Difference between revisions of "Part:BBa K1493501:Experience"
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[http://2015.igem.org/Team:Harvard_BioDesign Harvard BioDesign 2015] used the rhamnose promoter to express fimH adhesin in [https://parts.igem.org/wiki/index.php?title=Part:BBa_K1850000 BBa_K1850000] and BBa_K1850002-11. We were interested in maintaining very tight control over the expression of this protein to allow for control over the strength of binding and avoid gene-dosing issues. We selected the rhamnose promoter for this purpose, but were interested in a hypothesized characteristic of the promoter, which is that it is titratable on a colony level, but not on the level of a single cell. In other words, each single cell is either completely "on" or completely "off", but on a population level this ratio changes and appears titratable on the average. To that end we performed Fluorescence-activated cell sorting with using a flow cytometer, with variable inducer quantities, to characterize fluorescence for single cells. We found that the rhamnose promoter can either activate florescence completely or not at all, as shown in the bimodal fluorescence distribution of the samples: | [http://2015.igem.org/Team:Harvard_BioDesign Harvard BioDesign 2015] used the rhamnose promoter to express fimH adhesin in [https://parts.igem.org/wiki/index.php?title=Part:BBa_K1850000 BBa_K1850000] and BBa_K1850002-11. We were interested in maintaining very tight control over the expression of this protein to allow for control over the strength of binding and avoid gene-dosing issues. We selected the rhamnose promoter for this purpose, but were interested in a hypothesized characteristic of the promoter, which is that it is titratable on a colony level, but not on the level of a single cell. In other words, each single cell is either completely "on" or completely "off", but on a population level this ratio changes and appears titratable on the average. To that end we performed Fluorescence-activated cell sorting with using a flow cytometer, with variable inducer quantities, to characterize fluorescence for single cells. We found that the rhamnose promoter can either activate florescence completely or not at all, as shown in the bimodal fluorescence distribution of the samples: | ||
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<span class="harvardLogo"><a href="http://2015.igem.org/Team:Harvard_BioDesign"><img src="https://static.igem.org/mediawiki/2015/4/4c/Harvard_GFP_Rham.png" style="width:212px;height:144px;"/> | <span class="harvardLogo"><a href="http://2015.igem.org/Team:Harvard_BioDesign"><img src="https://static.igem.org/mediawiki/2015/4/4c/Harvard_GFP_Rham.png" style="width:212px;height:144px;"/> | ||
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This confirms the hypothesis that the rhamnose promoter is not titratable on a single cell level. We believe this information useful in understanding induction results for systems which depend on on close control of protein expression. | This confirms the hypothesis that the rhamnose promoter is not titratable on a single cell level. We believe this information useful in understanding induction results for systems which depend on on close control of protein expression. | ||
Revision as of 04:51, 25 September 2015
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[http://2015.igem.org/Team:Harvard_BioDesign Harvard BioDesign 2015] used the rhamnose promoter to express fimH adhesin in BBa_K1850000 and BBa_K1850002-11. We were interested in maintaining very tight control over the expression of this protein to allow for control over the strength of binding and avoid gene-dosing issues. We selected the rhamnose promoter for this purpose, but were interested in a hypothesized characteristic of the promoter, which is that it is titratable on a colony level, but not on the level of a single cell. In other words, each single cell is either completely "on" or completely "off", but on a population level this ratio changes and appears titratable on the average. To that end we performed Fluorescence-activated cell sorting with using a flow cytometer, with variable inducer quantities, to characterize fluorescence for single cells. We found that the rhamnose promoter can either activate florescence completely or not at all, as shown in the bimodal fluorescence distribution of the samples:
This confirms the hypothesis that the rhamnose promoter is not titratable on a single cell level. We believe this information useful in understanding induction results for systems which depend on on close control of protein expression.