Part:BBa_J10050:Experience

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UNIQ93275c2cf0e52222-partinfo-00000000-QINU

•••• ETH Zurich 2014

Characterization of weak promoter (BBa_J23113) and weak RBS (BBa_B0031) 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 pLas (BBa_R0079) followed by RBS (BBa_B0034) and superfolder green fluorescent protein (sfGFP). 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 (BBa_J23100) chosen from the Anderson promoter collection followed by one of the three different regulators (LuxR, LasR, and RhlR) used in the experiments in order to quantify crosstalk with pRhl. The detailed regulator construct design and full sequences (piG0040, piG0041, piG0042, piG0060) are [http://2014.igem.org/Team:ETH_Zurich/lab/sequences available here]. In the following, we describe the experimental set-up and all the different levels of crosstalk we have assessed. Figure 1 Overview of possible crosstalk of the RhlR/pRhl system with three different AHLs. Usually, C4-HSL binds to its corresponding regulator, RhlR, and activates the pRhl promoter (green). However, RhlR may also bind 3OC12-HSL (red) or 3OC6-HSL (light blue) and then unintentionally activate pRhl. Second Level crosstalk: other regulatory proteins, like LuxR and LasR, bind to their natural AHL substrate and activate the pRhl promoter In the conventional system C4-HSL binds to its corresponding regulator, RhlR, and activates the pRhl promoter (Figure 2, green). However, pRhl can potentially be activate by other regulators (LuxR, LasR), binding their corresponding regulator (figure 2, 3OC6-HSL in light blue, 3OC12-HSL in red). This leads then to unwanted gene expression (crosstalk). Figure 2 Overview of possible crosstalk of the RhlR/pRhl system with two additional regulators (LuxR and LasR). Usually, RhlR together with inducer C4-HSL activate their corresponding promoter pRhl (green). However, pRhl may also be activated by the LuxR regulator together with 3OC6-HSL (light blue) or by the LasR regulator together with 3OC12-HSL (red). Second order crosstalk: Combination of both cross-talk levels The second order crosstalk describes unintended activation of pRhl by a mixture of both the levels described above. The regulator and inducer are being different from RhlR and C4-HSL, respectively, and at the same time they do not belong to the same module. For example, the inducer 3OC6-HSL (light blue), usually binding to the regulator LuxR, could potentially interact with LasR regulator (red) and together activate pRhl (green). This kind of crosstalk is explained in Figure 3. Figure 3 Overview of possible crosstalk of the pRhl promoter with both the regulator and inducer being unrelated to the promoter and each other. Usually, RhlR together with inducer C4-HSL activate their corresponding promoter pRhl (green). However, pRhl may also be activated by another regulator together with an unrelated inducer. For example, the inducer 3OC6-HSL (light blue) may interact with the LasR regulator (red) and together activate pRhl (green). Results Table 1 Crosstalk matrix for the promoter prhl (BBa_I14017) In this set of experiments the promoter pRhl was tested for potential crosstalk. In the top left position we observe the induction of pRhl by C4-HSL bound to the regulator RhlR. The switching behaviour was observed at a C4-HSL concentration of 1 μM. In the case of 3OC12-HSL binding the RhlR regulator and subsequently the promoter pRhlinsignificant crosstalk has been observed. Severe crosstalk was observed in the case of 3OC6-HSL binding the RhlR regulator followed by induction of pRhl. The transition occurred at a concentration of the inducer molecule of 1 μM but compared to the reference curve a lower value of fluorescence per OD was observed (1000 a.u.). Another case of crosstalk with the pRhl was detected with 3OC12-HSL binding to the corresponding LasR regulator followed by inducing the promoter pRhl. Here switching occurred at a concentration 1 nM of 3OC12-HSL and reached fluorescence per OD of 750 a.u.. This is approximately 0.5 fold the value of the fluorescence per OD shown by the reference curve indicated in green. Modeling crosstalk Each experimental data set was fitted to an Hill function using the Least Absolute Residual method. The fitting of the graphs was performed using the following equation : rFluo = the relative fluorescence (absolute measured fluorescence value over OD)[a.u.] a = basal expression rate [a.u.](“leakiness”) b = maximum expression rate [a.u.]("full induction") n = Hill coefficient (“cooperativity”) Km = Half-maximal effective concentration (“sensitivity”) [AHL] = AHL concentration [nM] Parameters of HillFunction for crosstalk with Prhl (with 95% confidence bounds) C4-HSL 3OC6-HSL 3OC12-HSL RhlR a = 178.4 (174.9, 182) [a.u.] n = 1.053 (0.9489, 1.157) Km = 1969 (1625, 2313) [nM] b = 1736 (1629, 1842) [a.u.] a = 169.1 (155.2, 182.9) [a.u.] n = 0.507 (0.2303, 0.7837) Km = 1.08e8(0, 2.681e10) [nM] b = 9.708e4 (0, 1.192e7) [a.u.] a = 162.8 (150.4, 175.1) [a.u.] n = 0.404 (0, 0.998) Km = 9.627e8 (0, 7.824e11) [nM] b = 2.537e4 (0, 8.109e6) [a.u.] LuxR No crosstalk No crosstalk No crosstalk LasR No crosstalk No crosstalk a = 149.3 (140.6, 158.1) [a.u.] n = 1.366 (0.808, 1.923) Km = 1.674 (1.259, 2.09) [nM] b = 628.9 (599, 658.7) [a.u.]

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