Difference between revisions of "Part:BBa K1541009"

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sfGFP under promoter P(Rhl) with riboregulator RR12

This riboregulated promoter construct contains the quorum sensing promoter pRhl (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 superfolder green fluorecent protein (sfGFP). However, the promoter BBa_I14017 by itself shows leakiness. Together with the ribogegulator 12^{[http://www.pnas.org/content/109/15/5850.abstract|1]} the leakiness could be reduced. This riboregulator consists of two parts, the cis-repressor blocking the ribosome binding site (RBS) by folding into an inaccessible secondary structure ('lock') and a trans-activator RNA opening this structure and making the RBS accessible ('key').

Usage and Biology

Expression of sfGFP is induced when RhlR (BBa_C0171), bound to C4-HSL, activates the promoter pRhl (BBa_R0071). The cis-repressor element (crR12, 'lock') inhibits the translation of sfGFP, since the RBS (BBa_B0034) is blocked by secondary structures of the folded mRNA. The transcript of the trans-activating element (taR12, 'key', also under the control of pRhl (BBa_R0071)) binds to the transcript of the cis-repressive element, changing the secondary structure and as a result the RBS is not blocked anymore (for details, see Sequences). The two elements build a riboregulator ('key' and 'lock') that decreases the leakiness of pRhl (BBa_R0071). The systems functionality is depicted in figure 1.

Figure 1 Overview of the riboregulator construct together with superfolder green fluorescent protein (sfGFP) and the RhlR/pRhl system. First, a defined amount of the incoming signal molecule C4-HSL binds to RhlR. After dimerization, the complex binds to its corresponding promoter pRhl. Both, the trans-activator (taR12) and the cis-repressor (crR12) together with superfolder green fluorecent protein (sfGFP), are under control of pRhl. Upon induction with RhlR/C4-HSL, both are transcribed simultaneously and the taR12 (key symbol) opens up the crR12 sequence (lock) including the ribosome binding site of the sfGFP mRNA. This enables translation and sfGFP production (left-hand side). Low transcription (leakiness) gives only small concentrations of mRNA, as a result cis-repressor and trans-activator structures do not encounter each other and the translation is not enabled (right-hand side).

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 (crR12) version of RBS (BBa_B0034) and superfolder green fluorescent protein (sfGFP). The same plasmid contained the trans-activating RNA (taR12), 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 the constitutive promoter BBa_J23100 chosen from the Anderson promoter collection followed by rhlR (BBa_C0171). The detailed regulator construct design and full sequences (piG0110) are [http://2014.igem.org/Team:ETH_Zurich/lab/sequences available here]. As controls, the non-regulated construct was included (see figures 2 - 4), or only the cis-repressor used (figure 3). In addition, the initially used riboregulator sequences^{[http://www.pnas.org/content/109/15/5850.abstract [1]]} 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 2 and 4). For figures 2 and 3 the same construct with pLux (BBa_R0062) instead of pRhl (BBa_R0071) was used with RR12y^{[http://www.pnas.org/content/109/15/5850.abstract [1]]}.

Figure 2 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 3 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 4 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.

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 (taR12) sequence: Sequence length = 75. 25 A's, 13 C's, 15 G's, 22 U/T's

5' ACCCAAAUCC AGGAGGUGAU UGGUAGUGGU GGUUAAUGAA AAUUAACUUA CUACUACCAU AUAUCUCUAG CUAGA 3'

cis-repressor RNA sequence (crR12, including the RBS in 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'

The predicted RNA structures are shown in figure 5 and figure 6 for the cis-repressed RNA (crR12) sequence and the trans-activator RNA (taR12) sequence ([http://mfold.rna.albany.edu/?q=mfold/RNA-Folding-Form mfold web server]), repectively. In a addition, a prediction of the hybridization of taR12 with crR12 including the accessible RBS is shown in figure 7 ([http://mfold.rna.albany.edu/?q=DINAMelt DINAMelt web server]).

Figure 5 Prediction of the cis-repressed RNA structure. The 'locked' ribosome binding site (RBS) is indicated with uppercase letters in light blue. Predictions of the secondary structure of RNA were conducted with [http://mfold.rna.albany.edu/?q=mfold/RNA-Folding-Form the mfold web server] .

Figure 6 Prediction of the trans-activator RNA structure. Predictions of the secondary structure of RNA were conducted with [http://mfold.rna.albany.edu/?q=mfold/RNA-Folding-Form the mfold web server].

Figure 7 Prediction of the structure of trans-activator RNA together with the cis-repressed RNA . The now accessible ribosome binding site (RBS) is indicated with uppercase letters in light blue. Predictions of the secondary structure of RNA were conducted with [http://mfold.rna.albany.edu/?q=DINAMelt the DINAMelt web server].

References

[http://www.pnas.org/content/109/15/5850.abstract [1]] Callura, J. M., Cantor, C. R., Collins, J. J., Genetic switchboard for synthetic biology applications, PNAS, 2012