregistry.org/Part:BBa_K801064 BBa_K801064]] into the yeast genome.<br />
<br />
<div class="noborder" style="float:left"><br />
<html><a href="https://2012.igem.org/Team:TU_Munich/Project/Limonene"><img width="200px" src="https://static.igem.org/mediawiki/2012/3/33/TUM12_readmore.png"/></a></html><br />
</div><br />
</div><br />
<br />
<div class="bezel mfull"><br />
<br />
===Thaumatin===<br />
Thaumatin is a natural α+β-protein which is synthesized by the katamfe plant (''Thaumatococcus daniellii''). It is said to be 2,000 to 100,000 times sweeter than sucrose on molar basis, but the sweetness builds up slow and lasts long. It has been approved as a sweetener by the European Union (E957).<br />
<br />
Our aim is to have ''S. cerevisiae'' secrete functional thaumatin by expressing preprothaumatin – a principle which has been proven by [[http://www.ncbi.nlm.nih.gov/pubmed/6327079 Edens et al., 1984]].<br />
<hr><br />
[[file:TUM12_experiment_overwiew_Thaumati.png|500px|thumb|right| Main results from the Thaumatin subproject: Structure of Thaumatin (A), constructed BioBricks (B), profile of an ion exchange chromatography (IEC) used to detect our recombinant Thaumatin and a SDS-PAGE gel showing IEC elution fractions containing Thaumatin]]<br />
<center>'''Experimental results:'''</center><br><br />
The BioBrick for preprothaumatin [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801080 BBa_K801080] as well as an expression cassette [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801080 K801080] were successfully cloned, expressed in yeast, purified using an ion exchange chromatography (see figure C) and detected in the SDS-PAGE. Therefore, the expression of thaumatin in yeast could be demonstrated and functionality of the BioBrick is confirmed.<br />
<br />
<center>'''Conclusion and outlook:'''</center><br><br />
A proof of principle for the expression of thaumatin was achieved. Further goals are the increase of the expression of thaumatin and the investigation of the secretion.<br />
<br />
<div class="noborder" style="float:left"><br />
<html><a href="https://2012.igem.org/Team:TU_Munich/Project/Thaumatin"><img width="200px" src="https://static.igem.org/mediawiki/2012/3/33/TUM12_readmore.png"/></a></html><br />
</div><br />
</div><br />
<br />
<div class="bezel mfull"><br />
<br />
===Caffeine===<br />
Caffeine is a purine-alkaloid and its biosynthesis is known from coffee plants and tea plants. The molecule acts as a competitive antagonist of adenosine receptors and, therefore, increases indirectly neurotransmitter concentrations resulting in warding off drowsiness and restoring alertness. <br />
<br />
The idea is to perform a heterologous gene expression of the three enzymes 7-methylxanthosine synthase (CaXMT1), N-methyl nucleosidase (CaMXMT1) and caffeine synthase (CaDXMT1) required for caffeine biosynthesis in ''Saccharomyces cerevisiae''. <br />
<br />
<br />
<hr><br />
[[file:TUM12_experiment_overwiew_caffeine2.png|500px|thumb|right| Figure showing a schematic overview of the reaction in (A), a western blot against the Strep-tag II for GFP, [[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801070 BBa_K801070]] and [[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801071 BBa_K801071]] in (B) and finally the same western blot development for [[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801072 BBa_K801072]]]]<br />
<center>'''Experimental results:'''</center><br><br />
* Successful cloning of the three enzymes [http://partsregistry.org/Part:BBa_K801070 7-methylxanthosine synthase (CaXMT1)], [http://partsregistry.org/Part:BBa_K801071 N-methyl nucleosidase (CaMXMT1)] and the [http://partsregistry.org/Part:BBa_K801072 caffeine synthase (CaDXMT1)] into the shuttle vector pTUM104 and pSB1C3 each. <br />
* Successful assembly of the BioBricks to form expression cassettes consisting of promoter, gene and terminator: [http://partsregistry.org/Part:BBa_K801073 pTEF2-CaXMT1-tADH1], [http://partsregistry.org/Part:BBa_K801074 pTEF1-CaMXMT1-tADH1] and [http://partsregistry.org/Part:BBa_K801075 pTEF2-CaDXMT1-tADH1]) into pSB1C3.<br />
* Successful assembly of the expression cassettes of the three relevant enzymes forming a composite part of 6.4 kb capable of caffeine production in yeast ([http://partsregistry.org/Part:BBa_K801077 Caffeine Synthesis Pathway]) into pSB1C3.<br />
* Successful expression of CaXMT1, CaMXMT1 and CaDXMT1 in ''Saccharomyces cerevisiae'' INVSc1 in selective Sc minimal induction medium lacking uracil with 2 % galactose.<br />
<br />
<br />
<center>'''Conclusion and outlook:'''</center><br><br />
The homologue expression of the three required enzymes for caffeine synthesis in ''Saccharomyces cerevisiae'' INVSc1 transformed with pTUM102_CaXMT1, pTUM102_CaMXMT1 and pTUM102_CaDXMT1 was successful. Further testing of caffeine production using crude extracts from lysed transformed yeast cells and working on genome integration of the expression cassette containing all three enzymes flanked by promoter and terminator are in progress.<br />
<br />
<div class="noborder" style="float:left"><br />
<html><a href="https://2012.igem.org/Team:TU_Munich/Project/Caffeine"><img width="200px" src="https://static.igem.org/mediawiki/2012/3/33/TUM12_readmore.png"/></a></html><br />
</div><br />
</div><br />
<br />
<div class="bezel mfull"><br />
<br />
===Xanthohumol===<br />
Xanthohumol is known as a putative cancer chemopreventive agent due to its antioxidant activities [[http://www.ncbi.nlm.nih.gov/pubmed/10995285 Miranda et al., 2000]]. Our goal is a heterologous gene expression of all enzymes required for xanthohumol biosynthesis in ''S. cerevisiae''.<br />
<br />
The pathway for the production of this plant secondary metabolite is composed of five steps, starting with the conversion of phenylalanine and followed by four further enzymatic reactions.<br />
<hr><br />
[[file:TUM12_experiment_overwiew_xanto2.png|500px|thumb|right| Main results from the Xanthohumol subproject: Successful reconstruction of the metabolic pathway for Xanthohumol]]<br />
<center>'''Experimental results:'''</center><br><br />
The whole biosynthetic pathway for the production of xanthohumol was converted into BioBricks. Except for APT each of the enzymes were cloned in two versions one having the proposed consensus sequence for more efficient expression in yeast chassis and another for usage of these BioBricks in other chassis. All BioBricks were sequenced. Sequences can be found in the registry of standard biological parts:<br />
[http://partsregistry.org/Part:BBa_K801090 BBa_K801090], [http://partsregistry.org/Part:BBa_K801091 BBa_K801091], [http://partsregistry.org/Part:BBa_K801092 BBa_K801092], [http://partsregistry.org/Part:BBa_K801093 BBa_K801093], [http://partsregistry.org/Part:BBa_K801094 BBa_K801094], [http://partsregistry.org/Part:BBa_K801095 BBa_K801095], [http://partsregistry.org/Part:BBa_K801096 BBa_K801096], [http://partsregistry.org/Part:BBa_K801097 BBa_K801097] and [http://partsregistry.org/Part:BBa_K801098 BBa_K801098] <br />
<br />
<br />
<center>'''Conclusion and outlook:'''</center><br><br />
<br />
The construction of the xanthohumol pathway was achieved, whereas the expression and characterization have to be postponed after the European Jamboree or might be an interesting task for next year's iGEM teams.<br />
<br />
<div class="noborder" style="float:left"><br />
<html><a href="https://2012.igem.org/Team:TU_Munich/Project/Xanthohumol"><img width="200px" src="https://static.igem.org/mediawiki/2012/3/33/TUM12_readmore.png"/></a></html><br />
</div><br />
</div><br />
<br />
==Vector Design==<br />
----<br />
<div class="bezel mfull"><br />
===pTUM100===<br />
Designing an expression vector for yeast which is compatible to the iGEM cloning principles and standards was the main aim of this subproject. Based on the commercially available pYES2 vector we created vectors containing inducible and constitutive promoters in order to establish efficient possibilities to clone and express our enzymes.<br />
<hr><br />
[[file:TUM12_experiment_overwiew_vector.png|500px|thumb|right| Explanations on the figure:<br />
<br />
Figure A shows the new multiple cloning site (MCS) containing the RFC 10/25 restriction sides and the DNA sequence coding for the ''Strep''-tag II.<br />
<br />
Figure B gives an overview of all important functional elements located on the vector backbone. Upstream to the new MCS lies a T7 promoter primer binding site allowing easy forward sequencing of integrated gene constructs using the standard T7 primer. The URA 3 gene is a prototrophy marker used for the selection of transfected cells.<br />
<br />
Figure C to E present the successfully designed BioBricks: pTUM100 simply contains the new MCS, the transcription terminator and further elements required for cloning and transfection. pTUM102 to pTUM104 contain in addition the constitutive promoters pTef1, pTef2 and ADH. On pTUM104 is the galactose inducible promoter pGAL1 located. ]]<br />
<br />
<center>'''Experimental results:'''</center><br><br />
Using the pYES vector from Invitrogen we first deleted five forbidden restriction sites in the vector backbone via side directed mutagenesis. Furthermore, the original multiple cloning site was replaced by a multiple cloning site compatible with the RFC 10/25 cloning standards. To allow easy extraction and purification of proteins for ''in vitro'' applications the new multiple cloning site allows to express proteins with a ''Strep''-tag II. <br />
Exclusion of the galactose inducible promoter provided a powerful basis vector for the integration of user-defined promoters. This way the pTUM100 vector gives a valuable contribution to our and to further protein expression and promoter characterization experiments in ''Saccharomyces cerevisiae''.<br />
Moreover, we used the pTUM100 to integrate the three constitutive promoters Tef1, Tef2 and ADH which come all with different promoter intensities.<br />
<br />
<center>'''Outlook and conclusion:'''</center><br><br />
<br />
The galactose inducible expression system was a great aid for the majority of all subprojects. Especially the opportunity to purify and detect (via Western blot) proteins using the ''Strep''-tag II did facilitate our laboratory practice and accelerated our work progress.<br />
To cover even more demands we are planning to design a second vector template containing a His-tag.<br />
<br />
All BioBricks were sequenced. Sequences can be found in the registry of standard biological parts under the following entries:<br />
<br />
[http://partsregistry.org/Part:BBa_K801000 pTUM100 BBa_K801000], [http://partsregistry.org/Part:BBa_K801001 pTUM101 BBa_K801001], [http://partsregistry.org/Part:BBa_K801002 pTUM102 BBa_K801002], [[http://partsregistry.org/Part:BBa_K801003 pTUM103 BBa_K801003] and [[http://partsregistry.org/Part:BBa_K801004 pTUM104 BBa_K801004]] <br />
<br />
<br />
<div class="noborder" style="float:left"><br />
<html><a href="https://2012.igem.org/Team:TU_Munich/Project/Vector_Design"><img width="200px" src="https://static.igem.org/mediawiki/2012/3/33/TUM12_readmore.png"/></a></html><br />
</div><br />
</div><br />
<br />
==Regulation of Genexpression==<br />
----<br />
By developing inducible promoters and placing them upstream of our biosynthetic pathways we create the possibility to make ''S. cerevisiae'' dynamically respond to concentration changes in its medium as well as to external stimuli. <br />
<br />
An optimal inducing substance needs to be inexpensive, nontoxic and fully controllable in its application. Only substances with these characteristics allow to precisely regulate a system temporally, spatially and quantitatively. <br />
<br />
<br />
<br />
<div class="bezel mfull"><br />
===Ethanol-inducible promoter===<br />
The KlADH4-promoter from the yeast ''Kluyveromyces lactis'' regulates the expression of a mitochondrial alcohol dehydrogenase in an ethanol-dependent way. An alcohol-inducible promoter would be incredibly useful for anyone planning to brew a beer with a transgenic yeast - it would allow for the induction of the target genes after the main fermentation has finished and this way, the metabolic burden for the yeast cells could be lowered. All the transcription factors known to be involved in the regulation of the KlADH4-promoter in ''K. lactis'' also occur in ''S. cerevisiae'' [[http://www.ncbi.nlm.nih.gov/pubmed/10724480 Mazzoni et al., 2000]]. This is why we are confident that this promoter maintains its unique characteristics when transformed into ''S. cerevisiae''.<hr><br />
[[file:TUM12_experiment_overwiew_alcohol.png|400px|thumb|right| '''Top''': Emission spectrum, excitation spectrum and visible fluorescence of a yeast culture expressing eGFP under the control of the KlADH4-promoter at 1.72 Vol.-% ethanol. Significant ammounts of eGFP detectable '''Center''': Emission spectrum and excitation spectrum of a yeast culture carrying a plasmid in which eGFP-expression is controlled by the KlADH4-promoter cultivated with glycerol as only carbon source. Ethanol concentration: 0.21 Vol.-%. No eGFP detectable. '''Bottom''': Fluorescence per cell and ethanol concentration plotted against time after induction.]]<br />
<center>'''Experimental results:'''</center><br><br />
At this time our results concerning the KlADH4-promoter (originally from the yeast ''Kluyveromyces lactis'') suggest that this promoter is ethanol inducible in ''S. cerevisiae''. Further experiments are still being done to abolish residual ambiguities. The lowest ethanol concentration at which eGFP-expression was detected is 0.9 Vol.-%.<br />
<br />
<center>'''Conclusion and outlook:'''</center><br><br />
<br />
Because ''S. cerevisiae'' is such a good brewer, it was difficult to produce a stringent negative control in our characterization experiments. However, we finally figured out some experiments that allowed us to keep the ethanol concentration below 0.5 % v/v, which is a concentration at which induction is observed in ''K. lactis''. We are working hard on providing additional data, but we are confident that we will be able to provide clear evidence that this promoter is ethanol-inducible not only in ''K. lactis'', but also in ''S. cerevisiae''.<br />
<br />
<div class="noborder" style="float:left"><br />
<html><a href="https://2012.igem.org/Team:TU_Munich/Project/Ethanol_Inducible_Promoter"><img width="200px" src="https://static.igem.org/mediawiki/2012/3/33/TUM12_readmore.png"/></a></html><br />
</div><br />
</div><br />
<br />
<div class="bezel mfull"><br />
<br />
===Light-switchable promoter===<br />
The idea behind a light-switchable system is to create a gene expression system which can be induced and deactivated by light of a certain wavelength.<br />
<br />
This system is extremely attractive, as induction does not require the addition of a specific substance. This makes induction '''cheap, fast, precise''' and also compatible with the Bavarian purity law.<br />
<hr><br />
[[Image:TUM12_light.jpg|thumb|right|300px|Principle of light-dependent switching of gene-expression.]]<br />
<center>'''Experimental results:'''</center><br><br />
All fusion proteins for the two types of a light-switchable promoter system has been finished ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801039 BBa_K801039], [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801040 BBa_K801040] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801041 BBa_K801041]), also gene expression batteries coding for all components of each type of our light-switchable promoter system has been done ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801042 BBa_K801042] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801043 BBa_K801043]). Since lacking of a second functional yeast vector carrying another auxotrophy marker than URA3 of the pTUM plasmids, which is already reserved for the biosynthesis enzymes, proteins and also reporters, we were not able to clone the whole gene expression battery, into a yeast vector, in order to co-transfect the yeast with one plasmid with the reporter construct and the second plasmid coding for all the devices needed in a light-switchable promoter system.<br />
<br />
<center>'''Conclusion and outlook:'''</center><br><br />
<br />
To get gene expression casette for both of the light-switchable promoter systems ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801042 BBa_K801042] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801043 BBa_K801043]) into an yeast plasmid, we want to use pSB6A0 ([http://partsregistry.org/Part:BBa_K268000 BBa_K268000]) carrying a TRP1 auxotrophy marker. But we've discovered some discrepancies in this part, e.&nbsp;g. there is still at least one forbidden restriction site (XbaI or PstI, please see experience site of the part). So we have to correct the part by quickchange mutagenesis.<br />
<br />
After successful cloning and transfection of the gene expression batteries including there reporters, we will proceed with the characterization of the part.<br />
<br />
In parallel, we will implement also the whole biosynthesis pathway for the phycocyanobilin synthesis, donwstream of our both batteries for a light-switchable promoter system ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801042 BBa_K801042] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801043 BBa_K801043]); after that, we don't have to add phycocyanobilin in our medium anymore!<br />
<br />
If there is still time in the end, we will clone our system including the biosynthetic genes for PCB synthesis in the yeast integrative vector ([http://partsregistry.org/Part:BBa_K300001 BBa_K300001]) and will start a genome integration in order to create a new yeast strain with a fully functional light-switchable promoter system.<br />
<br />
<div class="noborder" style="float:left"><br />
<html><a href="https://2012.igem.org/Team:TU_Munich/Project/Light_Switchable_Promoter"><img width="200px" src="https://static.igem.org/mediawiki/2012/3/33/TUM12_readmore.png"/></a></html><br />
</div><br />
</div><br />
<br />
==Genome Integration==<br />
----<br />
<div class="bezel mfull"><br />
As we cannot obey the letter of the German purity law (there is a zero tolerance policy concerning transgenic ingredients), we try our best to meet the spirit. Thus, it is <b>unacceptable for us to work with antibiotics</b> to keep up the selective environment. Since we <b>cannot work with auxotrophies</b> in beer either, we have to make sure the yeasts do not lose the BioBricks. The most promising way to accomplish a long lasting presence of our constructs is to achieve <b>genome integration</b>.<br />
