Team:Calgary/Lab

Overview
Updates

Circuits

• Signalling Circuit
• Reporter Circuit
• Mutant Circuits
• Response Circuit

Quorum Sensing
Chassis Selection

Protocol & Safety
Notebook & Parts

As the fourth stop of our tour, and the first of the project segments, we have the wet lab portion: Biofilms & Bacterial Chatter. Here, you can read about the goals of the lab portion, and how we established our signalling system. You can also read about our application of quorum sensing through quorum quenching in the breakdown and prevention of biofilms. Our protocols can also be found here. Once you've read about our signalling system, you can head over to the next stop of our tour HERE for an exploration of characterizing this system.

The wetlab aspect for our project aims to engineer and characterize a novel Autoinducer-2 (AI-2) signalling system in E. coli for the study of quorum sensing in Gram-negative bacteria. Quorum sensing is a process where microorganisms use molecules to monitor their own population density and communicate with different species. Different applications have been developed through quorum sensing, including induction of virulence, biofilm formation and genetic competence. Our team decided to choose this signalling system because it has been shown that over 50% of bacteria whose genomes are sequenced to date are able to make this specific signal for pathogenic activity.

In Vibrio harveyi, the quorum sensing signal is induced by a molecule called AI-2. AI-2 is bound to the protein LuxP in the periplasm, and the LuxP-AI-2 complex interacts with another membrane-bound sensor histidine kinase, LuxQ. At low cell density, with low amounts of autoinducers, LuxQ sensor acts as a kinase, autophosphorylates and transfers the phosphate to the cytoplasmic protein LuxU. LuxU transfers the phosphate to the DNA binding response regulator protein LuxO. Phosphorylated LuxO with a transcription factor σ54, activates the transcription of genes encoding five regulatory small RNAs (sRNAs) termed Qrr1-5. The sRNAs bind to and destabilize the mRNA encoding the transcriptional activator, LuxR4, which is needed to activate the transcription of the luciferase operon, luxCDABE. Therefore, when there is a low cell density, the Lux R mRNA is degraded, thus the bacteria do not express bioluminescence. At higher cell density, when AI-2 concentration reaches the threshold, LuxQ sensor switches from a kinase to a phosphatase and removes the phosphate from Lux O via Lux U. Unphosphorylated LuxO cannot induce the expression of the sRNA, which in turn allows translation of LuxR mRNA, the production of Lux R and the expression of bioluminescence.

As a part of iGEM 2008 project, we have cloned, sequenced and standardized genes involved in the AI-2 signalling system in V. harveyi (LuxPQ, BBa_K131015; LuxOU, BBa_K131016; Pqrr4, BBa_K131017; LuxO D47E, BBa_K131022; LuxO D47A, BBa_K131023)6,7.

We will be engineering the complete AI-2 Signalling Circuit following Biobrick cloning methods. In order to accomplish this goal we need to:

1. Engineer variable σ70 promoter (Δσ70)
2. Clone the engineered Δσ70 promoter in front of the luxPQ construct.
3. Test robustness of the signalling system.

The major obstacle of this part of the project is ensuring that we have appropriate expression levels of the LuxP and LuxQ proteins. The creation of the engineered Δσ70 promoter driving the expression of LuxPQ will provide us with adequate levels of LuxP and LuxQ for efficient AI-2 signalling. In addition, this promoter will allow us to examine the robustness of AI-2 signalling with respect to LuxP and LuxQ levels.

An appropriate reporter circuit is designed to test to test the proper functioning of the Signalling Circuit. To this end, a reporter circuit regulated by AI-2 signalling was devised. In order to engineer the Reporter Circuit we will:

1. Perform site directed mutagenesis of luxCDABE (Luciferase genes) in order to remove XbaI restriction enzyme recognition sequence.
2. Create a reporter construct comprising of luxCDABE controlled by Pqrr4 promoter.

Before the Reporter Circuit may be used to test the proper functioning of the Signalling Circuit, the Reporter Circuit itself must be functionally verified. We will be using mutant forms of LuxO protein in order to test the behaviour of the Reporter Circuit. The LuxOD47A mutant protein mimics the inactive form of LuxO, and the LuxOD47E mutant protein mimics the phosphorylated active form of LuxO8. In order to express mutant proteins, a constitutively-active promoter and a ribosome-binding site (available from the iGEM Registry) will be cloned in front of LuxOD47E and LuxOD47A constructs using the BioBrick cloning method. Testing will be performed in the KT1144 E. coli strain containing the cosmid-encoded qrr4-gfp fusion construct9.

Once the Signalling circuit is tested, the addition of a response circuit will allow for our engineered E. coli to perform a target activity in the presence of AI-2. Different types of circuits have been considered, but this circuit will comprise of the cloned Pqrr4 promoter followed by a Registry available inverter and ending with the gene(s) of interest. The use of the Pqrr4 promoter and the inverter will allow for the induction of target activity following AI-2 exposure.