http://2013.igem.org/wiki/index.php?title=Team:Marburg/Project:lightcontrol&feed=atom&action=historyTeam:Marburg/Project:lightcontrol - Revision history2024-03-29T08:51:13ZRevision history for this page on the wikiMediaWiki 1.16.5http://2013.igem.org/wiki/index.php?title=Team:Marburg/Project:lightcontrol&diff=351335&oldid=prevRomanM89 at 20:25, 28 October 20132013-10-28T20:25:06Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Because plants and algae use sunlight as their primary energy source, they had to develop promoters, which respond to light. We therefore challenged the idea whether these light-inducible promoters would be suitable for regulating expression of target genes (Figure). The light-inducible promoter fcpB is used for the constitutive expression of proteins in genetically modified ''Phaeodactylum tricornutum'' algae. Unfortunately, this promoter is not well characterized until now. The fcpB-promoter is known to control the expression of fucoxanthin binding proteins. These proteins play an important role in the absorption of photosynthetic active sunlight. To protect the light harvesting complex (LHC) from dangerous high-energy sunlight these promoters are already induced by low levels of sunlight (Veith and Büchel, 2007, BBA). Therefore, we expected a promoter induction under blue, red and white light conditions and never under green light and in darkness due to the fact that photosynthesis occurs only when red or blue light is present and fucoxanthin binding proteins are necessary for light harvesting and photoprotection. That is the reason why we decided to study the promoter strength by radiating the cells with different transmission wavelengths (i.e. green (572 nm), blue (467 nm), red (672 nm), white light and darkness). Hence we used the light-inducible promoter for the expression of the reporter eGFP, which is advantageous because the expression level can be determined relatively easy. </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Because plants and algae use sunlight as their primary energy source, they had to develop promoters, which respond to light. We therefore challenged the idea whether these light-inducible promoters would be suitable for regulating expression of target genes (Figure). The light-inducible promoter fcpB is used for the constitutive expression of proteins in genetically modified ''Phaeodactylum tricornutum'' algae. Unfortunately, this promoter is not well characterized until now. The fcpB-promoter is known to control the expression of fucoxanthin binding proteins. These proteins play an important role in the absorption of photosynthetic active sunlight. To protect the light harvesting complex (LHC) from dangerous high-energy sunlight these promoters are already induced by low levels of sunlight (Veith and Büchel, 2007, BBA). Therefore, we expected a promoter induction under blue, red and white light conditions and never under green light and in darkness due to the fact that photosynthesis occurs only when red or blue light is present and fucoxanthin binding proteins are necessary for light harvesting and photoprotection. That is the reason why we decided to study the promoter strength by radiating the cells with different transmission wavelengths (i.e. green (572 nm), blue (467 nm), red (672 nm), white light and darkness). Hence we used the light-inducible promoter for the expression of the reporter eGFP, which is advantageous because the expression level can be determined relatively easy. </div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>A ''P. tricornutum'' culture was grown for six days under the specific light conditions described above. Afterwards, cultures were kept in darkness for two days to minimize the amount of existing GFP in the cells (<html><a href="https://2013.igem.org/Team:Marburg/Notebook:Ptricornutum">see the protocol</a></html>). The optical density was measured and GFP was quantified by Western blot analysis. </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>A ''P. tricornutum'' culture was grown for six days under the specific <ins class="diffchange diffchange-inline">white </ins>light conditions described above. Afterwards, cultures were kept in darkness for two days to minimize the amount of existing GFP in the cells (<html><a href="https://2013.igem.org/Team:Marburg/Notebook:Ptricornutum">see the protocol</a></html>). The optical density was measured and GFP was quantified by Western blot analysis. </div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>While determining the optical density, we found ''P. tricornutum'' growth under all light conditions except for darkness. Leblanc and coworkers showed that cryptochrome-, rhodopsin- and phytochrome-like receptors are present in marine diatoms indicating the ability to receive green light and to use green light for photosynthesis (Leblanc ''et al.'', 1999, Plant Mol Biol). Still, it was shown by Veith and coworkers that a fucoxanthin chlorophyll protein (FCP) complex, under the control of our light inducible promoter leads to a shift of the absorbance spectrum of PSI into the green spectrum after binding to fucoxanthin (Veith ''et al.'', 2007, BBA). </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>While determining the optical density, we found ''P. tricornutum'' growth under all light conditions except for darkness. Leblanc and coworkers showed that cryptochrome-, rhodopsin- and phytochrome-like receptors are present in marine diatoms indicating the ability to receive green light and to use green light for photosynthesis (Leblanc ''et al.'', 1999, Plant Mol Biol). Still, it was shown by Veith and coworkers that a fucoxanthin chlorophyll protein (FCP) complex, under the control of our light inducible promoter leads to a shift of the absorbance spectrum of PSI into the green spectrum after binding to fucoxanthin (Veith ''et al.'', 2007, BBA). </div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
</table>RomanM89http://2013.igem.org/wiki/index.php?title=Team:Marburg/Project:lightcontrol&diff=349485&oldid=prevRomanM89 at 18:19, 28 October 20132013-10-28T18:19:00Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>{{:Team:Marburg/Template:ContentTitleNav}}</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>{{:Team:Marburg/Template:ContentTitleNav}}</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Characterizing a new light-inducible promoter <html><a href="https://2013.igem.org/Team:Marburg/Project:RFP"><img src="https://static.igem.org/mediawiki/2013/1/13/Mr-igem-previous-arrow.png" alt="Previous" style="float:right;"></a></html></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Characterizing a new light-inducible promoter <html><a href="https://2013.igem.org/Team:Marburg/Project:RFP"><img src="https://static.igem.org/mediawiki/2013/1/13/Mr-igem-previous-arrow.png" alt="Previous" style="float:right;"></a></html></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>{{:Team:Marburg/Template:ContentStartNav}}The registry of standardized biological parts consists of plenty of well characterized promoters. Many of these promoters enable the production of proteins for example antibodies after induction with a specific substance. However, using an external inducer leads to several drawbacks: The inducer must be removed from the medium firstly to inactivate the promoter and accordingly stop the expression and secondly to simplify the purification of the desired protein. </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>{{:Team:Marburg/Template:ContentStartNav}}The <ins class="diffchange diffchange-inline"><html><a href="http://parts.igem.org/Main_Page"></ins>registry<ins class="diffchange diffchange-inline"></a></html> </ins>of standardized biological parts consists of plenty of well characterized promoters. Many of these promoters enable the production of proteins for example antibodies after induction with a specific substance. However, using an external inducer leads to several drawbacks: The inducer must be removed from the medium firstly to inactivate the promoter and accordingly stop the expression and secondly to simplify the purification of the desired protein. </div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Because plants and algae use sunlight as their primary energy source, they had to develop promoters, which respond to light. We therefore challenged the idea whether these light-inducible promoters would be suitable for regulating expression of target genes (Figure). The light-inducible promoter fcpB is used for the constitutive expression of proteins in genetically modified ''Phaeodactylum tricornutum'' algae. Unfortunately, this promoter is not well characterized until now. The fcpB-promoter is known to control the expression of fucoxanthin binding proteins. These proteins play an important role in the absorption of photosynthetic active sunlight. To protect the light harvesting complex (LHC) from dangerous high-energy sunlight these promoters are already induced by low levels of sunlight (Veith and Büchel, 2007, BBA). Therefore, we expected a promoter induction under blue, red and white light conditions and never under green light and in darkness due to the fact that photosynthesis occurs only when red or blue light is present and fucoxanthin binding proteins are necessary for light harvesting and photoprotection. That is the reason why we decided to study the promoter strength by radiating the cells with different transmission wavelengths (i.e. green (572 nm), blue (467 nm), red (672 nm), white light and darkness). Hence we used the light-inducible promoter for the expression of the reporter eGFP, which is advantageous because the expression level can be determined relatively