Part:BBa_C0179:Experience

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UNIQdbe4a71899c0bcc2-partinfo-00000000-QINU

•••• ETH Zurich 2014

Characterization of two-order crosstalk Background information Here, we focus on the characterization of crosstalk of BBa_C0179 LasR with different AHLs and further crosstalk of LasR-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 pLas due to LasR binding to inducers different from 3OC12-HSL or pLas itself binding to a regulator-inducer pair different from LasR-3OC12-HSL. First Level crosstalk: LasR binds to different HSL and activates the promoter Plas In the conventional system 3OC12-HSL binds to its corresponding regulator, LasR, and activates the pLas promoter (figure 2, red). However, LasR can potentially also bind other AHLs and then activate pLas (figure 2, 3OC6-HSL in light blue and C4-HSL in green). Second Level crosstalk: LasR binds to 3OC12-HSL, its natural HSL, and activates different promoter 400px Second order crosstalk: Combination of both cross-talk levels Results Table 1 Crosstalk matrix for the regulator LasR (BBa_C0179) In all the measurements conducted to create this matrix the regulator LasR 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 LasR (with 95% confidence bounds) 3OC12-HSL 3OC6-HSL C4-HSL Plas a = 5.274 (0, 15.51) [a.u.] n = 11.67 (0, 3.763e8) Km = 1.396 (0, 1.504e7) [nM] b = 189 (162.5, 215.6) [a.u.] No crosstalk No crosstalk Plux a = 362.4 (158.2,566.6) [a.u.] n = 0.7898 (0.3241,1.256) Km = 1.269 (0, 2.548) [nM] b = 1.253e4 (1.085e4, 1.422e4) [a.u.] No crosstalk No crosstalk Prhl 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.] No crosstalk No crosstalk

Antiquity

This review comes from the old result system and indicates that this part did not work in some test.

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 RhlR (C0171) continuously in our system. BBa K575032

••••• iGEM Dundee 2014

Dundee iGEM 2014 used this lasR coding sequence to build two composite parts termed BBa_K1315009 and BBa_K1315010. These were designed as biosensors for Pseudomonas aeruginosa AutoInducer-1 (PAI-1), and were to be used in a bio-electronic device to improve diagnostics for Cystic Fibrosis patients. Details of experimental work are logged on the experience pages of BBa_K1315009 and BBa_K1315010.