<partinfo>BBa_R0062 AddReview 4</partinfo> ETH Zurich 2014

Characterization of the promoter's basal leakiness to LuxR in the absence of 3OC6-HSL

The amount of regulator [http://parts.igem.org/Part:BBa_C0062:Experience LuxR (BBa_C0062)] in the system was shown to influence the pLuxR promoter's basal expression or leakiness. By using the three different constitutive promoters [http://parts.igem.org/Part:BBa_J23100:Experience BBa_J23100], [http://parts.igem.org/Part:BBa_J23109:Experience BBa_J23109], and [http://parts.igem.org/Part:BBa_J23111:Experience BBa_J23111] for the production of LuxR we have measured this effect in terms of fluorescence.

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 one contained the commonly used p15A origin of replication, a kanamycin resistance gene, [http://parts.igem.org/Part:BBa_R0062 pLuxR (BBa_R0062)], and [http://parts.igem.org/Part:BBa_B0034 RBS (BBa_B0034)]followed by superfolder green fluorescent protein (sfGFP). In general, for spacer and terminator sequences [http://parts.igem.org/Part:BBa_B0040 BBa_B0040)] and [http://parts.igem.org/Part:BBa_B0015 BBa_B0015) were used, respectively.

Systems considered

Modeling leakiness

Results

Characterization of the promoter's sensitivity to 3OC6-HSL depending on LuxR concentration

Background information

Systems considered

Modeling promoter's sensitivity

Results

Characterization of two-level crosstalk on the promoter

Background information

System considered

Modeling crosstalk

First-order crosstalk

First Level crosstalk: LuxR binds to different HSL and activates the promoter

Second Level crosstalk: other regulatory proteins, like LasR, bind to their natural HSL substrate and activates the promoter

Second order crosstalk: Combination of both cross-talk levels

Other regulatory proteins, like LasR, bind to different HSL and activates the promoter

Results