ETH Zurich 2014

Induction by the Supernatant of a LuxI Producing Culture

The production of an inducer for the 3OC6-HSL and LuxR dependent promoter R0062 by LuxI could be shown using the supernatant of a culture containing E. coli cells constitutively expressing LuxI to induce the 3OC6-HSL sensor construct with the reporter sfGFP in another culture.

Background information

LuxI synthesizes 3OC6-HSL which can bind to LuxR. The LuxR/3OC6-HSL complex can induce the promoter R0062.

In this experiment we used E. coli TOP10 strain transformed with a plasmid containing a pBBR1 origin of replication and a tetracycline resistance gene, the constitutive promoter J23100, an optimized RBS for this genetic context and the gene for the 3OC6-HSL synthetase LuxI. The RBS was optimised using the Salis Lab RBS Calculator. This strain was used as AHL producer strain.

As sensor strain 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 pLuxR (BBa_R0062) within a [http://2014.igem.org/Team:ETH_Zurich/expresults#Riboregulators riboregulator system] 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 one of three promoters chosen from the Anderson promoter collection followed by luxR (BBa_C0062). A third plasmid containing the pBBR1 or and a tetracycline resistance was introduced, since the LuxI expressing AHL producer strain contained a tetracycline resistance an therefore the supernatant also contained tetracycline. The detailed construct designs and full sequences (piG0041, piG0065) are [http://2014.igem.org/Team:ETH_Zurich/lab/sequences available here].

Experimental Setup

The above described E. coli TOP10 AHL producer strain was grown in Lysogeny Broth (LB) containing tetracycline (10 μg/mL) to an OD₆₀₀ of 1.2 (37 °C, 220 rpm). The supernatant was harvested by centrifugation at 20'000 g for 5 minutes. Dilutions of 100%, 85%, 70%, 55%, 40%, 25%, 10%, 5%, 1%, and 0% (v/v) supernatant/LB were prepared and kanamycin, ampicillin, and tetracycline were added to a final concentration of 50 μg/mL, 200 μg/mL, and 10 μg/mL respectively. The dilutions were aliquoted in triplicates in microtiter plate format on 96-well plates (200 μL volume).

The above described E. coli TOP10 sensor strain was 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 the prepared supernatant/LB mixtures 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 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-curve for 200 min past induction.

Results

Figure 1: Dose-response curve LuxI producer supernatant The supernatant culture of E. coli cells in LB medium constitutively expressing LuxI was used in different dilutions with fresh LB medium as growth medium for E. coli cell containing a construct expressing sfGFP under a Plux promoter and constitutively expressing the regulator LuxR. Fluorescence/OD₆₀₀ of sfGFP was measured 6 h after inoculation.

Antiquity

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