Difference between revisions of "Part:BBa K1541009"
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===Usage and Biology===
 
===Usage and Biology===
  
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===Background===
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Expression of sfGFP is induced when [https://parts.igem.org/Part:BBa_C0171 RhlR (BBa_C0171)], bound to [[AHL|4C-HSL]], activates the promoter [https://parts.igem.org/Part:BBa_C0062 pRhl (BBa_R0071)]. The ''cis''-repressive element (crR12y, 'lock') inhibits the translation of sfGFP, since the [https://parts.igem.org/Part:BBa_B0034 RBS (BBa_B0034)] is blocked by secondary structures of the mRNA. The transcript of the ''trans''-activating element (taR12y, 'key', also under the control of [https://parts.igem.org/Part:BBa_C0062 pRhl (BBa_R0071)]) binds to the transcript of the cis-repressive element, hence the RBS is not blocked anymore. The two elements build a riboregulator ('key' and 'lock') that decreases leakiness of [https://parts.igem.org/Part:BBa_C0062 pRhl (BBa_R0071)].
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===Background Information===
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We used an ''E. coli'' TOP10 strain transformed with two medium copy plasmids (about 15 to 20 copies per plasmid and cell). The first plasmid contained the commonly used p15A origin of replication, a kanamycin resistance gene, and promoter [https://parts.igem.org/Part:BBa_C0062 pRhl (BBa_R0071)  followed by a ''cis''-repressed (crR12y) [https://parts.igem.org/Part:BBa_B0034 RBS (BBa_B0034)] and superfolder green fluorescent protein (sfGFP). The same plasmid contained the ''trans''-activating RNA, also under control of the promoter [https://parts.igem.org/Part:BBa_C0062 pRhl (BBa_R0071).In general, for spacer and terminator sequences the parts [https://parts.igem.org/Part:BBa_B0040 BBa_B0040] and [https://parts.igem.org/Part:BBa_B0015 BBa_B0015] were used, respectively. The second plasmid contained the pBR322 origin (pMB1), which yields a stable two-plasmid system together with p15A, an ampicillin resistance gene, and a (strong) promoter [https://parts.igem.org/Part:BBa_J23100 rhlR (BBa_J23100)] chosen from the [https://parts.igem.org/Promoters/Catalog/Anderson Anderson promoter collection] followed by [https://parts.igem.org/Part:BBa_C0171 rhlR (BBa_C0171)].
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The detailed regulator construct design and full sequences (piG0110) is [http://2014.igem.org/Team:ETH_Zurich/lab/sequences available here].
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===Experimental Set-Up===
 
===Experimental Set-Up===

Revision as of 13:19, 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.

Usage and Biology

Expression of sfGFP is induced when RhlR (BBa_C0171), bound to 4C-HSL, activates the promoter pRhl (BBa_R0071). The cis-repressive element (crR12y, 'lock') inhibits the translation of sfGFP, since the RBS (BBa_B0034) is blocked by secondary structures of the mRNA. The transcript of the trans-activating element (taR12y, 'key', also under the control of pRhl (BBa_R0071)) binds to the transcript of the cis-repressive element, hence the RBS is not blocked anymore. The two elements build a riboregulator ('key' and 'lock') that decreases leakiness of pRhl (BBa_R0071).

Background Information

We used an E. coli TOP10 strain transformed with two medium copy plasmids (about 15 to 20 copies per plasmid and cell). The first plasmid contained the commonly used p15A origin of replication, a kanamycin resistance gene, and promoter pRhl (BBa_R0071) followed by a cis-repressed (crR12y) [https://parts.igem.org/Part:BBa_B0034 RBS (BBa_B0034) and superfolder green fluorescent protein (sfGFP). The same plasmid contained the trans-activating RNA, also under control of the promoter pRhl (BBa_R0071).In general, for spacer and terminator sequences the parts [https://parts.igem.org/Part:BBa_B0040 BBa_B0040 and BBa_B0015 were used, respectively. The second plasmid contained the pBR322 origin (pMB1), which yields a stable two-plasmid system together with p15A, an ampicillin resistance gene, and a (strong) promoter rhlR (BBa_J23100) chosen from the Anderson promoter collection followed by rhlR (BBa_C0171). The detailed regulator construct design and full sequences (piG0110) is [http://2014.igem.org/Team:ETH_Zurich/lab/sequences available here].


Experimental Set-Up