easy. </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Because plants and algae use sunlight as their primary energy source, they had to develop promoters, which respond to light. We therefore challenged the idea whether these light-inducible promoters would be suitable for regulating expression of target genes (Figure). The light-inducible promoter fcpB is used for the constitutive expression of proteins in genetically modified ''Phaeodactylum tricornutum'' algae. Unfortunately, this promoter is not well characterized until now. The fcpB-promoter is known to control the expression of fucoxanthin binding proteins. These proteins play an important role in the absorption of photosynthetic active sunlight. To protect the light harvesting complex (LHC) from dangerous high-energy sunlight these promoters are already induced by low levels of sunlight (Veith and Büchel, 2007, BBA). Therefore, we expected a promoter induction under blue, red and white light conditions and never under green light and in darkness due to the fact that photosynthesis occurs only when red or blue light is present and fucoxanthin binding proteins are necessary for light harvesting and photoprotection. That is the reason why we decided to study the promoter strength by radiating the cells with different transmission wavelengths (i.e. green (572 nm), blue (467 nm), red (672 nm), white light and darkness). Hence we used the light-inducible promoter for the expression of the reporter eGFP, which is advantageous because the expression level can be determined relatively easy. </div></td></tr>
</table>RomanM89http://2013.igem.org/wiki/index.php?title=Team:Marburg/Project:lightcontrol&diff=348939&oldid=prevDomenica at 17:46, 28 October 20132013-10-28T17:46:20Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>{{:Team:Marburg/Template:ContentStartNav}}The registry of standardized biological parts consists of plenty of well characterized promoters. Many of these promoters enable the production of proteins for example antibodies after induction with a specific substance. However, using an external inducer leads to several drawbacks: The inducer must be removed from the medium firstly to inactivate the promoter and accordingly stop the expression and secondly to simplify the purification of the desired protein. </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>{{:Team:Marburg/Template:ContentStartNav}}The registry of standardized biological parts consists of plenty of well characterized promoters. Many of these promoters enable the production of proteins for example antibodies after induction with a specific substance. However, using an external inducer leads to several drawbacks: The inducer must be removed from the medium firstly to inactivate the promoter and accordingly stop the expression and secondly to simplify the purification of the desired protein. </div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>Because plants and algae use sunlight as their primary energy source, they had to develop promoters, which respond to light. We therefore challenged the idea whether these light-inducible promoters would be suitable for regulating expression of target genes (Figure). The light-inducible promoter <del class="diffchange diffchange-inline">pfcpB </del>is used for the constitutive expression of proteins in genetically modified ''Phaeodactylum tricornutum'' algae. Unfortunately, this promoter is not well characterized until now. The fcpB-promoter is known to control the expression of fucoxanthin binding proteins. These proteins play an important role in the absorption of photosynthetic active sunlight. To protect the light harvesting complex (LHC) from dangerous high-energy sunlight these promoters are already induced by low levels of sunlight (Veith and Büchel, 2007, BBA). Therefore, we expected a promoter induction under blue, red and white light conditions and never under green light and in darkness due to the fact that photosynthesis occurs only when red or blue light is present and fucoxanthin binding proteins are necessary for light harvesting and photoprotection. That is the reason why we decided to study the promoter strength by radiating the cells with different <del class="diffchange diffchange-inline">Transmission </del>wavelengths (i.e. green (572 nm), blue (467 nm), red (672 nm), white light and darkness). Hence we used the light inducible promoter for the expression of the reporter eGFP, which is advantageous because the expression level can be determined relatively easy. </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>Because plants and algae use sunlight as their primary energy source, they had to develop promoters, which respond to light. We therefore challenged the idea whether these light-inducible promoters would be suitable for regulating expression of target genes (Figure). The light-inducible promoter <ins class="diffchange diffchange-inline">fcpB </ins>is used for the constitutive expression of proteins in genetically modified ''Phaeodactylum tricornutum'' algae. Unfortunately, this promoter is not well characterized until now. The fcpB-promoter is known to control the expression of fucoxanthin binding proteins. These proteins play an important role in the absorption of photosynthetic active sunlight. To protect the light harvesting complex (LHC) from dangerous high-energy sunlight these promoters are already induced by low levels of sunlight (Veith and Büchel, 2007, BBA). Therefore, we expected a promoter induction under blue, red and white light conditions and never under green light and in darkness due to the fact that photosynthesis occurs only when red or blue light is present and fucoxanthin binding proteins are necessary for light harvesting and photoprotection. That is the reason why we decided to study the promoter strength by radiating the cells with different <ins class="diffchange diffchange-inline">transmission </ins>wavelengths (i.e. green (572 nm), blue (467 nm), red (672 nm), white light and darkness). Hence we used the light<ins class="diffchange diffchange-inline">-</ins>inducible promoter for the expression of the reporter eGFP, which is advantageous because the expression level can be determined relatively easy. </div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>A ''P. tricornutum'' culture was grown for six days under the specific light conditions described above. Afterwards, cultures were kept in darkness for two days to minimize the amount of existing GFP in the cells (<html><a href="https://2013.igem.org/Team:Marburg/Notebook:Ptricornutum">see the protocol</a></html>). The optical density was measured and GFP was quantified by Western blot analysis. </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>A ''P. tricornutum'' culture was grown for six days under the specific light conditions described above. Afterwards, cultures were kept in darkness for two days to minimize the amount of existing GFP in the cells (<html><a href="https://2013.igem.org/Team:Marburg/Notebook:Ptricornutum">see the protocol</a></html>). The optical density was measured and GFP was quantified by Western blot analysis. </div></td></tr>
</table>Domenicahttp://2013.igem.org/wiki/index.php?title=Team:Marburg/Project:lightcontrol&diff=348824&oldid=prevRomanM89 at 17:38, 28 October 20132013-10-28T17:38:57Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>A ''P. tricornutum'' culture was grown for six days under the specific light conditions described above. Afterwards, cultures were kept in darkness for two days to minimize the amount of existing GFP in the cells (<html><a href="https://2013.igem.org/Team:Marburg/Notebook:Ptricornutum">see the protocol</a></html>). The optical density was measured and GFP was quantified by Western blot analysis. </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>A ''P. tricornutum'' culture was grown for six days under the specific light conditions described above. Afterwards, cultures were kept in darkness for two days to minimize the amount of existing GFP in the cells (<html><a href="https://2013.igem.org/Team:Marburg/Notebook:Ptricornutum">see the protocol</a></html>). The optical density was measured and GFP was quantified by Western blot analysis. </div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>While determining the optical density, we found ''P. tricornutum'' growth under all light conditions except for darkness. Leblanc and coworkers showed that cryptochrome-, rhodopsin- and phytochrome-like receptors are present in marine diatoms indicating the ability to receive green light and to use green light for photosynthesis (Leblanc ''et al.'', 1999, Plant Mol Biol). Still, it was shown by Veith and coworkers that a fucoxanthin chlorophyll protein (FCP) complex, under the control of our light inducible promoter leads to a shift of the absorbance spectrum of PSI into the green spectrum after binding to fucoxanthin (Veith ''et al.'', 2007, BBA). </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>While determining the optical density, we found ''P. tricornutum'' growth under all light conditions except for darkness. Leblanc and coworkers showed that cryptochrome-, rhodopsin- and phytochrome-like receptors are present in marine diatoms indicating the ability to receive green light and to use green light for photosynthesis (Leblanc ''et al.'', 1999, Plant Mol Biol). Still, it was shown by Veith and coworkers that a fucoxanthin chlorophyll protein (FCP) complex, under the control of our light inducible promoter leads to a shift of the absorbance spectrum of PSI into the green spectrum after binding to fucoxanthin (Veith ''et al.'', 2007, BBA). </div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;"></ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;"><html><a href="https://static.igem.org/mediawiki/2013/7/7c/Mr-light.png"><img src="https://static.igem.org/mediawiki/2013/a/a6/Mr-light-thumb.png" alt="light" /></a></html></ins></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>For the Western blot analysis, proteins were firstly extracted from the cells. As the standard method for cell disruption in ''P. tricornutum'' seemed too laborious and error-prone due to many intermediate steps, we decided to optimize the cell disruption in PHAECTORY. The methods to be tested were the disruption using glass beads, metal beads and ultrasound. As opposed to metal beads and ultrasound, glass beads did not lead to sufficient cell disruption. The first two methods yielded equal amounts of proteins. However the metal bead method seems to be preferable due to the fact that the ultrasonic method requires special equipment for the whole execution and the effects of the electromagnetic waves on the protein structure are not predictable. To minimize the protein decomposition by proteases the disruption was directly performed in SDS sample buffer containing 2-mercaptoethanol and immediately applied on a SDS gel (<html><a href="https://2013.igem.org/Team:Marburg/Notebook:September#26-09-2013">see protocol</a></html>). The eGFP in the whole protein extract was detected using antibodies against the codon-optimized eGFP from ''Arabidopsis thaliana''.