Part:BBa_C0179:Experience

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Process measurement characterisation of LasR by Shanghaitech iGEM 2017

Group: Shanghaitech 2017

The AHL receiver LasR from P.aeruginosa activates expression of GFP protein in response to 3OC12-HSL.

This Receiver can be easily replaced by other AHL receivers in our collection. A full collection could be found in: http://2017.igem.org/Team:Shanghaitech/Library

Fluorescent Response to cognate 3OC12-HSL

• To test this part, we used standard 3OC12HSL (HSL produced by LasI in P.aeruginosa) to determine the response curve.

Fig. 1 LasR-pLas-GFP‘s response to cognate 3OC12HSL

Orthogonality test against non-cognate inducers

• We have characterized crosstalk response of LasR to several non-cognate AHLs:

Fig. 2 Orthogonality test of LasR-pLas-GFP (i):Fluorescent response to cognate and non-cognate AHLs (ii)Dose-Response curves for cognate and non-cognate AHLs (iii-vi)Fluorescent response to non-cognate AHLs in compared with 3OC12-HSL

Furthermore, we test it under fluorescence microscope. Fig. 3 shows four testing samples.

Fig. 3 Orthogonality test under fluorescence microscope a) Interlab Study Test Device 1 BBa_J364000 b) Las molecule 3OC12-HSL as inducer c) Tra molecule 3OC8-HSL as inducer d) Rpa molecule Coumaroyl-HSL as inducer

It has shown that LasR is sensitive to it's cognate HSL and has obvious crosstalk with 3OC8-HSL in Tra System in relatively high concentration.

Usages in our Project

• We developed a measurement protocol using the fluorescent protein coupled AHL receiver germs to measure the actual AHLs concentration in high precision and sensitivity in compared with LC-MS.

AHL receiver from P.aeruginosa, actives expression of GFP protein.

User Reviews

UNIQ6c9821d347aa92a9-partinfo-00000001-QINU

•••• ETH Zurich 2014

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 one out of three cell-cell signaling promoters (pLux, pLas, or pRhl) 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 LasR (BBa_C0179). The detailed construct designs and full sequences (piG0040, piG0058, piG0059,piG0060) are [http://2014.igem.org/Team:ETH_Zurich/lab/sequences available here]. 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. Characterization of crosstalk Background information Here, we focus on the characterization of crosstalk of regulator LasR and promoter pLas with different AHLs and further crosstalk of LasR and inducer C4-HSL with the three promoters - pLux, pLas, and pRhl. Also we describe a combination of the two before mentioned types of crosstalk. 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 AHLs and activates the promoter pLas In the conventional system 3OC12-HSL binds to its corresponding regulator, LasR, and activates the pLas promoter (Figure 1, red). However, LasR can potentially also bind other AHLs and then activate pLas (Figure 1, 3OC6-HSL in light blue and C4-HSL in green). Figure 1 Overview of possible crosstalk of the LasR/pLas system with three different AHLs. Usually, 3OC12-HSL binds to its corresponding regulator, LasR, and activates the pLas promoter (red). However, LasR may also bind 3OC6-HSL (light blue) or C4-HSL (green) and then unintentionally activate pLas. Second Level crosstalk: LasR binds to 3OC12-HSL, its natural AHL, and activates promoters different from pLas In the conventional system 3OC12-HSL binds to its corresponding regulator, LasR, and activates the pLas promoter (Figure 2 middle, red). However, LasR can potentially also bind to additional cell-cell signaling promoters (Figure 2, pLux in light blue and pRhl in green) and then activate genes different from the pLas regulated gene of interest . Figure 2 Overview of possible crosstalk of the regulator LasR with three different cell-cell signaling promoters. Usually, 3OC12-HSL binds to its corresponding regulator, LasR, and activates the pLas promoter (red). However, LasR may also bind pLux (light blue) or pRhl (green) and then unintentionally activate 'off-target' gene expression. Second order crosstalk: Combination of both cross-talk levels The second order crosstalk describes unintended activation of a cell-cell signaling promoter by a mixture of both the levels described above. The regulator LasR binds an inducer different from 3OC12-HSL and then activates a promoter different from pLas. For example, the inducer 3OC6-HSL (light blue), usually binding to the regulator LuxR, could potentially interact with the LasR regulator (red) and together activate the promoter 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 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. Colored curves indicate the coventional inducer/regulator/promoter combination (e.g. 3OC12-HSL/LasR/pLas in red), black curves indicate crosstalk. 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.