Difference between revisions of "Part:BBa K1893003"

Latest revision as of 20:43, 28 October 2016

Rhl receiver with GFP reporter (RhlR+pRhl+GFP)

This composite part is based on the Rhl quorum-sensing system from Pseudomonas aeruginosa. It consists of a Rhl receiver device and a GFP reporter that is activated in the presence of a C4-AHL signal.

Usage and Biology

Quorum sensing is a naturally occurring mechanism that certain strains of bacteria use to regulate gene expression in response to their population density. These bacteria secrete autoinducer signalling molecules, such as N-acyl homoserine lactones (AHLs), that bind to transcription factors to alter gene expression.

In this case, the constitutively expressed RhlR transcriptional regulator (BBa_C0171) is activated by the binding of C4-AHL, an AHL quorum signal. The activated RhlR regulator binds to a RhlR-inducible promoter, pRhl, upstream of a GFP reporter. As a result, GFP is expressed when the receiver device is induced with C4 AHL. We designed this part to characterize the activation range of the Rhl receiver device. We also characterised the cross-talk of the Rhl receiver device with other quorum-sensing systems, in order to determine its orthogonality.

Characterisation data

The activation range of RhlR by its cognate inducer (C4-AHL) was characterised in TOP10 E.Coli cells. Cells transformed with the Rhl response device were cultured to the exponential phase and treated with appropriate concentrations of C4-AHL in 96-well microplates. These induced cells were grown in the microplates and their fluorescence and absorbance values (OD 600) were monitored over time using a microplate reader. The reported values for the normalised fluorescence represents the values recorded 180 minutes after AHL induction. The normalised fluorescence was calculated by dividing fluorescence values by absorbance values and correcting for LB autofluorescence.

In order to characterise the orthogonality of the Rhl system, we measured absorbance and fluorescence of the cells in the plate reader after treating them with varying concentrations of 3 different AHL signals (3O-C6 AHL of the Lux system, 3O-C12 AHL of the Las system, and 3O-C14 AHL of the Cin system). This allowed us to determine whether these non-cognate AHLs were capable of activating the RhlR response protein, and therefore the level of crosstalk between the quorum sensing systems.

Figure 1. Characterisation of the Rhl response device (BBa_K1893003). (A) Transfer function curve of normalised fluorescence against cognate inducer C4-AHL concentrations. (B) Heat map of normalised fluorescence of RhlR-GFP system over a range of AHL concentrations: (i) Binding of RhlR-GFP to its cognate AHL (C4 AHL). (ii) Binding of RhlR-GFP to 3 non-cognate AHLs (3O-C6 AHL, 3O-C12 AHL, 3OH-C14 AHL). (C) Transfer function curves of normalised fluorescence against non-cognate inducer AHL (C4 AHL) concentrations to investigate inducer AHL crosstalk: (i) C6-AHL (3O-C6 AHL) of the Lux system (ii) C12-AHL (3O-C12 AHL) of the Las system (iii) C14-AHL (3O-C14 AHL) of the Cin system. Experiments were performed in E. coli Top10 cell strain cultured at 37°C. Normalised fluorescence was calculated by dividing fluorescent signal by cell density (OD600). Fluorescence measurements were recorded at 180 minutes. Reported values represent the mean normalised fluorescence value from 3 technical repeats and error bars represent standard deviation of these.

Analysis

The results from Figure 1A show that the concentration range of AHL (C4 AHL) required for the activation of the Rhl response device was 100nM - 100uM. Furthermore, it can be seen from Figure 1C that RhlR does not appear to be activated by 3O-C14 AHL, suggesting that the Rhl quorum system is orthogonal with Cin. It is also seen that RhlR only exhibits slight activity at high concentrations at 3O-C12 AHL. RhlR is seen to be significantly activated by 3O-C6 AHL, suggesting that the Rhl and Lux quorum systems are not orthogonal.

Sequence and Features