<hr><br />
[[file:TUM12_experiment_overwiew_genome.png|500px|thumb|right| Plasmid backbone used for integration of our expression cassettes]]<br />
<center>'''Experimental results:'''</center><br><br />
First experiments to characterize the function of the yeast integration system were performed and the used selection marker was maintained in the yeast culture, although the selection pressure was switched off. This indicates that first integrations were achieved. <br />
<br />
<center>'''Conclusion and outlook:'''</center><br><br />
Maintaining the plasmids harboring our expression cassettes in the yeast cells during the brewing process is best possible using genome integration. This becomes increasingly interesting, when a yeast strain with different expression cassettes is to be created. Because this is intended for the next step of our project the integration of our expression cassettes becomes increasingly important.<br />
<br />
<div class="noborder" style="float:left"><br />
<html><a href="https://2012.igem.org/Team:TU_Munich/Project/Genome_Integration"><img width="200px" src="https://static.igem.org/mediawiki/2012/3/33/TUM12_readmore.png"/></a></html><br />
</div><br />
</div><br />
<br />
==Brewing our SynBio Beer==<br />
----<br />
<div class="bezel mfull"><br />
Contrary to popular opinion the chief ingredient of beer is not '''YPD''' but '''gyle''', a carefully prepared mixture of malt, hop and water. Although the name of the yeast strain commonly used in the lab, '''S. cerevisiae''', suggests that it is used in the beer brewing process, the yeast strains generally employed in brewing process have '''strongly adapted to gyle''', as they are reutilized in every succeeding brewing cycle.<br />
Hence some investigation on how our yeast '''performs''' in gyle was necessary.<br />
<br><hr><br />
[[file:TUM12_experiment_overwiew_Brewing.png|200px|thumb|right| Picture of the first SynBio Beer brewed during the iGEM competition in 2012]]<br />
<center>'''Experimental results:'''</center><br><br />
Our experiments show that the growth of several different yeast strains is '''not impaired in gyle'''!<br />
<br />
Expression assays proved the necessity of [[Team:TU_Munich/Project/Genome_Integration|'''genome integration''']] for a proper '''SynBio Beer'''.<br />
<br />
<center>'''Conclusion and outlook:'''</center><br><br />
As soon as cloning of integration vectors containing expression cassettes for our biosynthetic pathways are finished we will repeat the brewing experiments.<br />
<br />
<div class="noborder" style="float:left"><br />
<html><a href="https://2012.igem.org/Team:TU_Munich/Project/Brewing"><img width="200px" src="https://static.igem.org/mediawiki/2012/3/33/TUM12_readmore.png"/></a></html><br />
</div><br />
</div><br />
<br />
== References ==<br />
----<br />
* [[http://www.ncbi.nlm.nih.gov/pubmed/6327079 Edens et al., 1984]] Edens, L., Bom, I., Ledeboer, A. M., Maat, J., Toonen, M. Y., Visser, C., and Verrips, C. T. (1984). Synthesis and processing of the plant protein thaumatin in yeast. ''Cell'', 37(2):629–33.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/10995285 Miranda et al., 2000]] Miranda, C. L., Stevens, J. F., Ivanov, V., McCall, M., Frei, B., Deinzer, M. L., and Buhler, D. R. (2000). Antioxidant and prooxidant actions of prenylated and nonprenylated chalcones and flavanones in vitro. ''J Agric Food Chem'', 48(9):3876–84.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/10724480 Mazzoni et al., 2000]] Mazzoni, C., Santori, F., Saliola, M., and Falcone, C. (2000). Molecular analysis of uas(e), a cis element containing stress response elements responsible for ethanol induction of the kladh4 gene of ''kluyveromyces lactis''. ''Res Microbiol'', 151(1):19–28.</div>JaraOhttp://2012.igem.org/Team:TU_Munich/Project/OverviewTeam:TU Munich/Project/Overview2012-10-23T20:30:51Z<p>JaraO: /* pTUM100 */</p>
<hr />
<div>{{Team:TU_Munich/Header}}<br />
<br />
= Overview =<br />
<hr/><br />
<br />
== Vision ==<br />
----<br />
We, TU Munich’s 2012 iGEM team, strive to catalyze the diffusion process of knowledge about genetic engineering and synthetic biology among the general public. Using the example of iGEM’s first and finest SynBio Beer we involve, interest and inspire people to reconsider preconceived ideas and encourage them to openly engage in a broad discussion weighing pros and cons of genetic engineering in foodstuff. We sketch a future where new technology can be applied in a meaningful way to complement traditional foods or beverages.<br />
<br />
==Biosynthesis pathways==<br />
----<br />
<br />
<br />
<div class="bezel mfull"><br />
===Limonene===<br />
Limonene is a cyclic terpene and a major constituent of several citrus oils. D-Limonene has been used as a component of flavorings and fragrances. It is formed from geranyl pyrophosphate by limonene synthase.<br />
<br />
We successfully demonstrated the production of the flavoring substance limonene by expressing limonene synthase in ''S. cerevisiae'', which naturally synthesizes the educt geranyl pyrophosphate.<br />
<hr><br />
[[file:TUM12_experiment_overwiew_limonene.png|500px|thumb|right| Main results from our limonene subproject: reaction mechanism (A), constructed BioBricks (B) and proof of principle for the in vivo production of limonene]]<br />
<center>'''Experimental results:'''</center><br><br />
(+)-Limonene synthase 1 [[http://partsregistry.org/Part:BBa_K801065 BBa_K801065]] and (+)-limonene synthase 1 with yeast consensus sequence [[http://partsregistry.org/Part:BBa_K801060 BBa_K801060]] were successfully cloned into our new yeast expression vector pTUM100. Expression of recombinant limonene synthase in ''Saccharomyces cerevisiae'' was proven by western blotting. Subsequently the protein was purified using SA-chromatography and size exclusion chromatography. The functionality of the enzyme was verified by ''in vivo'' and ''in vitro'' detection of limonene via GC-MS. <br />
<br />
Furthermore, we established gene constructs of the limonene synthase coding sequence with different yeast specific promoters and terminators [[http://partsregistry.org/Part:BBa_K801062 BBa_K801062]], [[http://partsregistry.org/Part:BBa_K801063 BBa_K801063]], [[http://partsregistry.org/Part:BBa_K801064 BBa_K801064]]. <br />
<br />
Last but not least, we have brewed iGEM's first SynBio beer containing limonene.<br />
<br />
<center>'''Conclusion and outlook:'''</center><br><br />
<br />
We have achieved functional expression of ''Citrus limon'' limonene synthase and production of limonene in yeast. The last remaining step to a SynBio beer would be the integration of our gene constructs [[http://partsregistry.org/Part:BBa_K801062 BBa_K801062]], [[http://partsregistry.org/Part:BBa_K801063 BBa_K801063]], [[http://partsregistry.org/Part:BBa_K801064 BBa_K801064]] into the yeast genome.<br />
<br />
<div class="noborder" style="float:left"><br />
<html><a href="https://2012.igem.org/Team:TU_Munich/Project/Limonene"><img width="200px" src="https://static.igem.org/mediawiki/2012/3/33/TUM12_readmore.png"/></a></html><br />
</div><br />
</div><br />
<br />
<div class="bezel mfull"><br />
<br />
===Thaumatin===<br />
Thaumatin is a natural α+β-protein which is synthesized by the katamfe plant (''Thaumatococcus daniellii''). It is said to be 2,000 to 100,000 times sweeter than sucrose on molar basis, but the sweetness builds up slow and lasts long. It has been approved as a sweetener by the European Union (E957).<br />
<br />
Our aim is to have ''S. cerevisiae'' secrete functional thaumatin by expressing preprothaumatin – a principle which has been proven by [[http://www.ncbi.nlm.nih.gov/pubmed/6327079 Edens et al., 1984]].<br />
<hr><br />
[[file:TUM12_experiment_overwiew_Thaumati.png|500px|thumb|right| Main results from the Thaumatin subproject: Structure of Thaumatin (A), constructed BioBricks (B), profile of an ion exchange chromatography (IEC) used to detect our recombinant Thaumatin and a SDS-PAGE gel showing IEC elution fractions containing Thaumatin]]<br />
<center>'''Experimental results:'''</center><br><br />
The BioBrick for preprothaumatin [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801080 BBa_K801080] as well as an expression cassette [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801080 K801080] were successfully cloned, expressed in yeast, purified using an ion exchange chromatography (see figure C) and detected in the SDS-PAGE. Therefore, the expression of thaumatin in yeast could be demonstrated and functionality of the BioBrick is confirmed.<br />
<br />
<center>'''Conclusion and outlook:'''</center><br><br />
A proof of principle for the expression of thaumatin was achieved. Further goals are the increase of the expression of thaumatin and the investigation of the secretion.<br />
<br />
<div class="noborder" style="float:left"><br />
<html><a href="https://2012.igem.org/Team:TU_Munich/Project/Thaumatin"><img width="200px" src="https://static.igem.org/mediawiki/2012/3/33/TUM12_readmore.png"/></a></html><br />
</div><br />
</div><br />
<br />
<div class="bezel mfull"><br />
<br />
===Caffeine===<br />
Caffeine is a purine-alkaloid and its biosynthesis is known from coffee plants and tea plants. The molecule acts as a competitive antagonist of adenosine receptors and, therefore, increases indirectly neurotransmitter concentrations resulting in warding off drowsiness and restoring alertness. <br />
<br />
The idea is to perform a heterologous gene expression of the three enzymes 7-methylxanthosine synthase (CaXMT1), N-methyl nucleosidase (CaMXMT1) and caffeine synthase (CaDXMT1) required for caffeine biosynthesis in ''Saccharomyces cerevisiae''. <br />
<br />
<br />
<hr><br />
[[file:TUM12_experiment_overwiew_caffeine2.png|500px|thumb|right| Figure showing a schematic overview of the reaction in (A), a western blot against the Strep-tag II for GFP, [[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801070 BBa_K801070]] and [[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801071 BBa_K801071]] in (B) and finally the same western blot development for [[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801072 BBa_K801072]]]]<br />
<center>'''Experimental results:'''</center><br><br />
* Successful cloning of the three enzymes [http://partsregistry.org/Part:BBa_K801070 7-methylxanthosine synthase (CaXMT1)], [http://partsregistry.org/Part:BBa_K801071 N-methyl nucleosidase (CaMXMT1)] and the [http://partsregistry.org/Part:BBa_K801072 caffeine synthase (CaDXMT1)] into the shuttle vector pTUM104 and pSB1C3 each. <br />
* Successful assembly of the BioBricks to form expression cassettes consisting of promoter, gene and terminator: [http://partsregistry.org/Part:BBa_K801073 pTEF2-CaXMT1-tADH1], [http://partsregistry.org/Part:BBa_K801074 pTEF1-CaMXMT1-tADH1] and [http://partsregistry.org/Part:BBa_K801075 pTEF2-CaDXMT1-tADH1]) into pSB1C3.<br />
* Successful assembly of the expression cassettes of the three relevant enzymes forming a composite part of 6.4 kb capable of caffeine production in yeast ([http://partsregistry.org/Part:BBa_K801077 Caffeine Synthesis Pathway]) into pSB1C3.<br />
* Successful expression of CaXMT1, CaMXMT1 and CaDXMT1 in ''Saccharomyces cerevisiae'' INVSc1 in selective Sc minimal induction medium lacking uracil with 2 % galactose.<br />
<br />
<br />
<center>'''Conclusion and outlook:'''</center><br><br />
The homologue expression of the three required enzymes for caffeine synthesis in ''Saccharomyces cerevisiae'' INVSc1 transformed with pTUM102_CaXMT1, pTUM102_CaMXMT1 and pTUM102_CaDXMT1 was successful. Further testing of caffeine production using crude extracts from lysed transformed yeast cells and working on genome integration of the expression cassette containing all three enzymes flanked by promoter and terminator are in progress.<br />
<br />
<div class="noborder" style="float:left"><br />
<html><a href="https://2012.igem.org/Team:TU_Munich/Project/Caffeine"><img width="200px" src="https://static.igem.org/mediawiki/2012/3/33/TUM12_readmore.png"/></a></html><br />
</div><br />
</div><br />
<br />
<div class="bezel mfull"><br />
<br />
===Xanthohumol===<br />
Xanthohumol is known as a putative cancer chemopreventive agent due to its antioxidant activities [[http://www.ncbi.nlm.nih.gov/pubmed/10995285 Miranda et al., 2000]]. Our goal is a heterologous gene expression of all enzymes required for xanthohumol biosynthesis in ''S. cerevisiae''.<br />
<br />
The pathway for the production of this plant secondary metabolite is composed of five steps, starting with the conversion of phenylalanine and followed by four further enzymatic reactions.<br />
<hr><br />
[[file:TUM12_experiment_overwiew_xanto2.png|500px|thumb|right| Main results from the Xanthohumol subproject: Successful reconstruction of the metabolic pathway for Xanthohumol]]<br />
<center>'''Experimental results:'''</center><br><br />
The whole biosynthetic pathway for the production of xanthohumol was converted into BioBricks. Except for APT each of the enzymes were cloned in two versions one having the proposed consensus sequence for more efficient expression in yeast chassis and another for usage of these BioBricks in other chassis. All BioBricks were sequenced. Sequences can be found in the registry of standard biological parts:<br />
[http://partsregistry.org/Part:BBa_K801090 BBa_K801090], [http://partsregistry.org/Part:BBa_K801091 BBa_K801091], [http://partsregistry.org/Part:BBa_K801092 BBa_K801092], [http://partsregistry.org/Part:BBa_K801093 BBa_K801093], [http://partsregistry.org/Part:BBa_K801094 BBa_K801094], [http://partsregistry.org/Part:BBa_K801095 BBa_K801095], [http://partsregistry.org/Part:BBa_K801096 BBa_K801096], [http://partsregistry.org/Part:BBa_K801097 BBa_K801097] and [http://partsregistry.org/Part:BBa_K801098 BBa_K801098] <br />
<br />
<br />
<center>'''Conclusion and outlook:'''</center><br><br />
<br />
The construction of the xanthohumol pathway was achieved, whereas the expression and characterization have to be postponed after the European Jamboree or might be an interesting task for next year's iGEM teams.<br />
<br />
<div class="noborder" style="float:left"><br />
<html><a href="https://2012.igem.org/Team:TU_Munich/Project/Xanthohumol"><img width="200px" src="https://static.igem.org/mediawiki/2012/3/33/TUM12_readmore.png"/></a></html><br />
</div><br />
</div><br />
<br />
==Vector Design==<br />
----<br />
<div class="bezel mfull"><br />
===pTUM100===<br />
Designing an expression vector for yeast which is compatible to the iGEM cloning principles and standards was the main aim of this subproject. Based on the commercially available pYES2 vector we created vectors containing inducible and constitutive promoters in order to establish efficient possibilities to clone and express our enzymes.<br />
<hr><br />
[[file:TUM12_experiment_overwiew_vector.png|500px|thumb|right| Explanations on the figure:<br />
<br />
Figure A shows the new multiple cloning site (MCS) containing the RFC 10/25 restriction sides and the DNA sequence coding for the ''Strep''-tag II.<br />
<br />
Figure B gives an overview of all important functional elements located on the vector backbone. Upstream to the new MCS lies a T7 promoter primer binding site allowing easy forward sequencing of integrated gene constructs using the standard T7 primer. The URA 3 gene is a prototrophy marker used for the selection of transfected cells.<br />
<br />
Figure C to E present the successfully designed BioBricks: pTUM100 simply contains the new MCS, the transcription terminator and further elements required for cloning and transfection. pTUM102 to pTUM104 contain in addition the constitutive promoters pTef1, pTef2 and ADH. On pTUM104 is the galactose inducible promoter pGAL1 located. ]]<br />
<br />
<center>'''Experimental results:'''</center><br><br />
Using the pYES vector from Invitrogen we first deleted five forbidden restriction sites in the vector backbone via side directed mutagenesis. Furthermore, the original multiple cloning site was replaced by a multiple cloning site compatible with the RFC 10/25 cloning standards. To allow easy extraction and purification of proteins for in vitro applications the new multiple cloning site allows to express proteins with a ''Strep''-tag II. <br />
Exclusion of the galactose inducible promoter provided a powerful basis vector for the integration of user-defined promoters. This way the pTUM100 vector gives a valuable contribution to our and to further protein expression and promoter characterization experiments in ''Saccharomyces cerevisiae''.<br />
Moreover, we used the pTUM100 to integrate the three constitutive promoters Tef1, Tef2 and ADH which come all with different promoter intensities.<br />
<br />
<center>'''Outlook and conclusion:'''</center><br><br />
<br />
The galactose inducible expression system was a great aid for the majority of all subprojects. Especially the opportunity to purify and detect (via Western blot) proteins using the ''Strep''-tag II did facilitate our laboratory practice and accelerated our work progress.<br />
To cover even more demands we are planning to design a second vector template containing a His-tag.<br />
<br />
All BioBricks were sequenced. Sequences can be found in the registry of standard biological parts under the following entries:<br />
<br />
[http://partsregistry.org/Part:BBa_K801000 pTUM100 BBa_K801000], [http://partsregistry.org/Part:BBa_K801001 pTUM101 BBa_K801001], [http://partsregistry.org/Part:BBa_K801002 pTUM102 BBa_K801002], [[http://partsregistry.org/Part:BBa_K801003 pTUM103 BBa_K801003] and [[http://partsregistry.org/Part:BBa_K801004 pTUM104 BBa_K801004]] <br />
<br />
<br />
<div class="noborder" style="float:left"><br />
<html><a href="https://2012.igem.org/Team:TU_Munich/Project/Vector_Design"><img width="200px" src="https://static.igem.org/mediawiki/2012/3/33/TUM12_readmore.png"/></a></html><br />
</div><br />
</div><br />
<br />
==Regulation of Genexpression==<br />
----<br />
By developing inducible promoters and placing them upstream of our biosynthetic pathways we create the possibility to make ''S. cerevisiae'' dynamically respond to concentration changes in its medium as well as to external stimuli. <br />
<br />
An optimal inducing substance needs to be inexpensive, nontoxic and fully controllable in its application. Only substances with these characteristics allow to precisely regulate a system temporally, spatially and quantitatively. <br />
<br />
<br />
<br />
<div class="bezel mfull"><br />
