Part:BBa_C0171:Experience

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UNIQ1d0f693df2e055ef-partinfo-00000000-QINU

•••• ETH Zurich 2014

Characterization of two-order crosstalk on the promoter Background information Here, we focus on the characterization of crosstalk of BBa_C0171 RhlR with different AHLs and further crosstalk of RhlR-C4-HSL with the three promoters - pLux, pLas, and pRhl. In the following, we describe all the different levels of crosstalk we have assessed. First-order crosstalk In the first order crosstalk section we describe crosstalk of pRhl due to RhlR binding to inducers different from C4-HSL or pRhl itself binding a regulator-inducer pair different from RhlR-C4-HSL. First Level crosstalk: RhlR binds to different HSL and activates the promoter In the conventional system C4-HSL binds to its corresponding regulator, RhlR, and activates the pRhl promoter (figure 2, green). However, RhlR can potentially also bind other AHLs and then activate pRhl (figure 2, 3OC12-HSL in red and 3OC6-HSL in blue). Second Level crosstalk: other regulatory proteins, like LuxR, bind to their natural HSL substrate and activates the promoter Second order crosstalk: Combination of both cross-talk levels Results Table 1 Crosstalk matrix for the regulator LasR (BBa_C0171) In all the measurements conducted to create this matrix the regulator RhlR was the basis and was induced in six different variations shown. 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 Hill function for crosstalk with RhlR (with 95% confidence bounds) C4-HSL 3OC6-HSL 3OC12-HSL Prhl 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.] Plux a = 309.6 (283.6, 335.6) [a.u.] n = 0.741 (0.6463, 0.8357) Km = 2738 (1411, 4064) [nM] b = 1.22e4 (1.045e4,1.45e4) [a.u.] a = 313.2 (296.8, 329.5) [a.u.] n = 0.9427 (0.7565, 1.129) Km = 1210 (565, 1855) [nM] b = 8361 (7001, 9722) [a.u.] No crosstalk Plas No crosstalk No crosstalk No crosstalk

Antiquity

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UNIQ1d0f693df2e055ef-partinfo-00000003-QINU

No review score entered. Northwestern 2011

The 2011 Northwestern iGEM team used this part as a part of our Pseudomonas Aeruginosa biosensor. We were able to successfully express LasR (C0171) continuously in our system. BBa K575024