Difference between revisions of "Part:BBa K1541009"
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<partinfo>BBa_K1541009 short</partinfo> | <partinfo>BBa_K1541009 short</partinfo> | ||
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| + | This riboregulated promoter construct contains the quorum sensing promoter [https://parts.igem.org/Part:BBa_I14017 BBa_I14017] which can be activated in presence of [https://parts.igem.org/Part:BBa_C0171:Experience RhlR (BBa_C0171)] and C4-HSL, the product of the enzyme [https://parts.igem.org/Part:BBa_C0170 RhlI (BBa_C0170)], succeeded by the gene for sfGFP. However, the promoter [https://parts.igem.org/Part:BBa_I14017 BBa_I14017] by itself shows some leakiness. Together with the ribogegulator 12<sup>[[Team:ETH_Zurich/project/references#refCallura|[18]]]</sup> the leakiness could be reduced. | ||
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[[File:ETH_Zurich_2014_LuxWith_and_WORibo.png|thumb|350px|right|'''Figure 1 Improved signal-to-noise ratio and decreased basal GFP expression (leakiness) due to the use of a riboregulator in combination with a quorum-sensing module.''' The fluorescence per OD<sub>600</sub> is shown for the LuxR-system with a complete riboregulator over an inducer-range of 10<sup>-13</sup> M to 10<sup>-5</sup> M (dashed, light blue). An incomplete riboregulator without the ''trans''-activator shows the expected reduced sensitivity towards the inducer (dark blue). As a reference, a system with a [https://parts.igem.org/Part:BBa_B0034 non-regulated RBS (BBa_B0034)] is shown (light blue). Data points are mean values of triplicate measurements in 96-well microtiter plates 200 min after induction ± standard deviation. For the full data set and kinetics please [http://2014.igem.org/Team:ETH_Zurich/contact contact] us or visit the [http://2014.igem.org/Team:ETH_Zurich/data/raw raw data] page.]] | [[File:ETH_Zurich_2014_LuxWith_and_WORibo.png|thumb|350px|right|'''Figure 1 Improved signal-to-noise ratio and decreased basal GFP expression (leakiness) due to the use of a riboregulator in combination with a quorum-sensing module.''' The fluorescence per OD<sub>600</sub> is shown for the LuxR-system with a complete riboregulator over an inducer-range of 10<sup>-13</sup> M to 10<sup>-5</sup> M (dashed, light blue). An incomplete riboregulator without the ''trans''-activator shows the expected reduced sensitivity towards the inducer (dark blue). As a reference, a system with a [https://parts.igem.org/Part:BBa_B0034 non-regulated RBS (BBa_B0034)] is shown (light blue). Data points are mean values of triplicate measurements in 96-well microtiter plates 200 min after induction ± standard deviation. For the full data set and kinetics please [http://2014.igem.org/Team:ETH_Zurich/contact contact] us or visit the [http://2014.igem.org/Team:ETH_Zurich/data/raw raw data] page.]] | ||
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[[File:ETH_Zurich_2014_LuxWith_and_WOSites.png|thumb|350px|right|'''Figure 2 Confirmation of the improved signal-to-noise ratio and decreased basal GFP expression (leakiness) due to the use of a riboregulator without (w/o) EcoRI and XbaI restriction sites in combination with a quorum-sensing module.''' The fluorescence per OD<sub>600</sub> is shown for the LuxR-system with an unchanged riboregulator (dashed, light blue) and a regulator with a changed sequence due to EcoRI and XbaI restriction site removal (dashed, dark blue). The inducer range covers 10<sup>-13</sup> M to 10<sup>-5</sup> M. As a reference, a system with a [https://parts.igem.org/Part:BBa_B0034 non-regulated RBS (BBa_B0034)] is shown (light blue). Data points are mean values of triplicate measurements in 96-well microtiter plates 200 min after induction ± standard deviation. For the full data set and kinetics please [http://2014.igem.org/Team:ETH_Zurich/contact contact] us or visit the [http://2014.igem.org/Team:ETH_Zurich/data/raw raw data] page.]] | [[File:ETH_Zurich_2014_LuxWith_and_WOSites.png|thumb|350px|right|'''Figure 2 Confirmation of the improved signal-to-noise ratio and decreased basal GFP expression (leakiness) due to the use of a riboregulator without (w/o) EcoRI and XbaI restriction sites in combination with a quorum-sensing module.''' The fluorescence per OD<sub>600</sub> is shown for the LuxR-system with an unchanged riboregulator (dashed, light blue) and a regulator with a changed sequence due to EcoRI and XbaI restriction site removal (dashed, dark blue). The inducer range covers 10<sup>-13</sup> M to 10<sup>-5</sup> M. As a reference, a system with a [https://parts.igem.org/Part:BBa_B0034 non-regulated RBS (BBa_B0034)] is shown (light blue). Data points are mean values of triplicate measurements in 96-well microtiter plates 200 min after induction ± standard deviation. For the full data set and kinetics please [http://2014.igem.org/Team:ETH_Zurich/contact contact] us or visit the [http://2014.igem.org/Team:ETH_Zurich/data/raw raw data] page.]] | ||