===Ethanol-inducible promoter===<br />
The KlADH4-promoter from the yeast ''Kluyveromyces lactis'' regulates the expression of a mitochondrial alcohol dehydrogenase in an ethanol-dependent way. An alcohol-inducible promoter would be incredibly useful for anyone planning to brew a beer with a transgenic yeast - it would allow for the induction of the target genes after the main fermentation has finished and this way, the metabolic burden for the yeast cells could be lowered. All the transcription factors known to be involved in the regulation of the KlADH4-promoter in ''K. lactis'' also occur in ''S. cerevisiae'' [[http://www.ncbi.nlm.nih.gov/pubmed/10724480 Mazzoni et al., 2000]]. This is why we are confident that this promoter maintains its unique characteristics when transformed into ''S. cerevisiae''.<hr><br />
[[file:TUM12_experiment_overwiew_alcohol.png|400px|thumb|right| '''Top''': Emission spectrum, excitation spectrum and visible fluorescence of a yeast culture expressing eGFP under the control of the KlADH4-promoter at 1.72 Vol.-% ethanol. Significant ammounts of eGFP detectable '''Center''': Emission spectrum and excitation spectrum of a yeast culture carrying a plasmid in which eGFP-expression is controlled by the KlADH4-promoter cultivated with glycerol as only carbon source. Ethanol concentration: 0.21 Vol.-%. No eGFP detectable. '''Bottom''': Fluorescence per cell and ethanol concentration plotted against time after induction.]]<br />
<center>'''Experimental results:'''</center><br><br />
At this time our results concerning the KlADH4-promoter (originally from the yeast ''Kluyveromyces lactis'') suggest that this promoter is ethanol inducible in ''S. cerevisiae''. Further experiments are still being done to abolish residual ambiguities. The lowest ethanol concentration at which eGFP-expression was detected is 0.9 Vol.-%.<br />
<br />
<center>'''Conclusion and outlook:'''</center><br><br />
<br />
Because ''S. cerevisiae'' is such a good brewer, it was difficult to produce a stringent negative control in our characterization experiments. However, we finally figured out some experiments that allowed us to keep the ethanol concentration below 0.5 % v/v, which is a concentration at which induction is observed in ''K. lactis''. We are working hard on providing additional data, but we are confident that we will be able to provide clear evidence that this promoter is ethanol-inducible not only in ''K. lactis'', but also in ''S. cerevisiae''.<br />
<br />
<div class="noborder" style="float:left"><br />
<html><a href="https://2012.igem.org/Team:TU_Munich/Project/Ethanol_Inducible_Promoter"><img width="200px" src="https://static.igem.org/mediawiki/2012/3/33/TUM12_readmore.png"/></a></html><br />
</div><br />
</div><br />
<br />
<div class="bezel mfull"><br />
<br />
===Light-switchable promoter===<br />
The idea behind a light-switchable system is to create a gene expression system which can be induced and deactivated by light of a certain wavelength.<br />
<br />
This system is extremely attractive, as induction does not require the addition of a specific substance. This makes induction '''cheap, fast, precise''' and also compatible with the Bavarian purity law.<br />
<hr><br />
[[Image:TUM12_light.jpg|thumb|right|300px|Principle of light-dependent switching of gene-expression.]]<br />
<center>'''Experimental results:'''</center><br><br />
All fusion proteins for the two types of a light-switchable promoter system has been finished ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801039 BBa_K801039], [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801040 BBa_K801040] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801041 BBa_K801041]), also gene expression batteries coding for all components of each type of our light-switchable promoter system has been done ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801042 BBa_K801042] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801043 BBa_K801043]). Since lacking of a second functional yeast vector carrying another auxotrophy marker than URA3 of the pTUM plasmids, which is already reserved for the biosynthesis enzymes, proteins and also reporters, we were not able to clone the whole gene expression battery, into a yeast vector, in order to co-transfect the yeast with one plasmid with the reporter construct and the second plasmid coding for all the devices needed in a light-switchable promoter system.<br />
<br />
<center>'''Conclusion and outlook:'''</center><br><br />
<br />
To get gene expression casette for both of the light-switchable promoter systems ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801042 BBa_K801042] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801043 BBa_K801043]) into an yeast plasmid, we want to use pSB6A0 ([http://partsregistry.org/Part:BBa_K268000 BBa_K268000]) carrying a TRP1 auxotrophy marker. But we've discovered some discrepancies in this part, e.&nbsp;g. there is still at least one forbidden restriction site (XbaI or PstI, please see experience site of the part). So we have to correct the part by quickchange mutagenesis.<br />
<br />
After successful cloning and transfection of the gene expression batteries including there reporters, we will proceed with the characterization of the part.<br />
<br />
In parallel, we will implement also the whole biosynthesis pathway for the phycocyanobilin synthesis, donwstream of our both batteries for a light-switchable promoter system ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801042 BBa_K801042] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801043 BBa_K801043]); after that, we don't have to add phycocyanobilin in our medium anymore!<br />
<br />
If there is still time in the end, we will clone our system including the biosynthetic genes for PCB synthesis in the yeast integrative vector ([http://partsregistry.org/Part:BBa_K300001 BBa_K300001]) and will start a genome integration in order to create a new yeast strain with a fully functional light-switchable promoter system.<br />
<br />
<div class="noborder" style="float:left"><br />
<html><a href="https://2012.igem.org/Team:TU_Munich/Project/Light_Switchable_Promoter"><img width="200px" src="https://static.igem.org/mediawiki/2012/3/33/TUM12_readmore.png"/></a></html><br />
</div><br />
</div><br />
<br />
==Genome Integration==<br />
----<br />
<div class="bezel mfull"><br />
As we cannot obey the letter of the German purity law (there is a zero tolerance policy concerning transgenic ingredients), we try our best to meet the spirit. Thus, it is <b>unacceptable for us to work with antibiotics</b> to keep up the selective environment. Since we <b>cannot work with auxotrophies</b> in beer either, we have to make sure the yeasts do not lose the BioBricks. The most promising way to accomplish a long lasting presence of our constructs is to achieve <b>genome integration</b>.<br />
<hr><br />
[[file:TUM12_experiment_overwiew_genome.png|500px|thumb|right| Plasmid backbone used for integration of our expression cassettes]]<br />
<center>'''Experimental results:'''</center><br><br />
First experiments to characterize the function of the yeast integration system were performed and the used selection marker was maintained in the yeast culture, although the selection pressure was switched off. This indicates that first integrations were achieved. <br />
<br />
<center>'''Conclusion and outlook:'''</center><br><br />
Maintaining the plasmids harboring our expression cassettes in the yeast cells during the brewing process is best possible using genome integration. This becomes increasingly interesting, when a yeast strain with different expression cassettes is to be created. Because this is intended for the next step of our project the integration of our expression cassettes becomes increasingly important.<br />
<br />
<div class="noborder" style="float:left"><br />
<html><a href="https://2012.igem.org/Team:TU_Munich/Project/Genome_Integration"><img width="200px" src="https://static.igem.org/mediawiki/2012/3/33/TUM12_readmore.png"/></a></html><br />
</div><br />
</div><br />
<br />
==Brewing our SynBio Beer==<br />
----<br />
<div class="bezel mfull"><br />
Contrary to popular opinion the chief ingredient of beer is not '''YPD''' but '''gyle''', a carefully prepared mixture of malt, hop and water. Although the name of the yeast strain commonly used in the lab, '''S. cerevisiae''', suggests that it is used in the beer brewing process, the yeast strains generally employed in brewing process have '''strongly adapted to gyle''', as they are reutilized in every succeeding brewing cycle.<br />
Hence some investigation on how our yeast '''performs''' in gyle was necessary.<br />
<br><hr><br />
[[file:TUM12_experiment_overwiew_Brewing.png|200px|thumb|right| Picture of the first SynBio Beer brewed during the iGEM competition in 2012]]<br />
<center>'''Experimental results:'''</center><br><br />
Our experiments show that the growth of several different yeast strains is '''not impaired in gyle'''!<br />
<br />
Expression assays proved the necessity of [[Team:TU_Munich/Project/Genome_Integration|'''genome integration''']] for a proper '''SynBio Beer'''.<br />
<br />
<center>'''Conclusion and outlook:'''</center><br><br />
As soon as cloning of integration vectors containing expression cassettes for our biosynthetic pathways are finished we will repeat the brewing experiments.<br />
<br />
<div class="noborder" style="float:left"><br />
<html><a href="https://2012.igem.org/Team:TU_Munich/Project/Brewing"><img width="200px" src="https://static.igem.org/mediawiki/2012/3/33/TUM12_readmore.png"/></a></html><br />
</div><br />
</div><br />
<br />
== References ==<br />
----<br />
* [[http://www.ncbi.nlm.nih.gov/pubmed/6327079 Edens et al., 1984]] Edens, L., Bom, I., Ledeboer, A. M., Maat, J., Toonen, M. Y., Visser, C., and Verrips, C. T. (1984). Synthesis and processing of the plant protein thaumatin in yeast. ''Cell'', 37(2):629–33.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/10995285 Miranda et al., 2000]] Miranda, C. L., Stevens, J. F., Ivanov, V., McCall, M., Frei, B., Deinzer, M. L., and Buhler, D. R. (2000). Antioxidant and prooxidant actions of prenylated and nonprenylated chalcones and flavanones in vitro. ''J Agric Food Chem'', 48(9):3876–84.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/10724480 Mazzoni et al., 2000]] Mazzoni, C., Santori, F., Saliola, M., and Falcone, C. (2000). Molecular analysis of uas(e), a cis element containing stress response elements responsible for ethanol induction of the kladh4 gene of ''kluyveromyces lactis''. ''Res Microbiol'', 151(1):19–28.</div>JaraOhttp://2012.igem.org/Team:TU_Munich/Project/OverviewTeam:TU Munich/Project/Overview2012-10-23T20:24:07Z<p>JaraO: /* Thaumatin */</p>
<hr />
<div>{{Team:TU_Munich/Header}}<br />
<br />
= Overview =<br />
<hr/><br />
<br />
== Vision ==<br />
----<br />
We, TU Munich’s 2012 iGEM team, strive to catalyze the diffusion process of knowledge about genetic engineering and synthetic biology among the general public. Using the example of iGEM’s first and finest SynBio Beer we involve, interest and inspire people to reconsider preconceived ideas and encourage them to openly engage in a broad discussion weighing pros and cons of genetic engineering in foodstuff. We sketch a future where new technology can be applied in a meaningful way to complement traditional foods or beverages.<br />
<br />
==Biosynthesis pathways==<br />
----<br />
<br />
<br />
<div class="bezel mfull"><br />
===Limonene===<br />
Limonene is a cyclic terpene and a major constituent of several citrus oils. D-Limonene has been used as a component of flavorings and fragrances. It is formed from geranyl pyrophosphate by limonene synthase.<br />
<br />
We successfully demonstrated the production of the flavoring substance limonene by expressing limonene synthase in ''S. cerevisiae'', which naturally synthesizes the educt geranyl pyrophosphate.<br />
<hr><br />
[[file:TUM12_experiment_overwiew_limonene.png|500px|thumb|right| Main results from our limonene subproject: reaction mechanism (A), constructed BioBricks (B) and proof of principle for the in vivo production of limonene]]<br />
<center>'''Experimental results:'''</center><br><br />
(+)-Limonene synthase 1 [[http://partsregistry.org/Part:BBa_K801065 BBa_K801065]] and (+)-limonene synthase 1 with yeast consensus sequence [[http://partsregistry.org/Part:BBa_K801060 BBa_K801060]] were successfully cloned into our new yeast expression vector pTUM100. Expression of recombinant limonene synthase in ''Saccharomyces cerevisiae'' was proven by western blotting. Subsequently the protein was purified using SA-chromatography and size exclusion chromatography. The functionality of the enzyme was verified by ''in vivo'' and ''in vitro'' detection of limonene via GC-MS. <br />
<br />
Furthermore, we established gene constructs of the limonene synthase coding sequence with different yeast specific promoters and terminators [[http://partsregistry.org/Part:BBa_K801062 BBa_K801062]], [[http://partsregistry.org/Part:BBa_K801063 BBa_K801063]], [[http://partsregistry.org/Part:BBa_K801064 BBa_K801064]]. <br />
<br />
Last but not least, we have brewed iGEM's first SynBio beer containing limonene.<br />
<br />
<center>'''Conclusion and outlook:'''</center><br><br />
<br />
We have achieved functional expression of ''Citrus limon'' limonene synthase and production of limonene in yeast. The last remaining step to a SynBio beer would be the integration of our gene constructs [[http://partsregistry.org/Part:BBa_K801062 BBa_K801062]], [[http://partsregistry.org/Part:BBa_K801063 BBa_K801063]], [[http://partsregistry.org/Part:BBa_K801064 BBa_K801064]] into the yeast genome.<br />
<br />
<div class="noborder" style="float:left"><br />
<html><a href="https://2012.igem.org/Team:TU_Munich/Project/Limonene"><img width="200px" src="https://static.igem.org/mediawiki/2012/3/33/TUM12_readmore.png"/></a></html><br />
</div><br />
</div><br />
<br />
<div class="bezel mfull"><br />
<br />
===Thaumatin===<br />
Thaumatin is a natural α+β-protein which is synthesized by the katamfe plant (''Thaumatococcus daniellii''). It is said to be 2,000 to 100,000 times sweeter than sucrose on molar basis, but the sweetness builds up slow and lasts long. It has been approved as a sweetener by the European Union (E957).<br />
<br />
Our aim is to have ''S. cerevisiae'' secrete functional thaumatin by expressing preprothaumatin – a principle which has been proven by [[http://www.ncbi.nlm.nih.gov/pubmed/6327079 Edens et al., 1984]].<br />
<hr><br />
[[file:TUM12_experiment_overwiew_Thaumati.png|500px|thumb|right| Main results from the Thaumatin subproject: Structure of Thaumatin (A), constructed BioBricks (B), profile of an ion exchange chromatography (IEC) used to detect our recombinant Thaumatin and a SDS-PAGE gel showing IEC elution fractions containing Thaumatin]]<br />
<center>'''Experimental results:'''</center><br><br />
The BioBrick for preprothaumatin [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801080 BBa_K801080] as well as an expression cassette [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801080 K801080] were successfully cloned, expressed in yeast, purified using an ion exchange chromatography (see figure C) and detected in the SDS-PAGE. Therefore, the expression of thaumatin in yeast could be demonstrated and functionality of the BioBrick is confirmed.<br />
<br />
<center>'''Conclusion and outlook:'''</center><br><br />
A proof of principle for the expression of thaumatin was achieved. Further goals are the increase of the expression of thaumatin and the investigation of the secretion.<br />
<br />
<div class="noborder" style="float:left"><br />
<html><a href="https://2012.igem.org/Team:TU_Munich/Project/Thaumatin"><img width="200px" src="https://static.igem.org/mediawiki/2012/3/33/TUM12_readmore.png"/></a></html><br />
</div><br />
</div><br />
<br />
<div class="bezel mfull"><br />
<br />
===Caffeine===<br />
Caffeine is a purine-alkaloid and its biosynthesis is known from coffee plants and tea plants. The molecule acts as a competitive antagonist of adenosine receptors and, therefore, increases indirectly neurotransmitter concentrations resulting in warding off drowsiness and restoring alertness. <br />
<br />
The idea is to perform a heterologous gene expression of the three enzymes 7-methylxanthosine synthase (CaXMT1), N-methyl nucleosidase (CaMXMT1) and caffeine synthase (CaDXMT1) required for caffeine biosynthesis in ''Saccharomyces cerevisiae''. <br />
<br />
<br />
<hr><br />
[[file:TUM12_experiment_overwiew_caffeine2.png|500px|thumb|right| Figure showing a schematic overview of the reaction in (A), a western blot against the Strep-tag II for GFP, [[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801070 BBa_K801070]] and [[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801071 BBa_K801071]] in (B) and finally the same western blot development for [[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801072 BBa_K801072]]]]<br />
<center>'''Experimental results:'''</center><br><br />
* Successful cloning of the three enzymes [http://partsregistry.org/Part:BBa_K801070 7-methylxanthosine synthase (CaXMT1)], [http://partsregistry.org/Part:BBa_K801071 N-methyl nucleosidase (CaMXMT1)] and the [http://partsregistry.org/Part:BBa_K801072 caffeine synthase (CaDXMT1)] into the shuttle vector pTUM104 and pSB1C3 each. <br />
* Successful assembly of the BioBricks to form expression cassettes consisting of promoter, gene and terminator: [http://partsregistry.org/Part:BBa_K801073 pTEF2-CaXMT1-tADH1], [http://partsregistry.org/Part:BBa_K801074 pTEF1-CaMXMT1-tADH1] and [http://partsregistry.org/Part:BBa_K801075 pTEF2-CaDXMT1-tADH1]) into pSB1C3.<br />
* Successful assembly of the expression cassettes of the three relevant enzymes forming a composite part of 6.4 kb capable of caffeine production in yeast ([http://partsregistry.org/Part:BBa_K801077 Caffeine Synthesis Pathway]) into pSB1C3.<br />
* Successful expression of CaXMT1, CaMXMT1 and CaDXMT1 in ''Saccharomyces cerevisiae'' INVSc1 in selective Sc minimal induction medium lacking uracil with 2 % galactose.<br />
<br />
<br />
<center>'''Conclusion and outlook:'''</center><br><br />
The homologue expression of the three required enzymes for caffeine synthesis in ''Saccharomyces cerevisiae'' INVSc1 transformed with pTUM102_CaXMT1, pTUM102_CaMXMT1 and pTUM102_CaDXMT1 was successful. Further testing of caffeine production using crude extracts from lysed transformed yeast cells and working on genome integration of the expression cassette containing all three enzymes flanked by promoter and terminator are in progress.<br />
