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 the regulator LuxR. The LuxR/3OC6-HSL complex can then induce the promoter pLux).

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 from the Anderson collection, an optimized RBS for this genetic context and the gene for the 3OC6-HSL synthetase LuxI. The RBS was optimized using the [http://salis.psu.edu/software/RBSLibraryCalculatorSearchMode Salis Lab RBS Calculator]. This strain was used as a 3OC6-HSL producer strain. The detailed construct design and full sequence (piG0050max) is [http://2014.igem.org/Team:ETH_Zurich/lab/sequences available here].

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 pLux 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. A third plasmid containing the pBBR1 origin and a tetracycline resistance was introduced, since the LuxI expressing 3OC6-HSL-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), ampicillin (200 μg/mL), and tetracycline (10 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). From the the obtained kinetic data, we calculated mean values and plotted the dose-response-curve for 360 min past inoculation.

Results

It could be shown that the supernatant of the LuxI producer strain can induce the LuxR/pLux sensor strain. The ON/OFF ratio between 0% and 100% supernatant is approximately 33. The increasing fluorescence per OD₆₀₀ with increasing proportion of supernatant is shown in Figure 1.

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. Measurements were conducted in triplicates. The squares depict the mean value and the bars represent +/- the standard deviation of the measured triplicates.

Antiquity

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