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>For the Western blot analysis, proteins were firstly extracted from the cells. As the standard method for cell disruption in ''P. tricornutum'' seemed too laborious and error-prone due to many intermediate steps, we decided to optimize the cell disruption in PHAECTORY. The methods to be tested were the disruption using glass beads, metal beads and ultrasound. As opposed to metal beads and ultrasound, glass beads did not lead to sufficient cell disruption. The first two methods yielded equal amounts of proteins. However the metal bead method seems to be preferable due to the fact that the ultrasonic method requires special equipment for the whole execution and the effects of the electromagnetic waves on the protein structure are not predictable. To minimize the protein decomposition by proteases the disruption was directly performed in SDS sample buffer containing 2-mercaptoethanol and immediately applied on a SDS gel (<html><a href="https://2013.igem.org/Team:Marburg/Notebook:September#26-09-2013">see protocol</a></html>). The eGFP in the whole protein extract was detected using antibodies against the codon-optimized eGFP from ''Arabidopsis thaliana''.</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>For the evaluation of the Western blot both, normalization against optical density (OD) and whole cell protein were done. In the case of the optical density normalization, it is clearly evident that light of any wavelength leads to the activation of the light-inducible promoter and thus increases the amount of eGFP in PHAECTORY. There is almost no visible difference in the amount of eGFP in the cells irradiated by blue, green and white light. In contrast, red light leads to more than 1.5 times the amount of eGFP. Hence, red light is the most efficient way to activate the light-inducible promoter fcpB in spite of the selective light absorption of longer wavelengths in water.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>For the evaluation of the Western blot both, normalization against optical density (OD) and whole cell protein were done. In the case of the optical density normalization, it is clearly evident that light of any wavelength leads to the activation of the light-inducible promoter and thus increases the amount of eGFP in PHAECTORY. There is almost no visible difference in the amount of eGFP in the cells irradiated by blue, green and white light. In contrast, red light leads to more than 1.5 times the amount of eGFP. Hence, red light is the most efficient way to activate the light-inducible promoter fcpB in spite of the selective light absorption of longer wavelengths in water.</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del style="color: red; font-weight: bold; text-decoration: none;"></del></div></td><td colspan="2"> </td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del style="color: red; font-weight: bold; text-decoration: none;"><html><a href="https://static.igem.org/mediawiki/2013/7/7c/Mr-light.png"><img src="https://static.igem.org/mediawiki/2013/a/a6/Mr-light-thumb.png" alt="light" /></a></html></del></div></td><td colspan="2"> </td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>In the case of normalizing against whole cell protein, it can be observed that the highest amount of eGFP occurred in the cells exposed to darkness followed by the cells grown under green and red light conditions. This result does not agree with the one obtained by normalizing against the OD. Normalization against total cell’s protein amount might be hampered by the fact the amount of protein in the cell is not equal for the different light conditions tested. We therefore conclude that normalization against whole protein amount might not be appropriate. It could well be that in darkness the cells produce proteins, which are absent when grown under light or vice versa. However, further experiments have to be performed to understand these data. Taken together, we found that our promoter is light-inducible and can be activated by blue, green and red light whereof red light yields the best results.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>In the case of normalizing against whole cell protein, it can be observed that the highest amount of eGFP occurred in the cells exposed to darkness followed by the cells grown under green and red light conditions. This result does not agree with the one obtained by normalizing against the OD. Normalization against total cell’s protein amount might be hampered by the fact the amount of protein in the cell is not equal for the different light conditions tested. We therefore conclude that normalization against whole protein amount might not be appropriate. It could well be that in darkness the cells produce proteins, which are absent when grown under light or vice versa. However, further experiments have to be performed to understand these data. Taken together, we found that our promoter is light-inducible and can be activated by blue, green and red light whereof red light yields the best results.</div></td></tr>
</table>RomanM89http://2013.igem.org/wiki/index.php?title=Team:Marburg/Project:lightcontrol&diff=341639&oldid=prevRomanM89 at 23:47, 27 October 20132013-10-27T23:47:37Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Characterizing a new light-inducible promoter <html><a href="https://2013.igem.org/Team:Marburg/Project:RFP"><img src="https://static.igem.org/mediawiki/2013/1/13/Mr-igem-previous-arrow.png" alt="Previous" style="float:right;"></a></html></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Characterizing a new light-inducible promoter <html><a href="https://2013.igem.org/Team:Marburg/Project:RFP"><img src="https://static.igem.org/mediawiki/2013/1/13/Mr-igem-previous-arrow.png" alt="Previous" style="float:right;"></a></html></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>{{:Team:Marburg/Template:ContentStartNav}}</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>{{:Team:Marburg/Template:ContentStartNav}}<ins class="diffchange diffchange-inline">The registry of standardized biological parts consists of plenty of well characterized promoters. Many of these promoters enable the production of proteins for example antibodies after induction with a specific substance. However, using an external inducer leads to several drawbacks: The inducer must be removed from the medium firstly to inactivate the promoter and accordingly stop the expression and secondly to simplify the purification of the desired protein. </ins></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del class="diffchange diffchange-inline"><html><a href="https://static</del>.<del class="diffchange diffchange-inline">igem.org/mediawiki/2013/7/7c/Mr</del>-light.<del class="diffchange diffchange-inline">png"><img src="https://static</del>.<del class="diffchange diffchange-inline">igem.org/mediawiki/2013/a/a6/Mr</del>-light-<del class="diffchange diffchange-inline">thumb</del>.