<br />
<div class="noborder" style="float:left"><br />
<html><a href="https://2012.igem.org/Team:TU_Munich/Project/Caffeine"><img width="200px" src="https://static.igem.org/mediawiki/2012/3/33/TUM12_readmore.png"/></a></html><br />
</div><br />
</div><br />
<br />
<div class="bezel mfull"><br />
<br />
===Xanthohumol===<br />
Xanthohumol is known as a putative cancer chemopreventive agent due to its antioxidant activities [[http://www.ncbi.nlm.nih.gov/pubmed/10995285 Miranda et al., 2000]]. Our goal is a heterologous gene expression of all enzymes required for xanthohumol biosynthesis in ''S. cerevisiae''.<br />
<br />
The pathway for the production of this plant secondary metabolite is composed of five steps, starting with the conversion of phenylalanine and followed by four further enzymatic reactions.<br />
<hr><br />
[[file:TUM12_experiment_overwiew_xanto2.png|500px|thumb|right| Main results from the Xanthohumol subproject: Successful reconstruction of the metabolic pathway for Xanthohumol]]<br />
<center>'''Experimental results:'''</center><br><br />
The whole biosynthetic pathway for the production of xanthohumol was converted into BioBricks. Except for APT each of the enzymes were cloned in two versions one having the proposed consensus sequence for more efficient expression in yeast chassis and another for usage of these BioBricks in other chassis. All BioBricks were sequenced. Sequences can be found in the registry of standard biological parts:<br />
[http://partsregistry.org/Part:BBa_K801090 BBa_K801090], [http://partsregistry.org/Part:BBa_K801091 BBa_K801091], [http://partsregistry.org/Part:BBa_K801092 BBa_K801092], [http://partsregistry.org/Part:BBa_K801093 BBa_K801093], [http://partsregistry.org/Part:BBa_K801094 BBa_K801094], [http://partsregistry.org/Part:BBa_K801095 BBa_K801095], [http://partsregistry.org/Part:BBa_K801096 BBa_K801096], [http://partsregistry.org/Part:BBa_K801097 BBa_K801097] and [http://partsregistry.org/Part:BBa_K801098 BBa_K801098] <br />
<br />
<br />
<center>'''Conclusion and outlook:'''</center><br><br />
<br />
The construction of the xanthohumol pathway was achieved, whereas the expression and characterization have to be postponed after the European Jamboree or might be an interesting task for next year's iGEM teams.<br />
<br />
<div class="noborder" style="float:left"><br />
<html><a href="https://2012.igem.org/Team:TU_Munich/Project/Xanthohumol"><img width="200px" src="https://static.igem.org/mediawiki/2012/3/33/TUM12_readmore.png"/></a></html><br />
</div><br />
</div><br />
<br />
==Vector Design==<br />
----<br />
<div class="bezel mfull"><br />
===pTUM100===<br />
Designing an expression vector for yeast which is compatible to the iGEM cloning principles and standards was the main aim of this subproject. Based on the commercially available pYES2 vector we created vectors containing inducible and constitutive promoters in order to establish efficient possibilities to clone and express our enzymes.<br />
<hr><br />
[[file:TUM12_experiment_overwiew_vector.png|500px|thumb|right| Explanations on the figure:<br />
<br />
Figure A shows the new multiple cloning site (MCS) containing the RFC 10/25 restriction sides and the DNA sequence coding for the ''Strep''-tag II.<br />
<br />
Figure B gives an overview of all important functional elements located on the vector backbone. Upstream to the new MCS lies a T7 promoter primer binding site allowing easy forward sequencing of integrated gene constructs using the standard T7 primer. The URA 3 gene is a prototrophy marker used for the selection of transfected cells.<br />
<br />
Figure C to E present the successfully designed BioBricks: pTUM100 simply contains the new MCS, the transcription terminator and further elements required for cloning and transfection. pTUM102 to pTUM104 contain in addition the constitutive promoters pTef1, pTef2 and ADH. On pTUM104 is the galactose inducible promoter pGAL1 located. ]]<br />
<br />
<center>'''Experimental results:'''</center><br><br />
Using the pYES vector from Invitrogen we first deleted five forbidden restriction sites in the vector back bone via side directed mutagenesis. Furthermore, the original multiple cloning site was replaced by a multiple cloning site compatible with the RFC 10/25 cloning standards. To allow easy extraction and purification of proteins for in vitro applications the new multiple cloning site allows to express proteins with a ''Strep''-tag II. <br />
Exclusion of the galactose inducible promoter provided a powerful basis vector for the integration of user-defined promoters. This way the pTUM100 vector gives a valuable contribution to our and to further protein expression and promoter characterization experiments in ''Saccharomyces cerevisiae''.<br />
Moreover, we used the pTUM100 to integrate the three constitutive promoters Tef1, Tef2 and ADH which come all with different promoter intensities.<br />
<br />
<center>'''Outlook and conclusion:'''</center><br><br />
<br />
The galactose inducible expression system was a great aid for the majority of all subprojects. Especially the opportunity to purify and detect (via Western blot) proteins using the ''Strep''-tag II did facilitate our laboratory practice and accelerated our work progress.<br />
To cover even more demands we are planning to design a second vector template containing a His-tag.<br />
<br />
All BioBricks were sequenced. Sequences can be found in the registry of standard biological parts under the following entries:<br />
<br />
[http://partsregistry.org/Part:BBa_K801000 pTUM100 BBa_K801000], [http://partsregistry.org/Part:BBa_K801001 pTUM101 BBa_K801001], [http://partsregistry.org/Part:BBa_K801002 pTUM102 BBa_K801002], [[http://partsregistry.org/Part:BBa_K801003 pTUM103 BBa_K801003] and [[http://partsregistry.org/Part:BBa_K801004 pTUM104 BBa_K801004]] <br />
<br />
<br />
<div class="noborder" style="float:left"><br />
<html><a href="https://2012.igem.org/Team:TU_Munich/Project/Vector_Design"><img width="200px" src="https://static.igem.org/mediawiki/2012/3/33/TUM12_readmore.png"/></a></html><br />
</div><br />
</div><br />
<br />
==Regulation of Genexpression==<br />
----<br />
By developing inducible promoters and placing them upstream of our biosynthetic pathways we create the possibility to make ''S. cerevisiae'' dynamically respond to concentration changes in its medium as well as to external stimuli. <br />
<br />
An optimal inducing substance needs to be inexpensive, nontoxic and fully controllable in its application. Only substances with these characteristics allow to precisely regulate a system temporally, spatially and quantitatively. <br />
<br />
<br />
<br />
<div class="bezel mfull"><br />
===Ethanol-inducible promoter===<br />
The KlADH4-promoter from the yeast ''Kluyveromyces lactis'' regulates the expression of a mitochondrial alcohol dehydrogenase in an ethanol-dependent way. An alcohol-inducible promoter would be incredibly useful for anyone planning to brew a beer with a transgenic yeast - it would allow for the induction of the target genes after the main fermentation has finished and this way, the metabolic burden for the yeast cells could be lowered. All the transcription factors known to be involved in the regulation of the KlADH4-promoter in ''K. lactis'' also occur in ''S. cerevisiae'' [[http://www.ncbi.nlm.nih.gov/pubmed/10724480 Mazzoni et al., 2000]]. This is why we are confident that this promoter maintains its unique characteristics when transformed into ''S. cerevisiae''.<hr><br />
[[file:TUM12_experiment_overwiew_alcohol.png|400px|thumb|right| '''Top''': Emission spectrum, excitation spectrum and visible fluorescence of a yeast culture expressing eGFP under the control of the KlADH4-promoter at 1.72 Vol.-% ethanol. Significant ammounts of eGFP detectable '''Center''': Emission spectrum and excitation spectrum of a yeast culture carrying a plasmid in which eGFP-expression is controlled by the KlADH4-promoter cultivated with glycerol as only carbon source. Ethanol concentration: 0.21 Vol.-%. No eGFP detectable. '''Bottom''': Fluorescence per cell and ethanol concentration plotted against time after induction.]]<br />
<center>'''Experimental results:'''</center><br><br />
At this time our results concerning the KlADH4-promoter (originally from the yeast ''Kluyveromyces lactis'') suggest that this promoter is ethanol inducible in ''S. cerevisiae''. Further experiments are still being done to abolish residual ambiguities. The lowest ethanol concentration at which eGFP-expression was detected is 0.9 Vol.-%.<br />
<br />
<center>'''Conclusion and outlook:'''</center><br><br />
<br />
Because ''S. cerevisiae'' is such a good brewer, it was difficult to produce a stringent negative control in our characterization experiments. However, we finally figured out some experiments that allowed us to keep the ethanol concentration below 0.5 % v/v, which is a concentration at which induction is observed in ''K. lactis''. We are working hard on providing additional data, but we are confident that we will be able to provide clear evidence that this promoter is ethanol-inducible not only in ''K. lactis'', but also in ''S. cerevisiae''.<br />
<br />
<div class="noborder" style="float:left"><br />
<html><a href="https://2012.igem.org/Team:TU_Munich/Project/Ethanol_Inducible_Promoter"><img width="200px" src="https://static.igem.org/mediawiki/2012/3/33/TUM12_readmore.png"/></a></html><br />
</div><br />
</div><br />
<br />
<div class="bezel mfull"><br />
<br />
===Light-switchable promoter===<br />
The idea behind a light-switchable system is to create a gene expression system which can be induced and deactivated by light of a certain wavelength.<br />
<br />
This system is extremely attractive, as induction does not require the addition of a specific substance. This makes induction '''cheap, fast, precise''' and also compatible with the Bavarian purity law.<br />
<hr><br />
[[Image:TUM12_light.jpg|thumb|right|300px|Principle of light-dependent switching of gene-expression.]]<br />
<center>'''Experimental results:'''</center><br><br />
All fusion proteins for the two types of a light-switchable promoter system has been finished ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801039 BBa_K801039], [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801040 BBa_K801040] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801041 BBa_K801041]), also gene expression batteries coding for all components of each type of our light-switchable promoter system has been done ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801042 BBa_K801042] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801043 BBa_K801043]). Since lacking of a second functional yeast vector carrying another auxotrophy marker than URA3 of the pTUM plasmids, which is already reserved for the biosynthesis enzymes, proteins and also reporters, we were not able to clone the whole gene expression battery, into a yeast vector, in order to co-transfect the yeast with one plasmid with the reporter construct and the second plasmid coding for all the devices needed in a light-switchable promoter system.<br />
<br />
<center>'''Conclusion and outlook:'''</center><br><br />
<br />
To get gene expression casette for both of the light-switchable promoter systems ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801042 BBa_K801042] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801043 BBa_K801043]) into an yeast plasmid, we want to use pSB6A0 ([http://partsregistry.org/Part:BBa_K268000 BBa_K268000]) carrying a TRP1 auxotrophy marker. But we've discovered some discrepancies in this part, e.&nbsp;g. there is still at least one forbidden restriction site (XbaI or PstI, please see experience site of the part). So we have to correct the part by quickchange mutagenesis.<br />
<br />
After successful cloning and transfection of the gene expression batteries including there reporters, we will proceed with the characterization of the part.<br />
<br />
In parallel, we will implement also the whole biosynthesis pathway for the phycocyanobilin synthesis, donwstream of our both batteries for a light-switchable promoter system ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801042 BBa_K801042] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801043 BBa_K801043]); after that, we don't have to add phycocyanobilin in our medium anymore!<br />
<br />
If there is still time in the end, we will clone our system including the biosynthetic genes for PCB synthesis in the yeast integrative vector ([http://partsregistry.org/Part:BBa_K300001 BBa_K300001]) and will start a genome integration in order to create a new yeast strain with a fully functional light-switchable promoter system.<br />
<br />
<div class="noborder" style="float:left"><br />
<html><a href="https://2012.igem.org/Team:TU_Munich/Project/Light_Switchable_Promoter"><img width="200px" src="https://static.igem.org/mediawiki/2012/3/33/TUM12_readmore.png"/></a></html><br />
</div><br />
</div><br />
<br />
==Genome Integration==<br />
----<br />
<div class="bezel mfull"><br />
As we cannot obey the letter of the German purity law (there is a zero tolerance policy concerning transgenic ingredients), we try our best to meet the spirit. Thus, it is <b>unacceptable for us to work with antibiotics</b> to keep up the selective environment. Since we <b>cannot work with auxotrophies</b> in beer either, we have to make sure the yeasts do not lose the BioBricks. The most promising way to accomplish a long lasting presence of our constructs is to achieve <b>genome integration</b>.<br />
<hr><br />
[[file:TUM12_experiment_overwiew_genome.png|500px|thumb|right| Plasmid backbone used for integration of our expression cassettes]]<br />
<center>'''Experimental results:'''</center><br><br />
First experiments to characterize the function of the yeast integration system were performed and the used selection marker was maintained in the yeast culture, although the selection pressure was switched off. This indicates that first integrations were achieved. <br />
<br />
<center>'''Conclusion and outlook:'''</center><br><br />
Maintaining the plasmids harboring our expression cassettes in the yeast cells during the brewing process is best possible using genome integration. This becomes increasingly interesting, when a yeast strain with different expression cassettes is to be created. Because this is intended for the next step of our project the integration of our expression cassettes becomes increasingly important.<br />
<br />
<div class="noborder" style="float:left"><br />
<html><a href="https://2012.igem.org/Team:TU_Munich/Project/Genome_Integration"><img width="200px" src="https://static.igem.org/mediawiki/2012/3/33/TUM12_readmore.png"/></a></html><br />
</div><br />
</div><br />
<br />
==Brewing our SynBio Beer==<br />
----<br />
<div class="bezel mfull"><br />
Contrary to popular opinion the chief ingredient of beer is not '''YPD''' but '''gyle''', a carefully prepared mixture of malt, hop and water. Although the name of the yeast strain commonly used in the lab, '''S. cerevisiae''', suggests that it is used in the beer brewing process, the yeast strains generally employed in brewing process have '''strongly adapted to gyle''', as they are reutilized in every succeeding brewing cycle.<br />
Hence some investigation on how our yeast '''performs''' in gyle was necessary.<br />
<br><hr><br />
[[file:TUM12_experiment_overwiew_Brewing.png|200px|thumb|right| Picture of the first SynBio Beer brewed during the iGEM competition in 2012]]<br />
<center>'''Experimental results:'''</center><br><br />
Our experiments show that the growth of several different yeast strains is '''not impaired in gyle'''!<br />
<br />
Expression assays proved the necessity of [[Team:TU_Munich/Project/Genome_Integration|'''genome integration''']] for a proper '''SynBio Beer'''.<br />
<br />
<center>'''Conclusion and outlook:'''</center><br><br />
As soon as cloning of integration vectors containing expression cassettes for our biosynthetic pathways are finished we will repeat the brewing experiments.<br />
<br />
<div class="noborder" style="float:left"><br />
<html><a href="https://2012.igem.org/Team:TU_Munich/Project/Brewing"><img width="200px" src="https://static.igem.org/mediawiki/2012/3/33/TUM12_readmore.png"/></a></html><br />
</div><br />
</div><br />
<br />
== References ==<br />
----<br />
* [[http://www.ncbi.nlm.nih.gov/pubmed/6327079 Edens et al., 1984]] Edens, L., Bom, I., Ledeboer, A. M., Maat, J., Toonen, M. Y., Visser, C., and Verrips, C. T. (1984). Synthesis and processing of the plant protein thaumatin in yeast. ''Cell'', 37(2):629–33.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/10995285 Miranda et al., 2000]] Miranda, C. L., Stevens, J. F., Ivanov, V., McCall, M., Frei, B., Deinzer, M. L., and Buhler, D. R. (2000). Antioxidant and prooxidant actions of prenylated and nonprenylated chalcones and flavanones in vitro. ''J Agric Food Chem'', 48(9):3876–84.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/10724480 Mazzoni et al., 2000]] Mazzoni, C., Santori, F., Saliola, M., and Falcone, C. (2000). Molecular analysis of uas(e), a cis element containing stress response elements responsible for ethanol induction of the kladh4 gene of ''kluyveromyces lactis''. ''Res Microbiol'', 151(1):19–28.</div>JaraOhttp://2012.igem.org/Team:TU_Munich/Project/OverviewTeam:TU Munich/Project/Overview2012-10-23T20:21:49Z<p>JaraO: /* Limonene */</p>
<hr />
<div>{{Team:TU_Munich/Header}}<br />
<br />
= Overview =<br />
<hr/><br />
<br />
== Vision ==<br />
----<br />
We, TU Munich’s 2012 iGEM team, strive to catalyze the diffusion process of knowledge about genetic engineering and synthetic biology among the general public. Using the example of iGEM’s first and finest SynBio Beer we involve, interest and inspire people to reconsider preconceived ideas and encourage them to openly engage in a broad discussion weighing pros and cons of genetic engineering in foodstuff. We sketch a future where new technology can be applied in a meaningful way to complement traditional foods or beverages.<br />
<br />
==Biosynthesis pathways==<br />
----<br />
<br />
<br />
<div class="bezel mfull"><br />
===Limonene===<br />
Limonene is a cyclic terpene and a major constituent of several citrus oils. D-Limonene has been used as a component of flavorings and fragrances. It is formed from geranyl pyrophosphate by limonene synthase.<br />
