Team:Wash U/Biological Parts
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[[Image:Slide2.jpg|480px|left]] The first part of our characterization begins with the puc promoter. The puc promoter is what turns on the entire system naturally in ''Rhodobacter sphaeroides''. The puc promoter is what ultimately controls the number of LH2 light harvesting complexes, which is how our system will yield an increase in photosynthetic efficiency. It is important that we are able to compare the transcription rate of the puc promoter in the two systems so that we can determine exactly how much efficiency is gained by adding a red light sensor. By attaching Green Fluorescent Protein (GFP) to the promoter we can quantify the rate of transcription by measuring the emittance of green light using a fluorescence spectrophotometer. We would expect to see more fluorescence with more transcription and vis versa. | [[Image:Slide2.jpg|480px|left]] The first part of our characterization begins with the puc promoter. The puc promoter is what turns on the entire system naturally in ''Rhodobacter sphaeroides''. The puc promoter is what ultimately controls the number of LH2 light harvesting complexes, which is how our system will yield an increase in photosynthetic efficiency. It is important that we are able to compare the transcription rate of the puc promoter in the two systems so that we can determine exactly how much efficiency is gained by adding a red light sensor. By attaching Green Fluorescent Protein (GFP) to the promoter we can quantify the rate of transcription by measuring the emittance of green light using a fluorescence spectrophotometer. We would expect to see more fluorescence with more transcription and vis versa. | ||
<br><br><br><br><br><br><br><br><br><br><br>[[Image:Slide3.jpg|480px|left]] The next step in our characterization of our synthetic red light response system is to analyze the phosphorylation of ompR in ''Rhodobacter sphaeroides''. In our final system, we only want puc genes to be transcribed and expressed via the phosphorylation of ompR, not simply the puc promoter as it naturally occurs. By placing the ompR coding region downstream of the red light sensor and upstream of a terminator our modified system controls expression of the puc genes by the red light sensor in addition to the puc promoter. It should be impossible for the puc promoter to directly cause the transcription of puc genes due to the terminator, but instead, the puc genes must be activated via ompR phosphorylation. The end target of ompR transcription is the ompC promoter, located directly upstream of the puc genes. Placing GFP behind ompC tells us how often the promoter is transcribed and how often ompR is phoshorylated. | <br><br><br><br><br><br><br><br><br><br><br>[[Image:Slide3.jpg|480px|left]] The next step in our characterization of our synthetic red light response system is to analyze the phosphorylation of ompR in ''Rhodobacter sphaeroides''. In our final system, we only want puc genes to be transcribed and expressed via the phosphorylation of ompR, not simply the puc promoter as it naturally occurs. By placing the ompR coding region downstream of the red light sensor and upstream of a terminator our modified system controls expression of the puc genes by the red light sensor in addition to the puc promoter. It should be impossible for the puc promoter to directly cause the transcription of puc genes due to the terminator, but instead, the puc genes must be activated via ompR phosphorylation. The end target of ompR transcription is the ompC promoter, located directly upstream of the puc genes. Placing GFP behind ompC tells us how often the promoter is transcribed and how often ompR is phoshorylated. | ||
- | <br><br><br><br><br><br><br><br><br>[[Image:Slide4.jpg|480px|left]] Part three of our characterization measures the effectiveness of the red light sensor in | + | <br><br><br><br><br><br><br><br><br>[[Image:Slide4.jpg|480px|left]] Part three of our characterization measures the effectiveness of the red light sensor in downregulating the phosphorylation of ompR. This setup is identical to that of part two except we have introduced the red light sensor. Now, the rate of ompR autophosphorylation will be halted by binding to a domain on the light-activated EnvZ kinase analogue. GFP is still attached to the end product, the ompC promoter. By comparing the fluorescence of GFP in this scenario compared with the second scenario the decrease in rate of phosphorylation should be apparent due to the activity of the red light sensor. |
<br><br><br><br><br><br><br><br><br><br><br><br><br>[[Image:Slide5.jpg|480px|left]] This is the final construct that will be our actual functioning model in ''Rhodobacter sphaeroides''. This finished product will be compared to the wild type over various intensities of light and cell culture densities can be compared to see which strain, the wild type or the modified strain with the red light sensor was more efficient in harvesting light with varying intensities. | <br><br><br><br><br><br><br><br><br><br><br><br><br>[[Image:Slide5.jpg|480px|left]] This is the final construct that will be our actual functioning model in ''Rhodobacter sphaeroides''. This finished product will be compared to the wild type over various intensities of light and cell culture densities can be compared to see which strain, the wild type or the modified strain with the red light sensor was more efficient in harvesting light with varying intensities. | ||
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Revision as of 20:34, 20 July 2009