<del class="diffchange diffchange-inline">png" alt="light" /></</del>a<del class="diffchange diffchange-inline">></html></del></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline">Because plants and algae use sunlight as their primary energy source, they had to develop promoters, which respond to light</ins>. <ins class="diffchange diffchange-inline">We therefore challenged the idea whether these light</ins>-<ins class="diffchange diffchange-inline">inducible promoters would be suitable for regulating expression of target genes (Figure). The </ins>light<ins class="diffchange diffchange-inline">-inducible promoter pfcpB is used for the constitutive expression of proteins in genetically modified ''Phaeodactylum tricornutum'' algae</ins>. <ins class="diffchange diffchange-inline">Unfortunately, this promoter is not well characterized until now</ins>. <ins class="diffchange diffchange-inline">The fcpB</ins>-<ins class="diffchange diffchange-inline">promoter is known to control the expression of fucoxanthin binding proteins. These proteins play an important role in the absorption of photosynthetic active sunlight. To protect the </ins>light <ins class="diffchange diffchange-inline">harvesting complex (LHC) from dangerous high</ins>-<ins class="diffchange diffchange-inline">energy sunlight these promoters are already induced by low levels of sunlight (Veith and Büchel, 2007, BBA)</ins>. <ins class="diffchange diffchange-inline">Therefore, we expected </ins>a <ins class="diffchange diffchange-inline">promoter induction under blue, red and white light conditions and never under green light and in darkness due to the fact that photosynthesis occurs only when red or blue light is present and fucoxanthin binding proteins are necessary for light harvesting and photoprotection. That is the reason why we decided to study the promoter strength by radiating the cells with different Transmission wavelengths (i.e. green (572 nm), blue (467 nm), red (672 nm), white light and darkness). Hence we used the light inducible promoter for the expression of the reporter eGFP, which is advantageous because the expression level can be determined relatively easy. </ins></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del class="diffchange diffchange-inline">In </del>the <del class="diffchange diffchange-inline">case of normalizing against whole cell protein</del>, <del class="diffchange diffchange-inline">it can be observed that </del>the <del class="diffchange diffchange-inline">highest </del>amount of <del class="diffchange diffchange-inline">eGFP occurred </del>in the cells <del class="diffchange diffchange-inline">exposed to darkness followed by the cells grown under green and red light conditions</del>. <del class="diffchange diffchange-inline">This result does not agree with </del>the <del class="diffchange diffchange-inline">one obtained by normalizing against the OD</del>. <del class="diffchange diffchange-inline">Normalization against total cell’s protein amount might be hampered </del>by the <del class="diffchange diffchange-inline">fact the amount of protein in the cell is not equal for the different </del>light conditions <del class="diffchange diffchange-inline">tested</del>. <del class="diffchange diffchange-inline">We therefore conclude </del>that <del class="diffchange diffchange-inline">normalization against whole protein amount might not be appropriate. It could well be that in darkness the cells produce proteins</del>, <del class="diffchange diffchange-inline">which </del>are <del class="diffchange diffchange-inline">absent when grown under </del>light <del class="diffchange diffchange-inline">or vice versa</del>. <del class="diffchange diffchange-inline">However</del>, <del class="diffchange diffchange-inline">further experiments have to be performed to understand these data</del>. <del class="diffchange diffchange-inline">Taken together</del>, <del class="diffchange diffchange-inline">we found </del>that our <del class="diffchange diffchange-inline">promoter is </del>light<del class="diffchange diffchange-inline">-</del>inducible <del class="diffchange diffchange-inline">and can be activated by blue, green and red light whereof red light yields </del>the <del class="diffchange diffchange-inline">best results</del>.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline">A ''P. tricornutum'' culture was grown for six days under </ins>the <ins class="diffchange diffchange-inline">specific light conditions described above. Afterwards</ins>, <ins class="diffchange diffchange-inline">cultures were kept in darkness for two days to minimize </ins>the amount of <ins class="diffchange diffchange-inline">existing GFP </ins>in the cells <ins class="diffchange diffchange-inline">(<html><a href="https://2013</ins>.<ins class="diffchange diffchange-inline">igem.org/Team:Marburg/Notebook:Ptricornutum">see </ins>the <ins class="diffchange diffchange-inline">protocol</a></html>)</ins>. <ins class="diffchange diffchange-inline">The optical density was measured and GFP was quantified </ins>by <ins class="diffchange diffchange-inline">Western blot analysis. </ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline">While determining </ins>the <ins class="diffchange diffchange-inline">optical density, we found ''P. tricornutum'' growth under all </ins>light conditions <ins class="diffchange diffchange-inline">except for darkness</ins>. <ins class="diffchange diffchange-inline">Leblanc and coworkers showed </ins>that <ins class="diffchange diffchange-inline">cryptochrome-</ins>, <ins class="diffchange diffchange-inline">rhodopsin- and phytochrome-like receptors </ins>are <ins class="diffchange diffchange-inline">present in marine diatoms indicating the ability to receive green </ins>light <ins class="diffchange diffchange-inline">and to use green light for photosynthesis (Leblanc ''et al</ins>.<ins class="diffchange diffchange-inline">''</ins>, <ins class="diffchange diffchange-inline">1999, Plant Mol Biol)</ins>. <ins class="diffchange diffchange-inline">Still</ins>, <ins class="diffchange diffchange-inline">it was shown by Veith and coworkers </ins>that <ins class="diffchange diffchange-inline">a fucoxanthin chlorophyll protein (FCP) complex, under the control of </ins>our light inducible <ins class="diffchange diffchange-inline">promoter leads to a shift of </ins>the <ins class="diffchange diffchange-inline">absorbance spectrum of PSI into the green spectrum after binding to fucoxanthin (Veith ''et al.'', 2007, BBA)</ins>. </div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del class="diffchange diffchange-inline"><html><!--The registry of standardized biological parts consists of plenty of well characterized promoters. Many of these promoters enable </del>the <del class="diffchange diffchange-inline">production of </del>proteins for <del class="diffchange diffchange-inline">example antibodies after induction with a specific substance</del>. <del class="diffchange diffchange-inline">However</del>, <del class="diffchange diffchange-inline">using an external inducer leads </del>to <del class="diffchange diffchange-inline">several drawbacks: </del>The <del class="diffchange diffchange-inline">inducer must </del>be <del class="diffchange diffchange-inline">removed from </del>the <del class="diffchange diffchange-inline">medium firstly </del>to <del class="diffchange diffchange-inline">inactivate the promoter </del>and <del class="diffchange diffchange-inline">accordingly stop </del>the <del class="diffchange diffchange-inline">expression and secondly </del>to <del class="diffchange diffchange-inline">simplify </del>the <del class="diffchange diffchange-inline">purification </del>of the <del class="diffchange diffchange-inline">desired </del>protein.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline">For </ins>the <ins class="diffchange diffchange-inline">Western blot analysis, </ins>proteins <ins class="diffchange diffchange-inline">were firstly extracted from the cells. As the standard method </ins>for <ins class="diffchange diffchange-inline">cell disruption in ''P</ins>. <ins class="diffchange diffchange-inline">tricornutum'' seemed too laborious and error-prone due to many intermediate steps</ins>, <ins class="diffchange diffchange-inline">we decided </ins>to <ins class="diffchange diffchange-inline">optimize the cell disruption in PHAECTORY. </ins>The <ins class="diffchange diffchange-inline">methods to </ins>be <ins class="diffchange diffchange-inline">tested were </ins>the <ins class="diffchange diffchange-inline">disruption using glass beads, metal beads and ultrasound. As opposed </ins>to <ins class="diffchange diffchange-inline">metal beads </ins>and <ins class="diffchange diffchange-inline">ultrasound, glass beads did not lead to sufficient cell disruption. The first two methods yielded equal amounts of proteins. However </ins>the <ins class="diffchange diffchange-inline">metal bead method seems to be preferable due </ins>to the <ins class="diffchange diffchange-inline">fact that the ultrasonic method requires special equipment for the whole execution and the effects </ins>of the <ins class="diffchange diffchange-inline">electromagnetic waves on the </ins>protein <ins class="diffchange diffchange-inline">structure are not predictable. To minimize the protein decomposition by proteases the disruption was directly performed in SDS sample buffer containing 2-mercaptoethanol and immediately applied on a SDS gel (<html><a href="https://2013.igem.org/Team:Marburg/Notebook:September#26-09-2013">see protocol</a></html>). The eGFP in the whole protein extract was detected using antibodies against the codon-optimized eGFP from ''Arabidopsis thaliana''</ins>.