<br />
We successfully demonstrated the production of the flavoring substance limonene by expressing limonene synthase in ''S. cerevisiae'', which naturally synthesizes the educt geranyl pyrophosphate.<br />
<hr><br />
[[file:TUM12_experiment_overwiew_limonene.png|500px|thumb|right| Main results from our limonene subproject: reaction mechanism (A), constructed BioBricks (B) and proof of principle for the in vivo production of limonene]]<br />
<center>'''Experimental results:'''</center><br><br />
(+)-Limonene synthase 1 [[http://partsregistry.org/Part:BBa_K801065 BBa_K801065]] and (+)-limonene synthase 1 with yeast consensus sequence [[http://partsregistry.org/Part:BBa_K801060 BBa_K801060]] were successfully cloned into our new yeast expression vector pTUM100. Expression of recombinant limonene synthase in ''Saccharomyces cerevisiae'' was proven by western blotting. Subsequently the protein was purified using SA-chromatography and size exclusion chromatography. The functionality of the enzyme was verified by ''in vivo'' and ''in vitro'' detection of limonene via GC-MS. <br />
<br />
Furthermore, we established gene constructs of the limonene synthase coding sequence with different yeast specific promoters and terminators [[http://partsregistry.org/Part:BBa_K801062 BBa_K801062]], [[http://partsregistry.org/Part:BBa_K801063 BBa_K801063]], [[http://partsregistry.org/Part:BBa_K801064 BBa_K801064]]. <br />
<br />
Last but not least, we have brewed iGEM's first SynBio beer containing limonene.<br />
<br />
<center>'''Conclusion and outlook:'''</center><br><br />
<br />
We have achieved functional expression of ''Citrus limon'' limonene synthase and production of limonene in yeast. The last remaining step to a SynBio beer would be the integration of our gene constructs [[http://partsregistry.org/Part:BBa_K801062 BBa_K801062]], [[http://partsregistry.org/Part:BBa_K801063 BBa_K801063]], [[http://partsregistry.org/Part:BBa_K801064 BBa_K801064]] into the yeast genome.<br />
<br />
<div class="noborder" style="float:left"><br />
<html><a href="https://2012.igem.org/Team:TU_Munich/Project/Limonene"><img width="200px" src="https://static.igem.org/mediawiki/2012/3/33/TUM12_readmore.png"/></a></html><br />
</div><br />
</div><br />
<br />
<div class="bezel mfull"><br />
<br />
===Thaumatin===<br />
Thaumatin is a natural α+β-protein which is synthesized by the katamfe plant (''Thaumatococcus daniellii''). It is said to be 2,000 to 100,000 times sweeter than sucrose on molar basis, but the sweetness builds up slow and lasts long. It has been approved as a sweetener by the European Union (E957).<br />
<br />
Our aim is to have ''S. cerevisiae'' secrete functional thaumatin by expressing preprothaumatin – a principle which has been proven by [[http://www.ncbi.nlm.nih.gov/pubmed/6327079 Edens et al., 1984]].<br />
<hr><br />
[[file:TUM12_experiment_overwiew_Thaumati.png|500px|thumb|right| Main results from the Thaumatin subproject: Structure of Thaumatin (A), constructed BioBricks (B), profile of an ion exchange chromatography (IEC) used to detect our recombinant Thaumatin and a SDS-PAGE gel showing IEC elution fractions containing Thaumatin]]<br />
<center>'''Experimental results:'''</center><br><br />
The BioBrick for preprothaumatin [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801080 BBa_K801080] as well as a expression cassette [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801080 K801080] were successfully cloned, expressed in yeast, purified using an ion exchange chromatography (see figure C) and detected in the SDS-PAGE. Therefore, the expression of thaumatin in yeast could be demonstrated and functionality of the BioBrick is confirmed.<br />
<br />
<center>'''Conclusion and outlook:'''</center><br><br />
A proof of principle for the expression of thaumatin was achieved. Further goals are the increase of the expression of thaumatin and the investigation of the secretion.<br />
<br />
<div class="noborder" style="float:left"><br />
<html><a href="https://2012.igem.org/Team:TU_Munich/Project/Thaumatin"><img width="200px" src="https://static.igem.org/mediawiki/2012/3/33/TUM12_readmore.png"/></a></html><br />
</div><br />
</div><br />
<br />
<div class="bezel mfull"><br />
<br />
===Caffeine===<br />
Caffeine is a purine-alkaloid and its biosynthesis is known from coffee plants and tea plants. The molecule acts as a competitive antagonist of adenosine receptors and, therefore, increases indirectly neurotransmitter concentrations resulting in warding off drowsiness and restoring alertness. <br />
<br />
The idea is to perform a heterologous gene expression of the three enzymes 7-methylxanthosine synthase (CaXMT1), N-methyl nucleosidase (CaMXMT1) and caffeine synthase (CaDXMT1) required for caffeine biosynthesis in ''Saccharomyces cerevisiae''. <br />
<br />
<br />
<hr><br />
[[file:TUM12_experiment_overwiew_caffeine2.png|500px|thumb|right| Figure showing a schematic overview of the reaction in (A), a western blot against the Strep-tag II for GFP, [[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801070 BBa_K801070]] and [[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801071 BBa_K801071]] in (B) and finally the same western blot development for [[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801072 BBa_K801072]]]]<br />
<center>'''Experimental results:'''</center><br><br />
* Successful cloning of the three enzymes [http://partsregistry.org/Part:BBa_K801070 7-methylxanthosine synthase (CaXMT1)], [http://partsregistry.org/Part:BBa_K801071 N-methyl nucleosidase (CaMXMT1)] and the [http://partsregistry.org/Part:BBa_K801072 caffeine synthase (CaDXMT1)] into the shuttle vector pTUM104 and pSB1C3 each. <br />
* Successful assembly of the BioBricks to form expression cassettes consisting of promoter, gene and terminator: [http://partsregistry.org/Part:BBa_K801073 pTEF2-CaXMT1-tADH1], [http://partsregistry.org/Part:BBa_K801074 pTEF1-CaMXMT1-tADH1] and [http://partsregistry.org/Part:BBa_K801075 pTEF2-CaDXMT1-tADH1]) into pSB1C3.<br />
* Successful assembly of the expression cassettes of the three relevant enzymes forming a composite part of 6.4 kb capable of caffeine production in yeast ([http://partsregistry.org/Part:BBa_K801077 Caffeine Synthesis Pathway]) into pSB1C3.<br />
* Successful expression of CaXMT1, CaMXMT1 and CaDXMT1 in ''Saccharomyces cerevisiae'' INVSc1 in selective Sc minimal induction medium lacking uracil with 2 % galactose.<br />
<br />
<br />
<center>'''Conclusion and outlook:'''</center><br><br />
The homologue expression of the three required enzymes for caffeine synthesis in ''Saccharomyces cerevisiae'' INVSc1 transformed with pTUM102_CaXMT1, pTUM102_CaMXMT1 and pTUM102_CaDXMT1 was successful. Further testing of caffeine production using crude extracts from lysed transformed yeast cells and working on genome integration of the expression cassette containing all three enzymes flanked by promoter and terminator are in progress.<br />
<br />
<div class="noborder" style="float:left"><br />
<html><a href="https://2012.igem.org/Team:TU_Munich/Project/Caffeine"><img width="200px" src="https://static.igem.org/mediawiki/2012/3/33/TUM12_readmore.png"/></a></html><br />
</div><br />
</div><br />
<br />
<div class="bezel mfull"><br />
<br />
===Xanthohumol===<br />
Xanthohumol is known as a putative cancer chemopreventive agent due to its antioxidant activities [[http://www.ncbi.nlm.nih.gov/pubmed/10995285 Miranda et al., 2000]]. Our goal is a heterologous gene expression of all enzymes required for xanthohumol biosynthesis in ''S. cerevisiae''.<br />
<br />
The pathway for the production of this plant secondary metabolite is composed of five steps, starting with the conversion of phenylalanine and followed by four further enzymatic reactions.<br />
<hr><br />
[[file:TUM12_experiment_overwiew_xanto2.png|500px|thumb|right| Main results from the Xanthohumol subproject: Successful reconstruction of the metabolic pathway for Xanthohumol]]<br />
<center>'''Experimental results:'''</center><br><br />
The whole biosynthetic pathway for the production of xanthohumol was converted into BioBricks. Except for APT each of the enzymes were cloned in two versions one having the proposed consensus sequence for more efficient expression in yeast chassis and another for usage of these BioBricks in other chassis. All BioBricks were sequenced. Sequences can be found in the registry of standard biological parts:<br />
[http://partsregistry.org/Part:BBa_K801090 BBa_K801090], [http://partsregistry.org/Part:BBa_K801091 BBa_K801091], [http://partsregistry.org/Part:BBa_K801092 BBa_K801092], [http://partsregistry.org/Part:BBa_K801093 BBa_K801093], [http://partsregistry.org/Part:BBa_K801094 BBa_K801094], [http://partsregistry.org/Part:BBa_K801095 BBa_K801095], [http://partsregistry.org/Part:BBa_K801096 BBa_K801096], [http://partsregistry.org/Part:BBa_K801097 BBa_K801097] and [http://partsregistry.org/Part:BBa_K801098 BBa_K801098] <br />
<br />
<br />
<center>'''Conclusion and outlook:'''</center><br><br />
<br />
The construction of the xanthohumol pathway was achieved, whereas the expression and characterization have to be postponed after the European Jamboree or might be an interesting task for next year's iGEM teams.<br />
<br />
<div class="noborder" style="float:left"><br />
<html><a href="https://2012.igem.org/Team:TU_Munich/Project/Xanthohumol"><img width="200px" src="https://static.igem.org/mediawiki/2012/3/33/TUM12_readmore.png"/></a></html><br />
</div><br />
</div><br />
<br />
==Vector Design==<br />
----<br />
<div class="bezel mfull"><br />
===pTUM100===<br />
Designing an expression vector for yeast which is compatible to the iGEM cloning principles and standards was the main aim of this subproject. Based on the commercially available pYES2 vector we created vectors containing inducible and constitutive promoters in order to establish efficient possibilities to clone and express our enzymes.<br />
<hr><br />
[[file:TUM12_experiment_overwiew_vector.png|500px|thumb|right| Explanations on the figure:<br />
<br />
Figure A shows the new multiple cloning site (MCS) containing the RFC 10/25 restriction sides and the DNA sequence coding for the ''Strep''-tag II.<br />
<br />
Figure B gives an overview of all important functional elements located on the vector backbone. Upstream to the new MCS lies a T7 promoter primer binding site allowing easy forward sequencing of integrated gene constructs using the standard T7 primer. The URA 3 gene is a prototrophy marker used for the selection of transfected cells.<br />
<br />
Figure C to E present the successfully designed BioBricks: pTUM100 simply contains the new MCS, the transcription terminator and further elements required for cloning and transfection. pTUM102 to pTUM104 contain in addition the constitutive promoters pTef1, pTef2 and ADH. On pTUM104 is the galactose inducible promoter pGAL1 located. ]]<br />
<br />
<center>'''Experimental results:'''</center><br><br />
Using the pYES vector from Invitrogen we first deleted five forbidden restriction sites in the vector back bone via side directed mutagenesis. Furthermore, the original multiple cloning site was replaced by a multiple cloning site compatible with the RFC 10/25 cloning standards. To allow easy extraction and purification of proteins for in vitro applications the new multiple cloning site allows to express proteins with a ''Strep''-tag II. <br />
Exclusion of the galactose inducible promoter provided a powerful basis vector for the integration of user-defined promoters. This way the pTUM100 vector gives a valuable contribution to our and to further protein expression and promoter characterization experiments in ''Saccharomyces cerevisiae''.<br />
Moreover, we used the pTUM100 to integrate the three constitutive promoters Tef1, Tef2 and ADH which come all with different promoter intensities.<br />
<br />
<center>'''Outlook and conclusion:'''</center><br><br />
<br />
The galactose inducible expression system was a great aid for the majority of all subprojects. Especially the opportunity to purify and detect (via Western blot) proteins using the ''Strep''-tag II did facilitate our laboratory practice and accelerated our work progress.<br />
To cover even more demands we are planning to design a second vector template containing a His-tag.<br />
<br />
All BioBricks were sequenced. Sequences can be found in the registry of standard biological parts under the following entries:<br />
<br />
[http://partsregistry.org/Part:BBa_K801000 pTUM100 BBa_K801000], [http://partsregistry.org/Part:BBa_K801001 pTUM101 BBa_K801001], [http://partsregistry.org/Part:BBa_K801002 pTUM102 BBa_K801002], [[http://partsregistry.org/Part:BBa_K801003 pTUM103 BBa_K801003] and [[http://partsregistry.org/Part:BBa_K801004 pTUM104 BBa_K801004]] <br />
<br />
<br />
<div class="noborder" style="float:left"><br />
<html><a href="https://2012.igem.org/Team:TU_Munich/Project/Vector_Design"><img width="200px" src="https://static.igem.org/mediawiki/2012/3/33/TUM12_readmore.png"/></a></html><br />
</div><br />
</div><br />
<br />
==Regulation of Genexpression==<br />
----<br />
By developing inducible promoters and placing them upstream of our biosynthetic pathways we create the possibility to make ''S. cerevisiae'' dynamically respond to concentration changes in its medium as well as to external stimuli. <br />
<br />
An optimal inducing substance needs to be inexpensive, nontoxic and fully controllable in its application. Only substances with these characteristics allow to precisely regulate a system temporally, spatially and quantitatively. <br />
<br />
<br />
<br />
<div class="bezel mfull"><br />
===Ethanol-inducible promoter===<br />
The KlADH4-promoter from the yeast ''Kluyveromyces lactis'' regulates the expression of a mitochondrial alcohol dehydrogenase in an ethanol-dependent way. An alcohol-inducible promoter would be incredibly useful for anyone planning to brew a beer with a transgenic yeast - it would allow for the induction of the target genes after the main fermentation has finished and this way, the metabolic burden for the yeast cells could be lowered. All the transcription factors known to be involved in the regulation of the KlADH4-promoter in ''K. lactis'' also occur in ''S. cerevisiae'' [[http://www.ncbi.nlm.nih.gov/pubmed/10724480 Mazzoni et al., 2000]]. This is why we are confident that this promoter maintains its unique characteristics when transformed into ''S. cerevisiae''.<hr><br />
[[file:TUM12_experiment_overwiew_alcohol.png|400px|thumb|right| '''Top''': Emission spectrum, excitation spectrum and visible fluorescence of a yeast culture expressing eGFP under the control of the KlADH4-promoter at 1.72 Vol.-% ethanol. Significant ammounts of eGFP detectable '''Center''': Emission spectrum and excitation spectrum of a yeast culture carrying a plasmid in which eGFP-expression is controlled by the KlADH4-promoter cultivated with glycerol as only carbon source. Ethanol concentration: 0.21 Vol.-%. No eGFP detectable. '''Bottom''': Fluorescence per cell and ethanol concentration plotted against time after induction.]]<br />
<center>'''Experimental results:'''</center><br><br />
At this time our results concerning the KlADH4-promoter (originally from the yeast ''Kluyveromyces lactis'') suggest that this promoter is ethanol inducible in ''S. cerevisiae''. Further experiments are still being done to abolish residual ambiguities. The lowest ethanol concentration at which eGFP-expression was detected is 0.9 Vol.-%.<br />
<br />
<center>'''Conclusion and outlook:'''</center><br><br />
<br />
Because ''S. cerevisiae'' is such a good brewer, it was difficult to produce a stringent negative control in our characterization experiments. However, we finally figured out some experiments that allowed us to keep the ethanol concentration below 0.5 % v/v, which is a concentration at which induction is observed in ''K. lactis''. We are working hard on providing additional data, but we are confident that we will be able to provide clear evidence that this promoter is ethanol-inducible not only in ''K. lactis'', but also in ''S. cerevisiae''.<br />
<br />
<div class="noborder" style="float:left"><br />
<html><a href="https://2012.igem.org/Team:TU_Munich/Project/Ethanol_Inducible_Promoter"><img width="200px" src="https://static.igem.org/mediawiki/2012/3/33/TUM12_readmore.png"/></a></html><br />
</div><br />
</div><br />
<br />
<div class="bezel mfull"><br />
<br />
===Light-switchable promoter===<br />
The idea behind a light-switchable system is to create a gene expression system which can be induced and deactivated by light of a certain wavelength.<br />
<br />
This system is extremely attractive, as induction does not require the addition of a specific substance. This makes induction '''cheap, fast, precise''' and also compatible with the Bavarian purity law.<br />
<hr><br />
[[Image:TUM12_light.jpg|thumb|right|300px|Principle of light-dependent switching of gene-expression.]]<br />
<center>'''Experimental results:'''</center><br><br />
All fusion proteins for the two types of a light-switchable promoter system has been finished ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801039 BBa_K801039], [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801040 BBa_K801040] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801041 BBa_K801041]), also gene expression batteries coding for all components of each type of our light-switchable promoter system has been done ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801042 BBa_K801042] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801043 BBa_K801043]). Since lacking of a second functional yeast vector carrying another auxotrophy marker than URA3 of the pTUM plasmids, which is already reserved for the biosynthesis enzymes, proteins and also reporters, we were not able to clone the whole gene expression battery, into a yeast vector, in order to co-transfect the yeast with one plasmid with the reporter construct and the second plasmid coding for all the devices needed in a light-switchable promoter system.<br />
<br />
<center>'''Conclusion and outlook:'''</center><br><br />
<br />
To get gene expression casette for both of the light-switchable promoter systems ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801042 BBa_K801042] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801043 BBa_K801043]) into an yeast plasmid, we want to use pSB6A0 ([http://partsregistry.org/Part:BBa_K268000 BBa_K268000]) carrying a TRP1 auxotrophy marker. But we've discovered some discrepancies in this part, e.&nbsp;g. there is still at least one forbidden restriction site (XbaI or PstI, please see experience site of the part). So we have to correct the part by quickchange mutagenesis.<br />
