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

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.