</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del class="diffchange diffchange-inline">Because plants and algae use sunlight as primary energy source, they had to develop promoters, which respond to white light. We therefore challenged </del>the <del class="diffchange diffchange-inline">idea whether these light-inducible promoters would be suitable for regulating expression </del>of <del class="diffchange diffchange-inline">target genes </del>(<del class="diffchange diffchange-inline">Figure</del>). <del class="diffchange diffchange-inline">One </del>of the light-inducible <del class="diffchange diffchange-inline">promoters is used for </del>the <del class="diffchange diffchange-inline">constitutive expression </del>of <del class="diffchange diffchange-inline">proteins </del>in <del class="diffchange diffchange-inline">genetically modified ''Phaeodactylum tricornutum'' algae</del>. <del class="diffchange diffchange-inline">Regrettably this promoter, pfcpB, </del>is <del class="diffchange diffchange-inline">not well characterized until now. The fcpB-promoter is known to control </del>the <del class="diffchange diffchange-inline">expression </del>of <del class="diffchange diffchange-inline">Fucoxanthin binding proteins. These proteins play an important role </del>in the <del class="diffchange diffchange-inline">absorption of photosynthetic active sunlight. To protect the light harvesting complex (LHC) from dangerous high-energy sunlight these promoters are already induced </del>by <del class="diffchange diffchange-inline">low levels of sunlight (Veith and Büchel, 2007, BBA). Therefore, we expected a promoter induction under </del>blue, <del class="diffchange diffchange-inline">red </del>and white light <del class="diffchange diffchange-inline">conditions and never under green </del>light <del class="diffchange diffchange-inline">and in darkness due </del>to the <del class="diffchange diffchange-inline">fact that photosynthesis occurs only when </del>red <del class="diffchange diffchange-inline">or blue </del>light <del class="diffchange diffchange-inline">is present and fucoxanthin binding proteins are necessary for light harvesting and photoprotection. That </del>is the <del class="diffchange diffchange-inline">reason why we decided </del>to <del class="diffchange diffchange-inline">study the promoter strength by radiating the cells with different Transmission wavelengths (i.e. green (572 nm), blue (467 nm), red (672 nm), white light and darkness). Hence we used </del>the light inducible promoter <del class="diffchange diffchange-inline">for the expression </del>of the <del class="diffchange diffchange-inline">reporter eGFP, which is advantageous because the expression level can be determined relatively easy</del>.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline">For </ins>the <ins class="diffchange diffchange-inline">evaluation </ins>of <ins class="diffchange diffchange-inline">the Western blot both, normalization against optical density </ins>(<ins class="diffchange diffchange-inline">OD</ins>) <ins class="diffchange diffchange-inline">and whole cell protein were done</ins>. <ins class="diffchange diffchange-inline">In the case of the optical density normalization, it is clearly evident that light of any wavelength leads to the activation </ins>of the light-inducible <ins class="diffchange diffchange-inline">promoter and thus increases </ins>the <ins class="diffchange diffchange-inline">amount </ins>of <ins class="diffchange diffchange-inline">eGFP </ins>in <ins class="diffchange diffchange-inline">PHAECTORY</ins>. <ins class="diffchange diffchange-inline">There </ins>is <ins class="diffchange diffchange-inline">almost no visible difference in </ins>the <ins class="diffchange diffchange-inline">amount </ins>of <ins class="diffchange diffchange-inline">eGFP </ins>in the <ins class="diffchange diffchange-inline">cells irradiated </ins>by blue, <ins class="diffchange diffchange-inline">green </ins>and white light<ins class="diffchange diffchange-inline">. In contrast, red </ins>light <ins class="diffchange diffchange-inline">leads </ins>to <ins class="diffchange diffchange-inline">more than 1.5 times </ins>the <ins class="diffchange diffchange-inline">amount of eGFP. Hence, </ins>red light is the <ins class="diffchange diffchange-inline">most efficient way </ins>to <ins class="diffchange diffchange-inline">activate </ins>the light<ins class="diffchange diffchange-inline">-</ins>inducible promoter <ins class="diffchange diffchange-inline">fcpB in spite </ins>of the <ins class="diffchange diffchange-inline">selective light absorption of longer wavelengths in water</ins>.</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><html></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><html><ins class="diffchange diffchange-inline"><a href="https://static.igem.org/mediawiki/2013/7/7c/Mr-light.png"></ins><img src="https://static.igem.org/mediawiki/2013/<ins class="diffchange diffchange-inline">a</ins>/<ins class="diffchange diffchange-inline">a6</ins>/<ins class="diffchange diffchange-inline">Mr</ins>-<ins class="diffchange diffchange-inline">light-thumb</ins>.png" alt="<ins class="diffchange diffchange-inline">light</ins>" /><ins class="diffchange diffchange-inline"></a></ins></html></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><img src="https://static.igem.org/mediawiki/2013/<del class="diffchange diffchange-inline">c</del>/<del class="diffchange diffchange-inline">c6</del>/<del class="diffchange diffchange-inline">Lichtpromoter</del>-<del class="diffchange diffchange-inline">web</del>.png" alt="<del class="diffchange diffchange-inline">MTS</del>" /></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div> </div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div></html></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline">In the case of normalizing against whole cell protein, it can be observed that the highest amount of eGFP occurred in the cells exposed to darkness followed by the cells grown under green and red light conditions. This result does not agree with the one obtained by normalizing against the OD. Normalization against total cell’s protein amount might be hampered by the fact the amount of protein in the cell is not equal for the different light conditions tested. We therefore conclude that normalization against whole protein amount might not be appropriate. It could well be that in darkness the cells produce proteins, which are absent when grown under light or vice versa. However, further experiments have to be performed to understand these data. Taken together, we found that our promoter is light-inducible and can be activated by blue, green and red light whereof red light yields the best results.</ins></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del style="color: red; font-weight: bold; text-decoration: none;">A ''P. tricornutum'' culture was grown for six days under specific light conditions and afterwards for two days in darkness to minimize the amount of existing GFP in ''P. tricornutum'' (<html><a href="https://2013.igem.org/Team:Marburg/Notebook:Ptricornutum">see the protocol</a></html>). The cell density was adjusted to an optical density of 0,22 and GFP was quantified by <html><a href="https://2013.igem.org/Team:Marburg/Notebook:September#wb">Western Blot analysis</a></html>. While determining the optical density we found ''P. tricornutum'' growth under all light conditions, even under green light, which was in contrast to our assumption. Leblanc and coworkers showed that cryptochrome-, rhodopsin- and phytochrome-like receptors are present in marine diatoms indicating the ability to receive green light and to use green light for photosynthesis (Leblanc ''et al.'', 1999, Plant Mol Biol). Still it was shown by Veith and coworkers that a fucoxanthin chlorophyll protein (FCP) complex, under the control of our light inducible promoter, binding to fucoxanthin leads to a shift of the absorbance spectrum of PSI into the green spectrum (Veith ''et al.'', 2007, BBA).</del></div></td><td colspan="2"> </td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del style="color: red; font-weight: bold; text-decoration: none;">Also we found no expression of GFP under red light condition but this may be explained by the selective light absorbtion of longer wavelengths in water. Therefore, only blue and green spectra penetrate deeper water levels. Taken together, we found that our promoter can be activated by blue and green light. To this end, we did not observe any induction in red light. However, further experiments have to be performed to confirm these data.</del></div></td><td colspan="2"> </td></tr>
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</table>RomanM89http://2013.igem.org/wiki/index.php?title=Team:Marburg/Project:lightcontrol&diff=341637&oldid=prevRomanM89 at 23:47, 27 October 20132013-10-27T23:47:16Z<p></p>
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<td colspan='2' style="background-color: white; color:black;">Revision as of 23:47, 27 October 2013</td>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Characterizing a new light-inducible promoter <html><a href="https://2013.igem.org/Team:Marburg/Project:RFP"><img src="https://static.igem.org/mediawiki/2013/1/13/Mr-igem-previous-arrow.png" alt="Previous" style="float:right;"></a></html></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Characterizing a new light-inducible promoter <html><a href="https://2013.igem.org/Team:Marburg/Project:RFP"><img src="https://static.igem.org/mediawiki/2013/1/13/Mr-igem-previous-arrow.png" alt="Previous" style="float:right;"></a></html></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>{{:Team:Marburg/Template:ContentStartNav}}<del class="diffchange diffchange-inline">The registry of standardized biological parts consists of plenty of well characterized promoters. Many of these promoters enable the production of proteins for example antibodies after induction with a specific substance. However, using an external inducer leads to several drawbacks: The inducer must be removed from the medium firstly to inactivate the promoter and accordingly stop the expression and secondly to simplify the purification of the desired protein. </del></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>{{:Team:Marburg/Template:ContentStartNav}}</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div> </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del class="diffchange diffchange-inline">Because plants and algae use sunlight as their primary energy source, they had to develop promoters, which respond to light. We therefore challenged the idea whether these light-inducible promoters would be suitable for regulating expression of target genes (Figure). The light-inducible promoter pfcpB is used for the constitutive expression of proteins in genetically modified ''Phaeodactylum tricornutum'' algae. Unfortunately, this promoter is not well characterized until now. The fcpB-promoter is known to control the expression of fucoxanthin binding