<br />
After successful cloning and transfection of the gene expression batteries including there reporters, we will proceed with the characterization of the part.<br />
<br />
In parallel, we will implement also the whole biosynthesis pathway for the phycocyanobilin synthesis, donwstream of our both batteries for a light-switchable promoter system ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801042 BBa_K801042] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801043 BBa_K801043]); after that, we don't have to add phycocyanobilin in our medium anymore!<br />
<br />
If there is still time in the end, we will clone our system including the biosynthetic genes for PCB synthesis in the yeast integrative vector ([http://partsregistry.org/Part:BBa_K300001 BBa_K300001]) and will start a genome integration in order to create a new yeast strain with a fully functional light-switchable promoter system.<br />
<br />
<div class="noborder" style="float:left"><br />
<html><a href="https://2012.igem.org/Team:TU_Munich/Project/Light_Switchable_Promoter"><img width="200px" src="https://static.igem.org/mediawiki/2012/3/33/TUM12_readmore.png"/></a></html><br />
</div><br />
</div><br />
<br />
==Genome Integration==<br />
----<br />
<div class="bezel mfull"><br />
As we cannot obey the letter of the German purity law (there is a zero tolerance policy concerning transgenic ingredients), we try our best to meet the spirit. Thus, it is <b>unacceptable for us to work with antibiotics</b> to keep up the selective environment. Since we <b>cannot work with auxotrophies</b> in beer either, we have to make sure the yeasts do not lose the BioBricks. The most promising way to accomplish a long lasting presence of our constructs is to achieve <b>genome integration</b>.<br />
<hr><br />
[[file:TUM12_experiment_overwiew_genome.png|500px|thumb|right| Plasmid backbone used for integration of our expression cassettes]]<br />
<center>'''Experimental results:'''</center><br><br />
First experiments to characterize the function of the yeast integration system were performed and the used selection marker was maintained in the yeast culture, although the selection pressure was switched off. This indicates that first integrations were achieved. <br />
<br />
<center>'''Conclusion and outlook:'''</center><br><br />
Maintaining the plasmids harboring our expression cassettes in the yeast cells during the brewing process is best possible using genome integration. This becomes increasingly interesting, when a yeast strain with different expression cassettes is to be created. Because this is intended for the next step of our project the integration of our expression cassettes becomes increasingly important.<br />
<br />
<div class="noborder" style="float:left"><br />
<html><a href="https://2012.igem.org/Team:TU_Munich/Project/Genome_Integration"><img width="200px" src="https://static.igem.org/mediawiki/2012/3/33/TUM12_readmore.png"/></a></html><br />
</div><br />
</div><br />
<br />
==Brewing our SynBio Beer==<br />
----<br />
<div class="bezel mfull"><br />
Contrary to popular opinion the chief ingredient of beer is not '''YPD''' but '''gyle''', a carefully prepared mixture of malt, hop and water. Although the name of the yeast strain commonly used in the lab, '''S. cerevisiae''', suggests that it is used in the beer brewing process, the yeast strains generally employed in brewing process have '''strongly adapted to gyle''', as they are reutilized in every succeeding brewing cycle.<br />
Hence some investigation on how our yeast '''performs''' in gyle was necessary.<br />
<br><hr><br />
[[file:TUM12_experiment_overwiew_Brewing.png|200px|thumb|right| Picture of the first SynBio Beer brewed during the iGEM competition in 2012]]<br />
<center>'''Experimental results:'''</center><br><br />
Our experiments show that the growth of several different yeast strains is '''not impaired in gyle'''!<br />
<br />
Expression assays proved the necessity of [[Team:TU_Munich/Project/Genome_Integration|'''genome integration''']] for a proper '''SynBio Beer'''.<br />
<br />
<center>'''Conclusion and outlook:'''</center><br><br />
As soon as cloning of integration vectors containing expression cassettes for our biosynthetic pathways are finished we will repeat the brewing experiments.<br />
<br />
<div class="noborder" style="float:left"><br />
<html><a href="https://2012.igem.org/Team:TU_Munich/Project/Brewing"><img width="200px" src="https://static.igem.org/mediawiki/2012/3/33/TUM12_readmore.png"/></a></html><br />
</div><br />
</div><br />
<br />
== References ==<br />
----<br />
* [[http://www.ncbi.nlm.nih.gov/pubmed/6327079 Edens et al., 1984]] Edens, L., Bom, I., Ledeboer, A. M., Maat, J., Toonen, M. Y., Visser, C., and Verrips, C. T. (1984). Synthesis and processing of the plant protein thaumatin in yeast. ''Cell'', 37(2):629–33.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/10995285 Miranda et al., 2000]] Miranda, C. L., Stevens, J. F., Ivanov, V., McCall, M., Frei, B., Deinzer, M. L., and Buhler, D. R. (2000). Antioxidant and prooxidant actions of prenylated and nonprenylated chalcones and flavanones in vitro. ''J Agric Food Chem'', 48(9):3876–84.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/10724480 Mazzoni et al., 2000]] Mazzoni, C., Santori, F., Saliola, M., and Falcone, C. (2000). Molecular analysis of uas(e), a cis element containing stress response elements responsible for ethanol induction of the kladh4 gene of ''kluyveromyces lactis''. ''Res Microbiol'', 151(1):19–28.</div>JaraOhttp://2012.igem.org/Team:TU_Munich/Project/OverviewTeam:TU Munich/Project/Overview2012-10-23T20:20:37Z<p>JaraO: /* Limonene */</p>
<hr />
<div>{{Team:TU_Munich/Header}}<br />
<br />
= Overview =<br />
<hr/><br />
<br />
== Vision ==<br />
----<br />
We, TU Munich’s 2012 iGEM team, strive to catalyze the diffusion process of knowledge about genetic engineering and synthetic biology among the general public. Using the example of iGEM’s first and finest SynBio Beer we involve, interest and inspire people to reconsider preconceived ideas and encourage them to openly engage in a broad discussion weighing pros and cons of genetic engineering in foodstuff. We sketch a future where new technology can be applied in a meaningful way to complement traditional foods or beverages.<br />
<br />
==Biosynthesis pathways==<br />
----<br />
<br />
<br />
<div class="bezel mfull"><br />
===Limonene===<br />
Limonene is a cyclic terpene and a major constituent of several citrus oils. D-Limonene has been used as a component of flavorings and fragrances. It is formed from geranyl pyrophosphate by limonene synthase.<br />
<br />
We successfully demonstrated the production of the flavoring substance limonene by expressing limonene synthase in ''S. cerevisiae'', which naturally synthesizes the educt geranyl pyrophosphate.<br />
<hr><br />
[[file:TUM12_experiment_overwiew_limonene.png|500px|thumb|right| Main results from our limonene subproject: reaction mechanism (A), constructed BioBricks (B) and proof of principle for the in vivo production of limonene]]<br />
<center>'''Experimental results:'''</center><br><br />
(+)-Limonene synthase 1 [[http://partsregistry.org/Part:BBa_K801065 BBa_K801065]] and (+)-limonene synthase 1 with yeast consensus sequence [[http://partsregistry.org/Part:BBa_K801060 BBa_K801060]] were successfully cloned into our new yeast expression vector pTUM100. Expression of recombinant limonene synthase in ''Saccharomyces cerevisiae'' was proven by western blotting. Subsequently the protein was purified using SA-chromatography and size exclusion chromatography. The functionality of the enzyme was verified by ''in vivo'' and ''in vitro'' detection of limonene via GC-MS. <br />
<br />
Furthermore, we established gene constructs of the limonene synthase coding sequence with different yeast specific promoters and terminators [[http://partsregistry.org/Part:BBa_K801062 BBa_K801062]], [[http://partsregistry.org/Part:BBa_K801063 BBa_K801063]], [[http://partsregistry.org/Part:BBa_K801064 BBa_K801064]]. <br />
<br />
Last but not least, we have brewed iGEM's first SynBio beer containing limonene.<br />
<br />
<center>'''Conclusion and outlook:'''</center><br><br />
<br />
We have achieved functional expression of ''Citrus limon'' limonene synthase and production of limonene in yeast. The last remaining step to an SynBio beer would be the integrateion of our gene constructs [[http://partsregistry.org/Part:BBa_K801062 BBa_K801062]], [[http://partsregistry.org/Part:BBa_K801063 BBa_K801063]], [[http://partsregistry.org/Part:BBa_K801064 BBa_K801064]] into the yeast genome.<br />
<br />
<div class="noborder" style="float:left"><br />
<html><a href="https://2012.igem.org/Team:TU_Munich/Project/Limonene"><img width="200px" src="https://static.igem.org/mediawiki/2012/3/33/TUM12_readmore.png"/></a></html><br />
</div><br />
</div><br />
<br />
<div class="bezel mfull"><br />
<br />
===Thaumatin===<br />
Thaumatin is a natural α+β-protein which is synthesized by the katamfe plant (''Thaumatococcus daniellii''). It is said to be 2,000 to 100,000 times sweeter than sucrose on molar basis, but the sweetness builds up slow and lasts long. It has been approved as a sweetener by the European Union (E957).<br />
<br />
Our aim is to have ''S. cerevisiae'' secrete functional thaumatin by expressing preprothaumatin – a principle which has been proven by [[http://www.ncbi.nlm.nih.gov/pubmed/6327079 Edens et al., 1984]].<br />
<hr><br />
[[file:TUM12_experiment_overwiew_Thaumati.png|500px|thumb|right| Main results from the Thaumatin subproject: Structure of Thaumatin (A), constructed BioBricks (B), profile of an ion exchange chromatography (IEC) used to detect our recombinant Thaumatin and a SDS-PAGE gel showing IEC elution fractions containing Thaumatin]]<br />
<center>'''Experimental results:'''</center><br><br />
The BioBrick for preprothaumatin [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801080 BBa_K801080] as well as a expression cassette [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801080 K801080] were successfully cloned, expressed in yeast, purified using an ion exchange chromatography (see figure C) and detected in the SDS-PAGE. Therefore, the expression of thaumatin in yeast could be demonstrated and functionality of the BioBrick is confirmed.<br />
<br />
<center>'''Conclusion and outlook:'''</center><br><br />
A proof of principle for the expression of thaumatin was achieved. Further goals are the increase of the expression of thaumatin and the investigation of the secretion.<br />
<br />
<div class="noborder" style="float:left"><br />
<html><a href="https://2012.igem.org/Team:TU_Munich/Project/Thaumatin"><img width="200px" src="https://static.igem.org/mediawiki/2012/3/33/TUM12_readmore.png"/></a></html><br />
</div><br />
</div><br />
<br />
<div class="bezel mfull"><br />
<br />
===Caffeine===<br />
Caffeine is a purine-alkaloid and its biosynthesis is known from coffee plants and tea plants. The molecule acts as a competitive antagonist of adenosine receptors and, therefore, increases indirectly neurotransmitter concentrations resulting in warding off drowsiness and restoring alertness. <br />
<br />
The idea is to perform a heterologous gene expression of the three enzymes 7-methylxanthosine synthase (CaXMT1), N-methyl nucleosidase (CaMXMT1) and caffeine synthase (CaDXMT1) required for caffeine biosynthesis in ''Saccharomyces cerevisiae''. <br />
<br />
<br />
<hr><br />
[[file:TUM12_experiment_overwiew_caffeine2.png|500px|thumb|right| Figure showing a schematic overview of the reaction in (A), a western blot against the Strep-tag II for GFP, [[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801070 BBa_K801070]] and [[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801071 BBa_K801071]] in (B) and finally the same western blot development for [[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801072 BBa_K801072]]]]<br />
<center>'''Experimental results:'''</center><br><br />
* Successful cloning of the three enzymes [http://partsregistry.org/Part:BBa_K801070 7-methylxanthosine synthase (CaXMT1)], [http://partsregistry.org/Part:BBa_K801071 N-methyl nucleosidase (CaMXMT1)] and the [http://partsregistry.org/Part:BBa_K801072 caffeine synthase (CaDXMT1)] into the shuttle vector pTUM104 and pSB1C3 each. <br />
* Successful assembly of the BioBricks to form expression cassettes consisting of promoter, gene and terminator: [http://partsregistry.org/Part:BBa_K801073 pTEF2-CaXMT1-tADH1], [http://partsregistry.org/Part:BBa_K801074 pTEF1-CaMXMT1-tADH1] and [http://partsregistry.org/Part:BBa_K801075 pTEF2-CaDXMT1-tADH1]) into pSB1C3.<br />
* Successful assembly of the expression cassettes of the three relevant enzymes forming a composite part of 6.4 kb capable of caffeine production in yeast ([http://partsregistry.org/Part:BBa_K801077 Caffeine Synthesis Pathway]) into pSB1C3.<br />
* Successful expression of CaXMT1, CaMXMT1 and CaDXMT1 in ''Saccharomyces cerevisiae'' INVSc1 in selective Sc minimal induction medium lacking uracil with 2 % galactose.<br />
<br />
<br />
<center>'''Conclusion and outlook:'''</center><br><br />
The homologue expression of the three required enzymes for caffeine synthesis in ''Saccharomyces cerevisiae'' INVSc1 transformed with pTUM102_CaXMT1, pTUM102_CaMXMT1 and pTUM102_CaDXMT1 was successful. Further testing of caffeine production using crude extracts from lysed transformed yeast cells and working on genome integration of the expression cassette containing all three enzymes flanked by promoter and terminator are in progress.<br />
<br />
<div class="noborder" style="float:left"><br />
<html><a href="https://2012.igem.org/Team:TU_Munich/Project/Caffeine"><img width="200px" src="https://static.igem.org/mediawiki/2012/3/33/TUM12_readmore.png"/></a></html><br />
</div><br />
</div><br />
<br />
<div class="bezel mfull"><br />
<br />
===Xanthohumol===<br />
Xanthohumol is known as a putative cancer chemopreventive agent due to its antioxidant activities [[http://www.ncbi.nlm.nih.gov/pubmed/10995285 Miranda et al., 2000]]. Our goal is a heterologous gene expression of all enzymes required for xanthohumol biosynthesis in ''S. cerevisiae''.<br />
<br />
The pathway for the production of this plant secondary metabolite is composed of five steps, starting with the conversion of phenylalanine and followed by four further enzymatic reactions.<br />
<hr><br />
[[file:TUM12_experiment_overwiew_xanto2.png|500px|thumb|right| Main results from the Xanthohumol subproject: Successful reconstruction of the metabolic pathway for Xanthohumol]]<br />
<center>'''Experimental results:'''</center><br><br />
The whole biosynthetic pathway for the production of xanthohumol was converted into BioBricks. Except for APT each of the enzymes were cloned in two versions one having the proposed consensus sequence for more efficient expression in yeast chassis and another for usage of these BioBricks in other chassis. All BioBricks were sequenced. Sequences can be found in the registry of standard biological parts:<br />
[http://partsregistry.org/Part:BBa_K801090 BBa_K801090], [http://partsregistry.org/Part:BBa_K801091 BBa_K801091], [http://partsregistry.org/Part:BBa_K801092 BBa_K801092], [http://partsregistry.org/Part:BBa_K801093 BBa_K801093], [http://partsregistry.org/Part:BBa_K801094 BBa_K801094], [http://partsregistry.org/Part:BBa_K801095 BBa_K801095], [http://partsregistry.org/Part:BBa_K801096 BBa_K801096], [http://partsregistry.org/Part:BBa_K801097 BBa_K801097] and [http://partsregistry.org/Part:BBa_K801098 BBa_K801098] <br />
<br />
<br />
<center>'''Conclusion and outlook:'''</center><br><br />
<br />
The construction of the xanthohumol pathway was achieved, whereas the expression and characterization have to be postponed after the European Jamboree or might be an interesting task for next year's iGEM teams.<br />
<br />
<div class="noborder" style="float:left"><br />
<html><a href="https://2012.igem.org/Team:TU_Munich/Project/Xanthohumol"><img width="200px" src="https://static.igem.org/mediawiki/2012/3/33/TUM12_readmore.png"/></a></html><br />
</div><br />
</div><br />
<br />
==Vector Design==<br />
----<br />
<div class="bezel mfull"><br />
===pTUM100===<br />
Designing an expression vector for yeast which is compatible to the iGEM cloning principles and standards was the main aim of this subproject. Based on the commercially available pYES2 vector we created vectors containing inducible and constitutive promoters in order to establish efficient possibilities to clone and express our enzymes.<br />
<hr><br />
[[file:TUM12_experiment_overwiew_vector.png|500px|thumb|right| Explanations on the figure:<br />
<br />
Figure A shows the new multiple cloning site (MCS) containing the RFC 10/25 restriction sides and the DNA sequence coding for the ''Strep''-tag II.<br />
<br />
Figure B gives an overview of all important functional elements located on the vector backbone. Upstream to the new MCS lies a T7 promoter primer binding site allowing easy forward sequencing of integrated gene constructs using the standard T7 primer. The URA 3 gene is a prototrophy marker used for the selection of transfected cells.<br />
<br />
Figure C to E present the successfully designed BioBricks: pTUM100 simply contains the new MCS, the transcription terminator and further elements required for cloning and transfection. pTUM102 to pTUM104 contain in addition the constitutive promoters pTef1, pTef2 and ADH. On pTUM104 is the galactose inducible promoter pGAL1 located. ]]<br />
<br />
<center>'''Experimental results:'''</center><br><br />
Using the pYES vector from Invitrogen we first deleted five forbidden restriction sites in the vector back bone via side directed mutagenesis. Furthermore, the original multiple cloning site was replaced by a multiple cloning site compatible with the RFC 10/25 cloning standards. To allow easy extraction and purification of proteins for in vitro applications the new multiple cloning site allows to express proteins with a ''Strep''-tag II. <br />
Exclusion of the galactose inducible promoter provided a powerful basis vector for the integration of user-defined promoters. This way the pTUM100 vector gives a valuable contribution to our and to further protein expression and promoter characterization experiments in ''Saccharomyces cerevisiae''.<br />
