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Team:Marburg/Project:lightcontrol
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{{: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. | {{: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. | ||
| - | Because plants and algae use sunlight as 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). 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 | + | Because plants and algae use sunlight as 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). 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 excitation wavelengths (i.e. green (571 nm)-, blue (471 nm)-, red (673 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. |
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| - | A ''P. tricornutum'' culture was grown for seven days under specific light conditions and afterwards for four 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 | + | A ''P. tricornutum'' culture was grown for seven days under specific light conditions and afterwards for four 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). |
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. | 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. | ||
Revision as of 19:08, 4 October 2013
Characterizing a new light inducible promotor
Because plants and algae use sunlight as 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). 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 excitation wavelengths (i.e. green (571 nm)-, blue (471 nm)-, red (673 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.
A P. tricornutum culture was grown for seven days under specific light conditions and afterwards for four days in darkness to minimize the amount of existing GFP in P. tricornutum (see the protocol). The cell density was adjusted to an optical density of 0,22 and GFP was quantified by Western Blot analysis. 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).
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.