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[[File:ETH_Zurich_2014_RhlWith_and_WORibo.png|thumb|350px|left|'''Figure 3 Reduced basal GFP expression (leakiness) due to the use of a riboregulator in combination with a quorum-sensing module.''' The fluorescence per OD<sub>600</sub> is shown for the Rhl-system ([https://parts.igem.org/Part:BBa_I14017 pRhl (BBa_I14017)] and [https://parts.igem.org/Part:BBa_C0171 RhlR (BBa_C0171)]) with a riboregulator over an inducer-range of 10<sup>-4</sup> nM to 10<sup>4</sup> nM (dashed, light green). A riboregulated Rhl-system with removed EcoRI and XbaI restriction sites shows the expected reduced basal GFP expression and a reduced sensitivity towards the inducer (black). As a reference, the Rhl-system without a riboregulator is shown (light green, [https://parts.igem.org/Part:BBa_B0034 non-regulated RBS (BBa_B0034)]). Data points are mean values of triplicate measurements in 96-well microtiter plates 200 min after induction ± standard deviation. For the full data set and kinetics please [http://2014.igem.org/Team:ETH_Zurich/contact contact] us or visit the [http://2014.igem.org/Team:ETH_Zurich/data/raw raw data] page.]] | [[File:ETH_Zurich_2014_RhlWith_and_WORibo.png|thumb|350px|left|'''Figure 3 Reduced basal GFP expression (leakiness) due to the use of a riboregulator in combination with a quorum-sensing module.''' The fluorescence per OD<sub>600</sub> is shown for the Rhl-system ([https://parts.igem.org/Part:BBa_I14017 pRhl (BBa_I14017)] and [https://parts.igem.org/Part:BBa_C0171 RhlR (BBa_C0171)]) with a riboregulator over an inducer-range of 10<sup>-4</sup> nM to 10<sup>4</sup> nM (dashed, light green). A riboregulated Rhl-system with removed EcoRI and XbaI restriction sites shows the expected reduced basal GFP expression and a reduced sensitivity towards the inducer (black). As a reference, the Rhl-system without a riboregulator is shown (light green, [https://parts.igem.org/Part:BBa_B0034 non-regulated RBS (BBa_B0034)]). Data points are mean values of triplicate measurements in 96-well microtiter plates 200 min after induction ± standard deviation. For the full data set and kinetics please [http://2014.igem.org/Team:ETH_Zurich/contact contact] us or visit the [http://2014.igem.org/Team:ETH_Zurich/data/raw raw data] page.]] | ||
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===Usage and Biology=== | ===Usage and Biology=== | ||
| − | <!-- | + | ===Background=== |
| − | <span class='h3bb'>Sequence and Features</span> | + | |
| + | ===Experimental Set-UP=== | ||
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| + | <!--<span class='h3bb'>Sequence and Features</span> | ||
<partinfo>BBa_K1541009 SequenceAndFeatures</partinfo> | <partinfo>BBa_K1541009 SequenceAndFeatures</partinfo> | ||
Revision as of 12:51, 23 October 2014
sfGFP under promoter P(Rhl) with riboregulator RR12
This riboregulated promoter construct contains the quorum sensing promoter BBa_I14017 which can be activated in presence of RhlR (BBa_C0171) and C4-HSL, the product of the enzyme RhlI (BBa_C0170), succeeded by the gene for sfGFP. However, the promoter BBa_I14017 by itself shows some leakiness. Together with the ribogegulator 12[18] the leakiness could be reduced.
Figure 1 Improved signal-to-noise ratio and decreased basal GFP expression (leakiness) due to the use of a riboregulator in combination with a quorum-sensing module. The fluorescence per OD600 is shown for the LuxR-system with a complete riboregulator over an inducer-range of 10-13 M to 10-5 M (dashed, light blue). An incomplete riboregulator without the trans-activator shows the expected reduced sensitivity towards the inducer (dark blue). As a reference, a system with a non-regulated RBS (BBa_B0034) is shown (light blue). Data points are mean values of triplicate measurements in 96-well microtiter plates 200 min after induction ± standard deviation. For the full data set and kinetics please [http://2014.igem.org/Team:ETH_Zurich/contact contact] us or visit the [http://2014.igem.org/Team:ETH_Zurich/data/raw raw data] page.
Figure 2 Confirmation of the improved signal-to-noise ratio and decreased basal GFP expression (leakiness) due to the use of a riboregulator without (w/o) EcoRI and XbaI restriction sites in combination with a quorum-sensing module. The fluorescence per OD600 is shown for the LuxR-system with an unchanged riboregulator (dashed, light blue) and a regulator with a changed sequence due to EcoRI and XbaI restriction site removal (dashed, dark blue). The inducer range covers 10-13 M to 10-5 M. As a reference, a system with a non-regulated RBS (BBa_B0034) is shown (light blue). Data points are mean values of triplicate measurements in 96-well microtiter plates 200 min after induction ± standard deviation. For the full data set and kinetics please [http://2014.igem.org/Team:ETH_Zurich/contact contact] us or visit the [http://2014.igem.org/Team:ETH_Zurich/data/raw raw data] page.
Figure 3 Reduced basal GFP expression (leakiness) due to the use of a riboregulator in combination with a quorum-sensing module. The fluorescence per OD600 is shown for the Rhl-system (pRhl (BBa_I14017) and RhlR (BBa_C0171)) with a riboregulator over an inducer-range of 10-4 nM to 104 nM (dashed, light green). A riboregulated Rhl-system with removed EcoRI and XbaI restriction sites shows the expected reduced basal GFP expression and a reduced sensitivity towards the inducer (black). As a reference, the Rhl-system without a riboregulator is shown (light green, non-regulated RBS (BBa_B0034)). Data points are mean values of triplicate measurements in 96-well microtiter plates 200 min after induction ± standard deviation. For the full data set and kinetics please [http://2014.igem.org/Team:ETH_Zurich/contact contact] us or visit the [http://2014.igem.org/Team:ETH_Zurich/data/raw raw data] page.