Moreover, we used the pTUM100 to integrate the three constitutive promoters Tef1, Tef2 and ADH which come all with different promoter intensities.<br />
<br />
<center>'''Outlook and conclusion:'''</center><br><br />
<br />
The galactose inducible expression system was a great aid for the majority of all subprojects. Especially the opportunity to purify and detect (via Western blot) proteins using the ''Strep''-tag II did facilitate our laboratory practice and accelerated our work progress.<br />
To cover even more demands we are planning to design a second vector template containing a His-tag.<br />
<br />
All BioBricks were sequenced. Sequences can be found in the registry of standard biological parts under the following entries:<br />
<br />
[http://partsregistry.org/Part:BBa_K801000 pTUM100 BBa_K801000], [http://partsregistry.org/Part:BBa_K801001 pTUM101 BBa_K801001], [http://partsregistry.org/Part:BBa_K801002 pTUM102 BBa_K801002], [[http://partsregistry.org/Part:BBa_K801003 pTUM103 BBa_K801003] and [[http://partsregistry.org/Part:BBa_K801004 pTUM104 BBa_K801004]] <br />
<br />
<br />
<div class="noborder" style="float:left"><br />
<html><a href="https://2012.igem.org/Team:TU_Munich/Project/Vector_Design"><img width="200px" src="https://static.igem.org/mediawiki/2012/3/33/TUM12_readmore.png"/></a></html><br />
</div><br />
</div><br />
<br />
==Regulation of Genexpression==<br />
----<br />
By developing inducible promoters and placing them upstream of our biosynthetic pathways we create the possibility to make ''S. cerevisiae'' dynamically respond to concentration changes in its medium as well as to external stimuli. <br />
<br />
An optimal inducing substance needs to be inexpensive, nontoxic and fully controllable in its application. Only substances with these characteristics allow to precisely regulate a system temporally, spatially and quantitatively. <br />
<br />
<br />
<br />
<div class="bezel mfull"><br />
===Ethanol-inducible promoter===<br />
The KlADH4-promoter from the yeast ''Kluyveromyces lactis'' regulates the expression of a mitochondrial alcohol dehydrogenase in an ethanol-dependent way. An alcohol-inducible promoter would be incredibly useful for anyone planning to brew a beer with a transgenic yeast - it would allow for the induction of the target genes after the main fermentation has finished and this way, the metabolic burden for the yeast cells could be lowered. All the transcription factors known to be involved in the regulation of the KlADH4-promoter in ''K. lactis'' also occur in ''S. cerevisiae'' [[http://www.ncbi.nlm.nih.gov/pubmed/10724480 Mazzoni et al., 2000]]. This is why we are confident that this promoter maintains its unique characteristics when transformed into ''S. cerevisiae''.<hr><br />
[[file:TUM12_experiment_overwiew_alcohol.png|400px|thumb|right| '''Top''': Emission spectrum, excitation spectrum and visible fluorescence of a yeast culture expressing eGFP under the control of the KlADH4-promoter at 1.72 Vol.-% ethanol. Significant ammounts of eGFP detectable '''Center''': Emission spectrum and excitation spectrum of a yeast culture carrying a plasmid in which eGFP-expression is controlled by the KlADH4-promoter cultivated with glycerol as only carbon source. Ethanol concentration: 0.21 Vol.-%. No eGFP detectable. '''Bottom''': Fluorescence per cell and ethanol concentration plotted against time after induction.]]<br />
<center>'''Experimental results:'''</center><br><br />
At this time our results concerning the KlADH4-promoter (originally from the yeast ''Kluyveromyces lactis'') suggest that this promoter is ethanol inducible in ''S. cerevisiae''. Further experiments are still being done to abolish residual ambiguities. The lowest ethanol concentration at which eGFP-expression was detected is 0.9 Vol.-%.<br />
<br />
<center>'''Conclusion and outlook:'''</center><br><br />
<br />
Because ''S. cerevisiae'' is such a good brewer, it was difficult to produce a stringent negative control in our characterization experiments. However, we finally figured out some experiments that allowed us to keep the ethanol concentration below 0.5 % v/v, which is a concentration at which induction is observed in ''K. lactis''. We are working hard on providing additional data, but we are confident that we will be able to provide clear evidence that this promoter is ethanol-inducible not only in ''K. lactis'', but also in ''S. cerevisiae''.<br />
<br />
<div class="noborder" style="float:left"><br />
<html><a href="https://2012.igem.org/Team:TU_Munich/Project/Ethanol_Inducible_Promoter"><img width="200px" src="https://static.igem.org/mediawiki/2012/3/33/TUM12_readmore.png"/></a></html><br />
</div><br />
</div><br />
<br />
<div class="bezel mfull"><br />
<br />
===Light-switchable promoter===<br />
The idea behind a light-switchable system is to create a gene expression system which can be induced and deactivated by light of a certain wavelength.<br />
<br />
This system is extremely attractive, as induction does not require the addition of a specific substance. This makes induction '''cheap, fast, precise''' and also compatible with the Bavarian purity law.<br />
<hr><br />
[[Image:TUM12_light.jpg|thumb|right|300px|Principle of light-dependent switching of gene-expression.]]<br />
<center>'''Experimental results:'''</center><br><br />
All fusion proteins for the two types of a light-switchable promoter system has been finished ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801039 BBa_K801039], [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801040 BBa_K801040] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801041 BBa_K801041]), also gene expression batteries coding for all components of each type of our light-switchable promoter system has been done ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801042 BBa_K801042] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801043 BBa_K801043]). Since lacking of a second functional yeast vector carrying another auxotrophy marker than URA3 of the pTUM plasmids, which is already reserved for the biosynthesis enzymes, proteins and also reporters, we were not able to clone the whole gene expression battery, into a yeast vector, in order to co-transfect the yeast with one plasmid with the reporter construct and the second plasmid coding for all the devices needed in a light-switchable promoter system.<br />
<br />
<center>'''Conclusion and outlook:'''</center><br><br />
<br />
To get gene expression casette for both of the light-switchable promoter systems ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801042 BBa_K801042] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801043 BBa_K801043]) into an yeast plasmid, we want to use pSB6A0 ([http://partsregistry.org/Part:BBa_K268000 BBa_K268000]) carrying a TRP1 auxotrophy marker. But we've discovered some discrepancies in this part, e.&nbsp;g. there is still at least one forbidden restriction site (XbaI or PstI, please see experience site of the part). So we have to correct the part by quickchange mutagenesis.<br />
<br />
After successful cloning and transfection of the gene expression batteries including there reporters, we will proceed with the characterization of the part.<br />
<br />
In parallel, we will implement also the whole biosynthesis pathway for the phycocyanobilin synthesis, donwstream of our both batteries for a light-switchable promoter system ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801042 BBa_K801042] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801043 BBa_K801043]); after that, we don't have to add phycocyanobilin in our medium anymore!<br />
<br />
If there is still time in the end, we will clone our system including the biosynthetic genes for PCB synthesis in the yeast integrative vector ([http://partsregistry.org/Part:BBa_K300001 BBa_K300001]) and will start a genome integration in order to create a new yeast strain with a fully functional light-switchable promoter system.<br />
<br />
<div class="noborder" style="float:left"><br />
<html><a href="https://2012.igem.org/Team:TU_Munich/Project/Light_Switchable_Promoter"><img width="200px" src="https://static.igem.org/mediawiki/2012/3/33/TUM12_readmore.png"/></a></html><br />
</div><br />
</div><br />
<br />
==Genome Integration==<br />
----<br />
<div class="bezel mfull"><br />
As we cannot obey the letter of the German purity law (there is a zero tolerance policy concerning transgenic ingredients), we try our best to meet the spirit. Thus, it is <b>unacceptable for us to work with antibiotics</b> to keep up the selective environment. Since we <b>cannot work with auxotrophies</b> in beer either, we have to make sure the yeasts do not lose the BioBricks. The most promising way to accomplish a long lasting presence of our constructs is to achieve <b>genome integration</b>.<br />
<hr><br />
[[file:TUM12_experiment_overwiew_genome.png|500px|thumb|right| Plasmid backbone used for integration of our expression cassettes]]<br />
<center>'''Experimental results:'''</center><br><br />
First experiments to characterize the function of the yeast integration system were performed and the used selection marker was maintained in the yeast culture, although the selection pressure was switched off. This indicates that first integrations were achieved. <br />
<br />
<center>'''Conclusion and outlook:'''</center><br><br />
Maintaining the plasmids harboring our expression cassettes in the yeast cells during the brewing process is best possible using genome integration. This becomes increasingly interesting, when a yeast strain with different expression cassettes is to be created. Because this is intended for the next step of our project the integration of our expression cassettes becomes increasingly important.<br />
<br />
<div class="noborder" style="float:left"><br />
<html><a href="https://2012.igem.org/Team:TU_Munich/Project/Genome_Integration"><img width="200px" src="https://static.igem.org/mediawiki/2012/3/33/TUM12_readmore.png"/></a></html><br />
</div><br />
</div><br />
<br />
==Brewing our SynBio Beer==<br />
----<br />
<div class="bezel mfull"><br />
Contrary to popular opinion the chief ingredient of beer is not '''YPD''' but '''gyle''', a carefully prepared mixture of malt, hop and water. Although the name of the yeast strain commonly used in the lab, '''S. cerevisiae''', suggests that it is used in the beer brewing process, the yeast strains generally employed in brewing process have '''strongly adapted to gyle''', as they are reutilized in every succeeding brewing cycle.<br />
Hence some investigation on how our yeast '''performs''' in gyle was necessary.<br />
<br><hr><br />
[[file:TUM12_experiment_overwiew_Brewing.png|200px|thumb|right| Picture of the first SynBio Beer brewed during the iGEM competition in 2012]]<br />
<center>'''Experimental results:'''</center><br><br />
Our experiments show that the growth of several different yeast strains is '''not impaired in gyle'''!<br />
<br />
Expression assays proved the necessity of [[Team:TU_Munich/Project/Genome_Integration|'''genome integration''']] for a proper '''SynBio Beer'''.<br />
<br />
<center>'''Conclusion and outlook:'''</center><br><br />
As soon as cloning of integration vectors containing expression cassettes for our biosynthetic pathways are finished we will repeat the brewing experiments.<br />
<br />
<div class="noborder" style="float:left"><br />
<html><a href="https://2012.igem.org/Team:TU_Munich/Project/Brewing"><img width="200px" src="https://static.igem.org/mediawiki/2012/3/33/TUM12_readmore.png"/></a></html><br />
</div><br />
</div><br />
<br />
== References ==<br />
----<br />
* [[http://www.ncbi.nlm.nih.gov/pubmed/6327079 Edens et al., 1984]] Edens, L., Bom, I., Ledeboer, A. M., Maat, J., Toonen, M. Y., Visser, C., and Verrips, C. T. (1984). Synthesis and processing of the plant protein thaumatin in yeast. ''Cell'', 37(2):629–33.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/10995285 Miranda et al., 2000]] Miranda, C. L., Stevens, J. F., Ivanov, V., McCall, M., Frei, B., Deinzer, M. L., and Buhler, D. R. (2000). Antioxidant and prooxidant actions of prenylated and nonprenylated chalcones and flavanones in vitro. ''J Agric Food Chem'', 48(9):3876–84.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/10724480 Mazzoni et al., 2000]] Mazzoni, C., Santori, F., Saliola, M., and Falcone, C. (2000). Molecular analysis of uas(e), a cis element containing stress response elements responsible for ethanol induction of the kladh4 gene of ''kluyveromyces lactis''. ''Res Microbiol'', 151(1):19–28.</div>JaraOhttp://2012.igem.org/Team:TU_Munich/Human_Practice/MovieTeam:TU Munich/Human Practice/Movie2012-10-21T17:50:16Z<p>JaraO: </p>
<hr />
<div>{{Team:TU_Munich/Header}}<br />
<br />
=Movie=<br />
<hr/><br />
==Wissen+Konzepte==<br />
[[File:TUM_Moviemaking.png|200px|thumb|right|Movie Making]]<br />
"Wissen+Konzepte", a '''scientific online magazine''', contacted us via facebook. They asked us if we are interested in making a film about our team and our project. Of course we were really enthusiastic about this proposal. Consequently two nice ladies of the editorial staff of "Wissen+Konzepte" visited us on September 10th. <br />
<br />
First they took many pictures of our materials in the lab like petri dishes or yeast-medium. They also shot scenes in the lab when doing a SDS-Page. <br />
Afterwards someone of our team explained in front of the the camera our project. Especially the principles of cloning and the use of plasmids were explained using the analogy of a bus: The transfered genes represent the passengers. <br />
The third big scene was shot with another participant of our team. He talked about the societal perception of our project, the activities we organized in the context of "Human Practice" and about the Germany-wide Action Day. Besides he mentioned that we chose a topic that is rather controversial in Germany, but that the response to our work is mainly very positive. <br />
The next scene was an interview with Prof. Dr. Skerra who also talked about iGEM and synthetic biology. Last but not least the day ended with a scene in a beer garden. <br />
<br />
In total it was a lot of fun to make this movie!! The movie will present our project in an intuitive way so as to '''make it comprehensible to the greater public.''' That is the concept of Wissen+Konzepte.<br />
<br />
We are sorry that we cannot show you this film here since it is still in the postproduction.<br />
<br />
===Background about Wissen+Konzepte===<br />
"Wissen+Konzepte" is a scientific online magazine which reports about new scientific approaches in a simple way. Yet not only the science itself is important, but also the people who have the ideas to do it. Normally "Wissen+Konzepte" is doing corporate publishing for scientists.<br />
<br />
==Own "Production"==<br />
<hr/><br />
<br />
Enjoy our movie about TUM Brew, iGEM's first and finest SynBio beer. <br />
<br />
The movie was made by Matthias Moerch.<br />
<br />
<center><html><iframe src="http://player.vimeo.com/video/51804324?color=d92540" width="720" height="405" frameborder="0" webkitAllowFullScreen mozallowfullscreen allowFullScreen></iframe></html></center></div>JaraOhttp://2012.igem.org/Team:TU_Munich/Human_Practice/OverviewTeam:TU Munich/Human Practice/Overview2012-10-21T17:37:07Z<p>JaraO: /* Survey */</p>
<hr />
<div>{{Team:TU_Munich/Header}}<br />
<br />
= Overview =<br />
<hr/><br />
<br />
[[File:Gruppe_Human Practice_TUM12.jpg|350px||thumb||Responsible: Nadine Gerstenberg, David Wehner, Jara Obermann]]<br />
<div style="text-align:justify;"> Our project envisions genetically modified organisms in the scope of food production. In Germany genetic engineering especially in the realm of food production is a highly sensitive topic. The following video adequately summarizes the current situation in Germany using the example of BASF's decision to move their Plant Science headquarters from Limburgerhof, Germany, to Raleigh, North Carolina (USA) [http://www.youtube.com/watch?v=UqRbziwzVkk&feature=plcp]. <br><br />
<br />
<br />
<center><html><iframe width="560" height="315" src="http://www.youtube.com/embed/UqRbziwzVkk" frameborder="0" allowfullscreen></iframe></html></center><br />
<br />
<br />
More information on the development and on the current situation in Germany can be found in the [https://2012.igem.org/Team:TU_Munich/Human_Practice/Overview#References References] section.<br />
<br />
Considering the general rejection of genetic engineering and keeping in mind that Germany has a long tradition of brewing beer under the German purity law it is even more important to inform the general public about genetic engineering, synthetic biology and our project. While the general public broadly opposes genetic engineering, the scientific community tries to emphasizes the potential benefits of the technology. In a 2008 lecture Nobel Prize-Winner Christiane Nüsslein-Volhard made clear: "''Germany has so far failed to sufficiently accept that applying genetic engineering in plant breeding offers an as yet not fully tapped potential for organic farming, improved environmental protection, the conservation of biodiversity and health. Plants that are resistant towards moths, fungal infestation, viruses and nematodes need not be sprayed. Plants that are better adapted to unfavourable growth conditions, saline soil and karst and arid regions can thus be bred and grown to turn barren land back into fertile land'' ” [http://www.leopoldina.org/uploads/tx_leopublication/200910_NatEmpf_Gruene_Gentechnik-EN_01.pdf].<br />
<br />
Using the example iGEM's first SynBio Beer our team tried to bridge the gap between the scientific community and the general public by hosting a variety of “Human Practice” events. To inform younger members of our society about genetic engineering we visited a class of 10th and a class of 11th graders. An interview at the local student radio station gave us the opportunity to present our team and our mission to fellow students. In addition a comic was designed that illustrates our project in a playful way. Two versions of this comic were drawn: one for children and one for grown-ups. Our team never encouraged under-age alcohol consumption. The presented content was always adapted according to the respective audience.<br />
<br />
Our team also addressed the general public with a multitude of events. Laborwelt.de was our most important media partner. Moreover, other newspapers and websites reported about our team and project and a movie presented our project and explained the basic ideas of synthetic biology. A nationwide information day was organized in cooperation with other German iGEM teams. Our team also invited politicians and scientists to discuss the impact of genetic engineering in groceries.<br />