proteins. These proteins play an important role in the absorption of photosynthetic active sunlight. To protect the light harvesting complex (LHC) from dangerous high-energy sunlight these promoters are already induced by low levels of sunlight (Veith and Büchel, 2007, BBA). Therefore, we expected a promoter induction under blue, red and white light conditions and never under green light and in darkness due to the fact that photosynthesis occurs only when red or blue light is present and fucoxanthin binding proteins are necessary for light harvesting and photoprotection. That is the reason why we decided to study the promoter strength by radiating the cells with different Transmission wavelengths (i.e. green (572 nm), blue (467 nm), red (672 nm), white light and darkness). Hence we used the light inducible promoter for the expression of the reporter eGFP, which is advantageous because the expression level can be determined relatively easy. </del></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div> </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del class="diffchange diffchange-inline">A ''P. tricornutum'' culture was grown for six days under the specific light conditions described above. Afterwards, cultures were kept in darkness for two days to minimize the amount of existing GFP in the cells (<html><a href="https://2013.igem.org/Team:Marburg/Notebook:Ptricornutum">see the protocol</a></html>). The optical density was measured and GFP was quantified by Western blot analysis. </del></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del class="diffchange diffchange-inline">While determining the optical density, we found ''P. tricornutum'' growth under all light conditions except for darkness. Leblanc and coworkers showed that cryptochrome-, rhodopsin- and phytochrome-like receptors are present in marine diatoms indicating the ability to receive green light and to use green light for photosynthesis (Leblanc ''et al.'', 1999, Plant Mol Biol). Still, it was shown by Veith and coworkers that a fucoxanthin chlorophyll protein (FCP) complex, under the control of our light inducible promoter leads to a shift of the absorbance spectrum of PSI into the green spectrum after binding to fucoxanthin (Veith ''et al.'', 2007, BBA). </del></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div> </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del class="diffchange diffchange-inline">For the Western blot analysis, proteins were firstly extracted from the cells. As the standard method for cell disruption in ''P. tricornutum'' seemed too laborious and error-prone due to many intermediate steps, we decided to optimize the cell disruption in PHAECTORY. The methods to be tested were the disruption using glass beads, metal beads and ultrasound. As opposed to metal beads and ultrasound, glass beads did not lead to sufficient cell disruption. The first two methods yielded equal amounts of proteins. However the metal bead method seems to be preferable due to the fact that the ultrasonic method requires special equipment for the whole execution and the effects of the electromagnetic waves on the protein structure are not predictable. To minimize the protein decomposition by proteases the disruption was directly performed in SDS sample buffer containing 2-mercaptoethanol and immediately applied on a SDS gel (<html><a href="https://2013.igem.org/Team:Marburg/Notebook:September#26-09-2013">see protocol</a></html>). The eGFP in the whole protein extract was detected using antibodies against the codon-optimized eGFP from ''Arabidopsis thaliana''.</del></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div> </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del class="diffchange diffchange-inline">For the evaluation of the Western blot both, normalization against optical density (OD) and whole cell protein were done. In the case of the optical density normalization, it is clearly evident that light of any wavelength leads to the activation of the light-inducible promoter and thus increases the amount of eGFP in PHAECTORY. There is almost no visible difference in the amount of eGFP in the cells irradiated by blue, green and white light. In contrast, red light leads to more than 1.5 times the amount of eGFP. Hence, red light is the most efficient way to activate the light-inducible promoter fcpB in spite of the selective light absorption of longer wavelengths in water. </del></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div> </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;"><html><a href="https://static.igem.org/mediawiki/2013/7/7c/Mr-light.png"><img src="https://static.igem.org/mediawiki/2013/a/a6/Mr-light-thumb.png" alt="light" /></a></html></ins></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>In the case of normalizing against whole cell protein, it can be observed that the highest amount of eGFP occurred in the cells exposed to darkness followed by the cells grown under green and red light conditions. This result does not agree with the one obtained by normalizing against the OD. Normalization against total cell’s protein amount might be hampered by the fact the amount of protein in the cell is not equal for the different light conditions tested. We therefore conclude that normalization against whole protein amount might not be appropriate. It could well be that in darkness the cells produce proteins, which are absent when grown under light or vice versa. However, further experiments have to be performed to understand these data. Taken together, we found that our promoter is light-inducible and can be activated by blue, green and red light whereof red light yields the best results.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>In the case of normalizing against whole cell protein, it can be observed that the highest amount of eGFP occurred in the cells exposed to darkness followed by the cells grown under green and red light conditions. This result does not agree with the one obtained by normalizing against the OD. Normalization against total cell’s protein amount might be hampered by the fact the amount of protein in the cell is not equal for the different light conditions tested. We therefore conclude that normalization against whole protein amount might not be appropriate. It could well be that in darkness the cells produce proteins, which are absent when grown under light or vice versa. However, further experiments have to be performed to understand these data. Taken together, we found that our promoter is light-inducible and can be activated by blue, green and red light whereof red light yields the best results.</div></td></tr>
</table>RomanM89http://2013.igem.org/wiki/index.php?title=Team:Marburg/Project:lightcontrol&diff=341633&oldid=prevRomanM89 at 23:46, 27 October 20132013-10-27T23:46:44Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>For the evaluation of the Western blot both, normalization against optical density (OD) and whole cell protein were done. In the case of the optical density normalization, it is clearly evident that light of any wavelength leads to the activation of the light-inducible promoter and thus increases the amount of eGFP in PHAECTORY. There is almost no visible difference in the amount of eGFP in the cells irradiated by blue, green and white light. In contrast, red light leads to more than 1.5 times the amount of eGFP. Hence, red light is the most efficient way to activate the light-inducible promoter fcpB in spite of the selective light absorption of longer wavelengths in water. </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>For the evaluation of the Western blot both, normalization against optical density (OD) and whole cell protein were done. In the case of the optical density normalization, it is clearly evident that light of any wavelength leads to the activation of the light-inducible promoter and thus increases the amount of eGFP in PHAECTORY. There is almost no visible difference in the amount of eGFP in the cells irradiated by blue, green and white light. In contrast, red light leads to more than 1.5 times the amount of eGFP. Hence, red light is the most efficient way to activate the light-inducible promoter fcpB in spite of the selective light absorption of longer wavelengths in water. </div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del class="diffchange diffchange-inline"><html><a href="https://static.igem.org/mediawiki/2013/7/7c/Mr-light.png"><img src="https://static.igem.org/mediawiki/2013/a/a6/Mr-light-thumb.png" alt="light" /></a></html></del></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div> </div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>In the case of normalizing against whole cell protein, it can be observed that the highest amount of eGFP occurred in the cells exposed to darkness followed by the cells grown under green and red light conditions. This result does not agree with the one obtained by normalizing against the OD. Normalization against total cell’s protein amount might be hampered by the fact the amount of protein in the cell is not equal for the different light conditions tested. We therefore conclude that normalization against whole protein amount might not be appropriate. It could well be that in darkness the cells produce proteins, which are absent when grown under light or vice versa. However, further experiments have to be performed to understand these data. Taken together, we found that our promoter is light-inducible and can be activated by blue, green and red light whereof red light yields the best results.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>In the case of normalizing against whole cell protein, it can be observed that the highest amount of eGFP occurred in the cells exposed to darkness followed by the cells grown under green and red light conditions. This result does not agree with the one obtained by normalizing against the OD. Normalization against total cell’s protein amount might be hampered by the fact the amount of protein in the cell is not equal for the different light conditions tested. We therefore conclude that normalization against whole protein amount might not be appropriate. It could well be that in darkness the cells produce proteins, which are absent when grown under light or vice versa. However, further experiments have to be performed to understand these data. Taken together, we found that our promoter is light-inducible and can be activated by blue, green and red light whereof red light yields the best results.</div></td></tr>
