Part:BBa_K1541009

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.

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).

Figure 1 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.

Figure 2 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 3 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.

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) version of 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 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]. As controls, the non-regulated construct was included (see figures 1 - 3), or only the cis-repressor used (figure 2). In addition, the initially used riboregulator sequences^[18] contained forbidden restriction sites (EcoRI and XbaI). The removal of the restriction sites achieved by blunting and ligation (Klenow and T4 DNA polymerase) had no significant influence on the result, as compared to the original riboregulator sequence, however the sensitivity was slightly reduced (see figures 1 and 3). For figures 2 and 3 the same construct with pLux (BBa_R0062) instead of pRhl (BBa_R0071) was used. The submission of this new part is in preparation.

Experimental Set-Up

The above described E. coli TOP10 strains were grown overnight in Lysogeny Broth (LB) containing kanamycin (50 μg/mL) and ampicillin (200 μg/mL) to an OD₆₀₀ of about 1.5 (37 °C, 220 rpm). As a reference, a preculture of the same strain lacking the sfGFP gene was included for each assay. The cultures were then diluted 1:40 in fresh LB containing the appropriate antibiotics and measured in triplicates in microtiter plate format on 96-well plates (200 μL culture volume) for 10 h at 37 °C with a Tecan infinite M200 PRO plate reader (optical density measured at 600 nm; fluorescence with an excitation wavelength of 488 nm and an emission wavelength of 530 nm). After 200 min we added the following concentrations of inducers (3OC6-HSL, 3OC12-HSL, and C4-HSL): 10^-4 nM and 10⁴ nM (from 100 mM stocks in DMSO). Attention: All the dilutions of 3OC12-HSL should be made in DMSO in order to avoid precipitation. In addition, in one triplicate only H₂O was added as a control. From the the obtained kinetic data, we calculated mean values and plotted the dose-response-curves for 200 min past induction.

Sequences

trans-activator RNA sequence: Sequence length = 75. 22 A's, 15 C's, 13 G's, 25 U/T's

5' uggguuuagg uccuccacua accaucacca ccaauuacuu uuaauugaau gaugauggua uauagagauc gaucu 3'

cis-repressor RNA sequence (including the RBS, uppercase letters): Sequence length = 56. 18 A's, 9 C's, 12 G's, 17 U/T's

5' gaauuaauuc uaccauucac cucuuggauu uggguauuAA AGAGGAGAAA gguacc 3'

Figure 4 For questions or comments, please [http://2014.igem.org/Team:ETH_Zurich/contact contact]. Predictions of the secondary structure of RNA were conducted with [http://mfold.rna.albany.edu/?q=mfold/RNA-Folding-Form the mfold web server] and [http://mfold.rna.albany.edu/?q=DINAMelt the DINAMelt web server].