</div><br />
<br />
==GMO in Food==<br />
<hr/><br />
<br />
Genetic Engineering or Genetic Modification of food is an issue that is present in the media all over the world [http://www.globalissues.org/issue/188/genetically-engineered-food]. There are a lot of people who are critical and skeptical about genetically modified (GM) food. The opponents of genetic engineering are wondering if food produced from genetically engineered organisms is safe. They are afraid of eating GM food because they think that the food can interfere their normal bodily functions. Yet a lot of food that we eat every day contains genetically modified ingredients and we do not know about it [http://www.globalissues.org/issue/188/genetically-engineered-food [3]]. Consequently labeling GM food is another important issue that is often raised in controversies over genetic engineering of food. Especially in Germany these controversies are omnipresent and have led to many disputes, protests and restrictions. <br />
<br />
Unfortunately many people who debate about genetic engineering have only little background knowledge. Consequently the content of the discussion is often unobjective and emotional. A negative effect of GM food on humans is not approved sufficiently yet. Supporters of GM food say that genetic engineering of crops for example is needed to address the world’s food needs. In this case the sustainability of food security and availability is an important topic because the population increases [http://www.globalissues.org/issue/188/genetically-engineered-food [3]]. Biotechnological methods could help increase food production since generating genetically modified crops can provide protection from pathogens. Besides fewer insecticides and other chemicals are needed. Another aspect in generating GM food is to modify the nutritional value of the food. One example is the so called “Golden Rice” that has a higher concentration of vitamin A than normal rice. <br />
<br />
Green genetic engineering is not only a scientific issue, but also a political, social and commercial one. That is why our project addresses the populace. It is an important issue for almost everybody in the population and it is a really present and much debated topic. <br />
In Germany a lot of research is done in the field of green genetic engineering. However, the step to the application of GM food is still difficult. That is why the iGEM TU Munich Team wants to inform people about genetic engineering in general and to moderate the concerns about it. Besides we want to lay the foundations for a new generation of functional food by producing iGEM’s first and finest SynBio Beer. <br />
<br />
<br />
<br />
<br />
<br />
<div class="mleft bezel"><br />
<br />
== School Visit ==<br />
<hr/><br />
<br />
[[File:TUM School Visit.JPG|150px|right]]<br />
<br />
On July 24th, two of us visited the Joseph-Bernahrt-Gymnasium to give exciting talks about synthetic biology, iGEM and our project. In addition students could get their hands dirty trying to reveal some of the genetic secrets of bananas, pineapples and "gummi bears".<br />
<br />
<div class="noborder" style="float:left"><br />
<html><a href="https://2012.igem.org/Team:TU_Munich/Human_Practice/School_Visit"><img width="200px" src="https://static.igem.org/mediawiki/2012/3/33/TUM12_readmore.png"/></a></html><br />
</div><br />
</div><br />
<br />
<div class="mright bezel"><br />
== Public Relations ==<br />
<hr/><br />
<br />
[[File:TUM_Public_Relations.jpg|300px|right]]<br />
<br />
Throughout the entire project we used different media channels to inform the general public. Our cooperation partner was Laborwelt.de. In addition we kept our followers up to date using a variety of social media.<br />
<br />
<div class="noborder" style="float:left"><br />
<html><a href="https://2012.igem.org/Team:TU_Munich/Human Practice/Public"><img width="200px" src="https://static.igem.org/mediawiki/2012/3/33/TUM12_readmore.png"/></a></html><br />
</div><br />
</div><br />
<br />
<div class="mleft bezel"><br />
<br />
== Comic ==<br />
<hr/><br />
<br />
[[File:TUM_Super_Yeast.jpg|120px|right]]<br />
<br />
Our team designed a comic introducing our hero "Super Yeast". In an entertaining way the comic explains the idea of synthetic biology and our project. We created two versions: one for children and one for grown-ups. Have a look and find out what our hero experiences!<br />
<br />
<div class="noborder" style="float:left"><br />
<br />
<html><a href="https://2012.igem.org/Team:TU_Munich/Human_Practice/Comic_children"><img width="200px" src="https://static.igem.org/mediawiki/2012/3/33/TUM12_readmore.png"/></a></html><br />
</div><br />
</div><br />
<br />
<div class="mright bezel"><br />
<br />
== Information Day ==<br />
<hr/><br />
<br />
[[File:TUM_Information Day.jpg|150px|right]]<br />
<br />
On August 25th, German iGEM teams informed the general public about synthetic biology and iGEM. This nationwide information day was a joint effort to inspire people throughout Germany and spread the word about synthetic biology.<br><br />
<br />
<div class="noborder" style="float:left"><br />
<html><a href="https://2012.igem.org/Team:TU_Munich/Human Practice/Information_Day"><img width="200px" src="https://static.igem.org/mediawiki/2012/3/33/TUM12_readmore.png"/></a></html><br />
</div><br />
</div><br />
<br />
<div class="mleft bezel"><br />
<br />
== Politics ==<br />
<hr/><br />
<br />
[[File:TUM_Politics.jpg|160px|right]]<br />
<br />
On September 18th, we invited the general public to an evening full of presentations and an interesting panel discussion. Experts gave comprehensive talks and during the panel discussion scientists and politicians debated on genetic engineering in food.<br />
<br />
<div class="noborder" style="float:left"><br />
<html><a href="https://2012.igem.org/Team:TU_Munich/Human_Practice/Politics"><img width="200px" src="https://static.igem.org/mediawiki/2012/3/33/TUM12_readmore.png"/></a></html><br />
</div><br />
</div><br />
<br />
<div class="mright bezel"><br />
<br />
== Movie ==<br />
<hr/><br />
<br />
[[File:TUM_Brew.jpg|200px|thumb|right]]<br />
<br />
In total we produced two movies. One in cooperation with an online magazine called "Wissen + Konzepte" to highlight the scientific part of our project and another one to present our team and our project.<br />
<br />
<div class="noborder" style="float:left"><br />
<html><a href="https://2012.igem.org/Team:TU_Munich/Human_Practice/Movie"><img width="200px" src="https://static.igem.org/mediawiki/2012/3/33/TUM12_readmore.png"/></a></html><br />
</div><br />
</div><br />
<br />
<div class="mleft bezel"><br />
<br />
== Radio Interview ==<br />
<hr/><br />
<br />
[[File:TUM_Radiointerview.jpg|150px|right]]<br />
<br />
On August 19th, three of us gave an interview at a local radio station. Nadine, Fabian and David talked about synthetic biology, iGEM and our project.<br />
<br />
<div class="noborder" style="float:left"><br />
<html><a href="https://2012.igem.org/Team:TU_Munich/Human_Practice/Radio"><img width="200px" src="https://static.igem.org/mediawiki/2012/3/33/TUM12_readmore.png"/></a></html><br />
</div><br />
</div><br />
<br />
<div class="mleft bezel"><br />
<br />
== Survey ==<br />
<hr/><br />
<br />
[[File:Food.png|120px|right]]<br />
<br />
In cooperation with "TUM:Junge Akademie", a collegiate association of our university, we got the chance to take a look at the results of a survey about "genetic engineering". The survey helped us to get a picture of the attitude of the German population towards genetic engineering in food. Read more and see the results of the survey. <br />
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<div class="noborder" style="float:left"><br />
<html><a href="https://2012.igem.org/Team:TU_Munich/Human_Practice/Survey"><img width="200px" src="https://static.igem.org/mediawiki/2012/3/33/TUM12_readmore.png"/></a></html><br />
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==References==<br />
<hr/><br />
* [1] http://www.youtube.com/watch?v=UqRbziwzVkk&feature=plcp<br />
* [2] http://www.leopoldina.org/uploads/tx_leopublication/200910_NatEmpf_Gruene_Gentechnik-EN_01.pdf<br />
* [3] http://www.globalissues.org/issue/188/genetically-engineered-food<br />
<br />
Additional information:<br />
*http://www.universityworldnews.com/article.php?story=20090528173643889<br />
*http://www.study-in.de/en/community/tandem-reporters/--16453<br />
*http://oneco.biofach.de/en/news/?focus=ce188f4d-46b2-4f0c-9b55-64cc1ecadf40<br />
*http://www.care2.com/greenliving/germany-decides-genetically-modified-foods-are-not-welcome.html<br />
*http://expertlywrapped.wordpress.com/2012/01/25/basf-plant-science-leaves-europe-for-us/<br />
*http://insight.kellogg.northwestern.edu/index.php/Kellogg/article/the_fall_of_german_biotech<br />
*http://www.bvl.bund.de/EN/06_Genetic_Engineering/genetic_engineering_node.html<br />
*http://www.gmo-compass.org/eng/news/546.german_constitutional_court_confirms_biotech_law.html<br />
*http://www.bfn.de/0301_gentechnik+M52087573ab0.html<br />
*[[File:TUM_biotech in Germany.pdf]]</div>JaraOhttp://2012.igem.org/Team:TU_Munich/Human_Practice/MovieTeam:TU Munich/Human Practice/Movie2012-10-21T10:50:38Z<p>JaraO: /* TUM: Junge Akademie */</p>
<hr />
<div>{{Team:TU_Munich/Header}}<br />
<br />
=Survey=<br />
<hr/><br />
==Results==<br />
<br />
Here you can see the results of the survey about genetic engineering in general and especially in food. <br />
<br />
1200 people took part in this survey and the results showed us, that in Germany, there is still a sceptical and controversial opinion about genetic engineering. Most people accept genetic engineering in medical and pharmaceutical issues, but in food genetic engineering is not tolerated. Yet, unfortunalety, many people don't have a well-founded background about genetic engineering and consequently they are sceptical about it. <br />
That's why an important part of our project was to inform people about the opportunities of genetic engineering.<br />
<br />
<br />
[[File:Age.png|400px|thumb|left]]<br />
[[File:Concerns1.png|400px|thumb|right]]<br />
[[File:Environmental_techniques.png|400px|thumb|left]]<br />
[[File:Farming1.png|400px|thumb|right]]<br />
[[File:Food.png|400px|thumb|left]]<br />
[[File:Gender.png|400px|thumb|right]]<br />
[[File:Healthier.png|400px|thumb|left]]<br />
[[File:Industrial_processes.png|400px|thumb|right]]<br />
[[File:TUMKnowledge.png|400px|thumb|left]]<br />
[[File:TUMMedia.png|400px|thumb|right]]<br />
[[File:TUMMedicine.png|400px|thumb|left]]<br />
[[File:Overall.png|400px|thumb|right]]<br />
[[File:Politics.png|400px|thumb|left]]<br />
[[File:Price.png|400px|thumb|right]]<br />
[[File:Regulations.png|400px|thumb|left]]<br />
[[File:Taste.png|400px|thumb|right]]<br />
[[File:Therapeutical_issues.png|400px|thumb|left]]<br />
[[File:Unbenannt.png|400px|thumb|right]]<br />
<br />
==TUM: Junge Akademie==<br />
<br />
We cooperated with "TUM: Junge Akademie", a collegiate association of our university. They created a homepage to inform people about genetic engineering. <br />
Besides the survey, they interviewed experts about the issue. One of our big goals was to inform people about genetic engineering and to moderate the sceptical opinion of many people. That's why the cooperation with "TUM: Junge Akademie" was a perfect addition to our project.</div>JaraOhttp://2012.igem.org/Team:TU_Munich/Human_Practice/MovieTeam:TU Munich/Human Practice/Movie2012-10-21T10:46:40Z<p>JaraO: /* Results */</p>
<hr />
<div>{{Team:TU_Munich/Header}}<br />
<br />
=Survey=<br />
<hr/><br />
==Results==<br />
<br />
Here you can see the results of the survey about genetic engineering in general and especially in food. <br />
<br />
1200 people took part in this survey and the results showed us, that in Germany, there is still a sceptical and controversial opinion about genetic engineering. Most people accept genetic engineering in medical and pharmaceutical issues, but in food genetic engineering is not tolerated. Yet, unfortunalety, many people don't have a well-founded background about genetic engineering and consequently they are sceptical about it. <br />
That's why an important part of our project was to inform people about the opportunities of genetic engineering.<br />
<br />
<br />
[[File:Age.png|400px|thumb|left]]<br />
[[File:Concerns1.png|400px|thumb|right]]<br />
[[File:Environmental_techniques.png|400px|thumb|left]]<br />
[[File:Farming1.png|400px|thumb|right]]<br />
[[File:Food.png|400px|thumb|left]]<br />
[[File:Gender.png|400px|thumb|right]]<br />
[[File:Healthier.png|400px|thumb|left]]<br />
[[File:Industrial_processes.png|400px|thumb|right]]<br />
[[File:TUMKnowledge.png|400px|thumb|left]]<br />
[[File:TUMMedia.png|400px|thumb|right]]<br />
[[File:TUMMedicine.png|400px|thumb|left]]<br />
[[File:Overall.png|400px|thumb|right]]<br />
[[File:Politics.png|400px|thumb|left]]<br />
[[File:Price.png|400px|thumb|right]]<br />
[[File:Regulations.png|400px|thumb|left]]<br />
[[File:Taste.png|400px|thumb|right]]<br />
[[File:Therapeutical_issues.png|400px|thumb|left]]<br />
[[File:Unbenannt.png|400px|thumb|right]]<br />
<br />
==TUM: Junge Akademie==<br />
<br />
We cooperated with "TUM: Junge Akademie", a collegiate association of our university. They created a homepage to inform people about genetic engineering. <br />
Besides the survey, they interviewed experts about the issue. You can see the movies here.</div>JaraOhttp://2012.igem.org/File:Healthier.pngFile:Healthier.png2012-10-21T10:37:06Z<p>JaraO: </p>
<hr />
<div></div>JaraOhttp://2012.igem.org/Team:TU_Munich/Human_Practice/MovieTeam:TU Munich/Human Practice/Movie2012-10-21T10:36:37Z<p>JaraO: /* Results */</p>
<hr />
<div>{{Team:TU_Munich/Header}}<br />
<br />
=Survey=<br />
<hr/><br />
==Results==<br />
<br />
Here you can see the results of the survey about genetic engineering in general and especially in food. <br />
<br />
<br />
1200 people took part in this survey and the results showed us, that in Germany, there is still a sceptical and controversial opinion about genetic engineering. Most people accept genetic engineering in medical an pharmaceutical issues, but in food genetic engineering is not tolerated. Yet, unfortunalety, many people don't have a well-founded background about genetic engineering and consequently they are sceptical about it. <br />
That's why an important part of our project was to inform people about the opportunities of genetic engineering.<br />
<br />
<br />
[[File:Age.png|400px|thumb|left]]<br />
[[File:Concerns1.png|400px|thumb|right]]<br />
[[File:Environmental_techniques.png|400px|thumb|left]]<br />
[[File:Farming1.png|400px|thumb|right]]<br />
[[File:Food.png|400px|thumb|left]]<br />
[[File:Gender.png|400px|thumb|right]]<br />
[[File:Healthier.png|400px|thumb|left]]<br />
[[File:Industrial_processes.png|400px|thumb|right]]<br />
[[File:TUMKnowledge.png|400px|thumb|left]]<br />
[[File:TUMMedia.png|400px|thumb|right]]<br />
[[File:TUMMedicine.png|400px|thumb|left]]<br />
[[File:Overall.png|400px|thumb|right]]<br />
[[File:Politics.png|400px|thumb|left]]<br />
[[File:Price.png|400px|thumb|right]]<br />
[[File:Regulations.png|400px|thumb|left]]<br />
[[File:Taste.png|400px|thumb|right]]<br />
[[File:Therapeutical_issues.png|400px|thumb|left]]<br />
[[File:Unbenannt.png|400px|thumb|right]]</div>JaraOhttp://2012.igem.org/Team:TU_Munich/Human_Practice/MovieTeam:TU Munich/Human Practice/Movie2012-10-21T10:34:56Z<p>JaraO: /* Results */</p>
<hr />
<div>{{Team:TU_Munich/Header}}<br />
<br />
=Survey=<br />
<hr/><br />
==Results==<br />
<br />
Here you can see the results of the survey about genetic engineering in general and especially in food. <br />
<br />
<br />
1200 people took part in this survey and the results showed us, that in Germany, there is still a sceptical and controversial opinion about genetic engineering. Most people accept genetic engineering in medical an pharmaceutical issues, but in food genetic engineering is not tolerated. Yet, unfortunalety, many people don't have a well-founded background about genetic engineering and consequently they are sceptical about it. <br />
That's why an important part of our project was to inform people about the opportunities of genetic engineering.<br />
<br />
<br />
[[File:Age.png|400px|thumb|left]]<br />
[[File:Concerns1.png|400px|thumb|right]]<br />
[[File:Environmental_techniques.png|400px|thumb|left]]<br />
[[File:Farming1.png|400px|thumb|right]]<br />
[[File:Food.png|400px|thumb|left]]<br />
[[File:Gender.png|400px|thumb|right]]<br />
[[File:Healthier.png|400px|thumb|left]]<br />
[[File:Industrial_processes.png|400px|thumb|right]]<br />
[[File:TUMKnowledge.png|400px|thumb|left]]<br />
[[File:TUMMedia.png|400px|thumb|right]]<br />
[[File:TUMMedicine.png|400px|thumb|left]]<br />
[[File:Overall.png|400px|thumb|right]]</div>JaraOhttp://2012.igem.org/Team:TU_Munich/Human_Practice/MovieTeam:TU Munich/Human Practice/Movie2012-10-21T10:34:32Z<p>JaraO: /* Results */</p>
<hr />
<div>{{Team:TU_Munich/Header}}<br />
<br />
=Survey=<br />
<hr/><br />
==Results==<br />
<br />
Here you can see the results of the survey about genetic engineering in general and especially in food. <br />
<br />
<br />
1200 people took part in this survey and the results showed us, that in Germany, there is still a sceptical and controversial opinion about genetic engineering. Most people accept genetic engineering in medical an pharmaceutical issues, but in food genetic engineering is not tolerated. Yet, unfortunalety, many people don't have a well-founded background about genetic engineering and consequently they are sceptical about it. <br />
That's why an important part of our project was to inform people about the opportunities of genetic engineering.<br />
<br />
<br />
[[File:Age.png|400px|thumb|left]]<br />
[[File:Concerns1.png|400px|thumb|right]]<br />
[[File:Environmental_techniques.png|400px|thumb|left]]<br />
[[File:Farming1.png|400px|thumb|right]]<br />
[[File:Food.png|400px|thumb|left]]<br />
[[File:Gender.png|400px|thumb|right]]<br />
[[File:Healthier.png|400px|thumb|left]]<br />
[[File:Industrial_processes.png|400px|thumb|right]]<br />
[[File:Knowledge.png|400px|thumb|left]]<br />
[[File:Media.png|400px|thumb|right]]<br />
[[File:Medicine.png|400px|thumb|left]]<br />
[[File:Overall.png|400px|thumb|right]]</div>JaraO