</table>RomanM89http://2013.igem.org/wiki/index.php?title=Team:Marburg/Project:lightcontrol&diff=341628&oldid=prevRomanM89 at 23:46, 27 October 20132013-10-27T23:46:00Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Characterizing a new light-inducible promoter <html><a href="https://2013.igem.org/Team:Marburg/Project:RFP"><img src="https://static.igem.org/mediawiki/2013/1/13/Mr-igem-previous-arrow.png" alt="Previous" style="float:right;"></a></html></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Characterizing a new light-inducible promoter <html><a href="https://2013.igem.org/Team:Marburg/Project:RFP"><img src="https://static.igem.org/mediawiki/2013/1/13/Mr-igem-previous-arrow.png" alt="Previous" style="float:right;"></a></html></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>{{:Team:Marburg/Template:ContentStartNav}}The registry of standardized biological parts consists of plenty of well characterized promoters. Many of these promoters enable the production of proteins for example antibodies after induction with a specific substance. However, using an external inducer leads to several drawbacks: The inducer must be removed from the medium firstly to inactivate the promoter and accordingly stop the expression and secondly to simplify the purification of the desired protein.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>{{:Team:Marburg/Template:ContentStartNav}}<ins class="diffchange diffchange-inline">The registry of standardized biological parts consists of plenty of well characterized promoters. Many of these promoters enable the production of proteins for example antibodies after induction with a specific substance. However, using an external inducer leads to several drawbacks: The inducer must be removed from the medium firstly to inactivate the promoter and accordingly stop the expression and secondly to simplify the purification of the desired protein. </ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div> </div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline">Because plants and algae use sunlight as their primary energy source, they had to develop promoters, which respond to light. We therefore challenged the idea whether these light-inducible promoters would be suitable for regulating expression of target genes (Figure). The light-inducible promoter pfcpB is used for the constitutive expression of proteins in genetically modified ''Phaeodactylum tricornutum'' algae. Unfortunately, this promoter is not well characterized until now. The fcpB-promoter is known to control the expression of fucoxanthin binding proteins. These proteins play an important role in the absorption of photosynthetic active sunlight. To protect the light harvesting complex (LHC) from dangerous high-energy sunlight these promoters are already induced by low levels of sunlight (Veith and Büchel, 2007, BBA). Therefore, we expected a promoter induction under blue, red and white light conditions and never under green light and in darkness due to the fact that photosynthesis occurs only when red or blue light is present and fucoxanthin binding proteins are necessary for light harvesting and photoprotection. That is the reason why we decided to study the promoter strength by radiating the cells with different Transmission wavelengths (i.e. green (572 nm), blue (467 nm), red (672 nm), white light and darkness). Hence we used the light inducible promoter for the expression of the reporter eGFP, which is advantageous because the expression level can be determined relatively easy. </ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div> </div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline">A ''P. tricornutum'' culture was grown for six days under the specific light conditions described above. Afterwards, cultures were kept in darkness for two days to minimize the amount of existing GFP in the cells (<html><a href="https://2013.igem.org/Team:Marburg/Notebook:Ptricornutum">see the protocol</a></html>). The optical density was measured and GFP was quantified by Western blot analysis. </ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline">While determining the optical density, we found ''P. tricornutum'' growth under all light conditions except for darkness. Leblanc and coworkers showed that cryptochrome-, rhodopsin- and phytochrome-like receptors are present in marine diatoms indicating the ability to receive green light and to use green light for photosynthesis (Leblanc ''et al.'', 1999, Plant Mol Biol). Still, it was shown by Veith and coworkers that a fucoxanthin chlorophyll protein (FCP) complex, under the control of our light inducible promoter leads to a shift of the absorbance spectrum of PSI into the green spectrum after binding to fucoxanthin (Veith ''et al.'', 2007, BBA). </ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div> </div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline">For the Western blot analysis, proteins were firstly extracted from the cells. As the standard method for cell disruption in ''P. tricornutum'' seemed too laborious and error-prone due to many intermediate steps, we decided to optimize the cell disruption in PHAECTORY. The methods to be tested were the disruption using glass beads, metal beads and ultrasound. As opposed to metal beads and ultrasound, glass beads did not lead to sufficient cell disruption. The first two methods yielded equal amounts of proteins. However the metal bead method seems to be preferable due to the fact that the ultrasonic method requires special equipment for the whole execution and the effects of the electromagnetic waves on the protein structure are not predictable. To minimize the protein decomposition by proteases the disruption was directly performed in SDS sample buffer containing 2-mercaptoethanol and immediately applied on a SDS gel (<html><a href="https://2013.igem.org/Team:Marburg/Notebook:September#26-09-2013">see protocol</a></html>). The eGFP in the whole protein extract was detected using antibodies against the codon-optimized eGFP from ''Arabidopsis thaliana''.</ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div> </div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline">For the evaluation of the Western blot both, normalization against optical density (OD) and whole cell protein were done. In the case of the optical density normalization, it is clearly evident that light of any wavelength leads to the activation of the light-inducible promoter and thus increases the amount of eGFP in PHAECTORY. There is almost no visible difference in the amount of eGFP in the cells irradiated by blue, green and white light. In contrast, red light leads to more than 1.5 times the amount of eGFP. Hence, red light is the most efficient way to activate the light-inducible promoter fcpB in spite of the selective light absorption of longer wavelengths in water. </ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div> </div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline"><html><a href="https://static.igem.org/mediawiki/2013/7/7c/Mr-light.png"><img src="https://static.igem.org/mediawiki/2013/a/a6/Mr-light-thumb.png" alt="light" /></a></html></ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div> </div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline">In the case of normalizing against whole cell protein, it can be observed that the highest amount of eGFP occurred in the cells exposed to darkness followed by the cells grown under green and red light conditions. This result does not agree with the one obtained by normalizing against the OD. Normalization against total cell’s protein amount might be hampered by the fact the amount of protein in the cell is not equal for the different light conditions tested. We therefore conclude that normalization against whole protein amount might not be appropriate. It could well be that in darkness the cells produce proteins, which are absent when grown under light or vice versa. However, further experiments have to be performed to understand these data. Taken together, we found that our promoter is light-inducible and can be activated by blue, green and red light whereof red light yields the best results.</ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div> </div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline"><html><!--</ins>The registry of standardized biological parts consists of plenty of well characterized promoters. Many of these promoters enable the production of proteins for example antibodies after induction with a specific substance. However, using an external inducer leads to several drawbacks: The inducer must be removed from the medium firstly to inactivate the promoter and accordingly stop the expression and secondly to simplify the purification of the desired protein.