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Team:Groningen/project characterisation promoters hybb
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=hybB promoter= | =hybB promoter= | ||
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| + | We have chosen the hybB Cold Shock Promoter ([http://partsregistry.org/Part:BBa_J45503 BBa_J45503]) as our system's input. A cold shock seemed to be a good input method, as it is easy to cool down the cells and heat them up again, thus allowing for short impulses of lower temperature that would facilitate short-term expression needed for a functional circuit. | ||
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| + | After cloning, we encountered problems with the activation of the PhybB promotor cloned upstream gfp, designed to measure the promoter activity ([http://partsregistry.org/Part:BBa_K607039 BBa_K607039]). Previous teams claim that the part is functional and that it is active in temperatures below 30 degrees. This was not our experience. The construct, despite having proper sequence and being present in the cells during the measurements (done in presence of antibiotic), did not show any activity neither in low temperature nor in anaerobic conditions, which should also activate it ([http://www.ncbi.nlm.nih.gov/pubmed/10537212?dopt=Abstract Richard DJ, 1999]). After obtaining the negative results, we have also tested two another strains: BW25113 and TOP10. In neither of them the promoter was active. | ||
<gallery> | <gallery> | ||
| - | File:hybb1.png|'''Figure 1.''' Fluorescence intensity measured in DH5alpha cells carrying a pSB1C3 plasmid with | + | File:hybb1.png|'''Figure 1.''' Fluorescence intensity measured in DH5alpha cells carrying a pSB1C3 plasmid with [http://partsregistry.org/Part:BBa_K607039 BBa_K607039] construct, growing at 27 degrees. |
| - | File:hybb2.png|'''Figure 2.''' Fluorescence intensity measured in | + | File:hybb2.png|'''Figure 2.''' Fluorescence intensity measured in BW25113 cells carrying a pSB1C3 plasmid with [http://partsregistry.org/Part:BBa_K607039 BBa_K607039] construct, growing at 27 degrees. |
| - | File:hybb3.png|'''Figure 3.''' Fluorescence intensity measured in TOP10 cells carrying a pSB1C3 plasmid with | + | File:hybb3.png|'''Figure 3.''' Fluorescence intensity measured in TOP10 cells carrying a pSB1C3 plasmid with [http://partsregistry.org/Part:BBa_K607039 BBa_K607039] construct, growing at 27 degrees. |
| - | File:hybb4.png|'''Figure 4.''' Fluorescence intensity measured in DH5alpha cells carrying a pSB1C3 plasmid with | + | File:hybb4.png|'''Figure 4.''' Fluorescence intensity measured in DH5alpha cells carrying a pSB1C3 plasmid with [http://partsregistry.org/Part:BBa_K607039 BBa_K607039] construct, growing at 37 degrees without oxygen. |
File:hybb5.png|'''Figure 5.''' Fluorescence intensity measured in wild-type cells, growing at 27 degrees. | File:hybb5.png|'''Figure 5.''' Fluorescence intensity measured in wild-type cells, growing at 27 degrees. | ||
</gallery> | </gallery> | ||
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| + | We believe that the problems with the promoter might be a result of a wrong sequence listed in the Parts Registry. [http://ecocyc.org/ECOLI/NEW-IMAGE?type=OPERON&object=TU00289 Ecocyc.org entry] shows that the promoter is much shorter than the version listed in the Parts Registry. Utilizing the proper, shorter sequence might cause that it will be possible to activate the promoter. | ||
{{FooterGroningen2011}} | {{FooterGroningen2011}} | ||
Latest revision as of 15:49, 21 September 2011
hybB promoter
We have chosen the hybB Cold Shock Promoter ([http://partsregistry.org/Part:BBa_J45503 BBa_J45503]) as our system's input. A cold shock seemed to be a good input method, as it is easy to cool down the cells and heat them up again, thus allowing for short impulses of lower temperature that would facilitate short-term expression needed for a functional circuit.
After cloning, we encountered problems with the activation of the PhybB promotor cloned upstream gfp, designed to measure the promoter activity ([http://partsregistry.org/Part:BBa_K607039 BBa_K607039]). Previous teams claim that the part is functional and that it is active in temperatures below 30 degrees. This was not our experience. The construct, despite having proper sequence and being present in the cells during the measurements (done in presence of antibiotic), did not show any activity neither in low temperature nor in anaerobic conditions, which should also activate it ([http://www.ncbi.nlm.nih.gov/pubmed/10537212?dopt=Abstract Richard DJ, 1999]). After obtaining the negative results, we have also tested two another strains: BW25113 and TOP10. In neither of them the promoter was active.
 Figure 1. Fluorescence intensity measured in DH5alpha cells carrying a pSB1C3 plasmid with [http://partsregistry.org/Part:BBa_K607039 BBa_K607039] construct, growing at 27 degrees. |
 Figure 2. Fluorescence intensity measured in BW25113 cells carrying a pSB1C3 plasmid with [http://partsregistry.org/Part:BBa_K607039 BBa_K607039] construct, growing at 27 degrees. |
 Figure 3. Fluorescence intensity measured in TOP10 cells carrying a pSB1C3 plasmid with [http://partsregistry.org/Part:BBa_K607039 BBa_K607039] construct, growing at 27 degrees. |
 Figure 4. Fluorescence intensity measured in DH5alpha cells carrying a pSB1C3 plasmid with [http://partsregistry.org/Part:BBa_K607039 BBa_K607039] construct, growing at 37 degrees without oxygen. |
 Figure 5. Fluorescence intensity measured in wild-type cells, growing at 27 degrees. |
We believe that the problems with the promoter might be a result of a wrong sequence listed in the Parts Registry. [http://ecocyc.org/ECOLI/NEW-IMAGE?type=OPERON&object=TU00289 Ecocyc.org entry] shows that the promoter is much shorter than the version listed in the Parts Registry. Utilizing the proper, shorter sequence might cause that it will be possible to activate the promoter.