</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Because plants and algae use sunlight as primary energy source, they had to develop promoters, which respond to white light. We therefore challenged the idea whether these light-inducible promoters would be suitable for regulating expression of target genes (Figure). One of the light-inducible promoters is used for the constitutive expression of proteins in genetically modified ''Phaeodactylum tricornutum'' algae. Regrettably this promoter, pfcpB, is not well characterized until now. The fcpB-promoter is known to control the expression of Fucoxanthin binding proteins. These proteins play an important role in the absorption of photosynthetic active sunlight. To protect the light harvesting complex (LHC) from dangerous high-energy sunlight these promoters are already induced by low levels of sunlight (Veith and Büchel, 2007, BBA). Therefore, we expected a promoter induction under blue, red and white light conditions and never under green light and in darkness due to the fact that photosynthesis occurs only when red or blue light is present and fucoxanthin binding proteins are necessary for light harvesting and photoprotection. That is the reason why we decided to study the promoter strength by radiating the cells with different Transmission wavelengths (i.e. green (572 nm), blue (467 nm), red (672 nm), white light and darkness). Hence we used the light inducible promoter for the expression of the reporter eGFP, which is advantageous because the expression level can be determined relatively easy.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Because plants and algae use sunlight as primary energy source, they had to develop promoters, which respond to white light. We therefore challenged the idea whether these light-inducible promoters would be suitable for regulating expression of target genes (Figure). One of the light-inducible promoters is used for the constitutive expression of proteins in genetically modified ''Phaeodactylum tricornutum'' algae. Regrettably this promoter, pfcpB, is not well characterized until now. The fcpB-promoter is known to control the expression of Fucoxanthin binding proteins. These proteins play an important role in the absorption of photosynthetic active sunlight. To protect the light harvesting complex (LHC) from dangerous high-energy sunlight these promoters are already induced by low levels of sunlight (Veith and Büchel, 2007, BBA). Therefore, we expected a promoter induction under blue, red and white light conditions and never under green light and in darkness due to the fact that photosynthesis occurs only when red or blue light is present and fucoxanthin binding proteins are necessary for light harvesting and photoprotection. That is the reason why we decided to study the promoter strength by radiating the cells with different Transmission wavelengths (i.e. green (572 nm), blue (467 nm), red (672 nm), white light and darkness). Hence we used the light inducible promoter for the expression of the reporter eGFP, which is advantageous because the expression level can be determined relatively easy.</div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Also we found no expression of GFP under red light condition but this may be explained by the selective light absorbtion of longer wavelengths in water. Therefore, only blue and green spectra penetrate deeper water levels. Taken together, we found that our promoter can be activated by blue and green light. To this end, we did not observe any induction in red light. However, further experiments have to be performed to confirm these data.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Also we found no expression of GFP under red light condition but this may be explained by the selective light absorbtion of longer wavelengths in water. Therefore, only blue and green spectra penetrate deeper water levels. Taken together, we found that our promoter can be activated by blue and green light. To this end, we did not observe any induction in red light. However, further experiments have to be performed to confirm these data.</div></td></tr>
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</table>RomanM89http://2013.igem.org/wiki/index.php?title=Team:Marburg/Project:lightcontrol&diff=336183&oldid=prevKuehnma at 11:02, 27 October 20132013-10-27T11:02:10Z<p></p>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>Characterizing a new light-inducible promoter</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>Characterizing a new light-inducible promoter <ins class="diffchange diffchange-inline"><html><a href="https://2013.igem.org/Team:Marburg/Project:RFP"><img src="https://static.igem.org/mediawiki/2013/1/13/Mr-igem-previous-arrow.png" alt="Previous" style="float:right;"></a></html></ins></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>{{:Team:Marburg/Template:ContentStartNav}}The registry of standardized biological parts consists of plenty of well characterized promoters. Many of these promoters enable the production of proteins for example antibodies after induction with a specific substance. However, using an external inducer leads to several drawbacks: The inducer must be removed from the medium firstly to inactivate the promoter and accordingly stop the expression and secondly to simplify the purification of the desired protein.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>{{:Team:Marburg/Template:ContentStartNav}}The registry of standardized biological parts consists of plenty of well characterized promoters. Many of these promoters enable the production of proteins for example antibodies after induction with a specific substance. However, using an external inducer leads to several drawbacks: The inducer must be removed from the medium firstly to inactivate the promoter and accordingly stop the expression and secondly to simplify the purification of the desired protein.</div></td></tr>
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</table>Kuehnmahttp://2013.igem.org/wiki/index.php?title=Team:Marburg/Project:lightcontrol&diff=331608&oldid=prevDomenica at 14:01, 26 October 20132013-10-26T14:01:54Z<p></p>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>A ''P. tricornutum'' culture was grown for <del class="diffchange diffchange-inline">seven </del>days under specific light conditions and afterwards for <del class="diffchange diffchange-inline">four </del>days in darkness to minimize the amount of existing GFP in ''P. tricornutum'' (<html><a href="https://2013.igem.org/Team:Marburg/Notebook:Ptricornutum">see the protocol</a></html>). The cell density was adjusted to an optical density of 0,22 and GFP was quantified by <html><a href="https://2013.igem.org/Team:Marburg/Notebook:September#wb">Western Blot analysis</a></html>. While determining the optical density we found ''P. tricornutum'' growth under all light conditions, even under green light, which was in contrast to our assumption. Leblanc and coworkers showed that cryptochrome-, rhodopsin- and phytochrome-like receptors are present in marine diatoms indicating the ability to receive green light and to use green light for photosynthesis (Leblanc ''et al.'', 1999, Plant Mol Biol). Still it was shown by Veith and coworkers that a fucoxanthin chlorophyll protein (FCP) complex, under the control of our light inducible promoter, binding to fucoxanthin leads to a shift of the absorbance spectrum of PSI into the green spectrum (Veith ''et al.'', 2007, BBA).</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>A ''P. tricornutum'' culture was grown for <ins class="diffchange diffchange-inline">six </ins>days under specific light conditions and afterwards for <ins class="diffchange diffchange-inline">two </ins>days in darkness to minimize the amount of existing GFP in ''P. tricornutum'' (<html><a href="https://2013.igem.org/Team:Marburg/Notebook:Ptricornutum">see the protocol</a></html>). The cell density was adjusted to an optical density of 0,22 and GFP was quantified by <html><a href="https://2013.igem.org/Team:Marburg/Notebook:September#wb">Western Blot analysis</a></html>. While determining the optical density we found ''P. tricornutum'' growth under all light conditions, even under green light, which was in contrast to our assumption. Leblanc and coworkers showed that cryptochrome-, rhodopsin- and phytochrome-like receptors are present in marine diatoms indicating the ability to receive green light and to use green light for photosynthesis (Leblanc ''et al.'', 1999, Plant Mol Biol). Still it was shown by Veith and coworkers that a fucoxanthin chlorophyll protein (FCP) complex, under the control of our light inducible promoter, binding to fucoxanthin leads to a shift of the absorbance spectrum of PSI into the green spectrum (Veith ''et al.'', 2007, BBA).</div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Also we found no expression of GFP under red light condition but this may be explained by the selective light absorbtion of longer wavelengths in water. Therefore, only blue and green spectra penetrate deeper water levels. Taken together, we found that our promoter can be activated by blue and green light. To this end, we did not observe any induction in red light. However, further experiments have to be performed to confirm these data.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Also we found no expression of GFP under red light condition but this may be explained by the selective light absorbtion of longer wavelengths in water. Therefore, only blue and green spectra penetrate deeper water levels. Taken together, we found that our promoter can be activated by blue and green light. To this end, we did not observe any induction in red light. However, further experiments have to be performed to confirm these data.</div></td></tr>
</table>Domenica