Team:BIOTEC Dresden/Sensor

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Aim

The aim of our team is to find and characterize a reporter system for AHL that is as sensitive as possible. This system is supposed to produce a reporter, like GFP, in an E. coli cell when the LuxI-fusion protein produces AHL. AHL is a small molecule that is able to diffuse through the cell wall of the bacteria and inside can induce transcription of a targeted gene. The general setup of a reporter system is as follows:

LuxR is constitutively expressed under control of a pTet promoter. LuxR binds to AHL when it is available and the resulting complex activates a promoter called LuxPR or LuxPL. In the simplest case, this induction leads to the expression of a reporter molecule like GFP. In order to create a signal-amplification, we created parts that have either LuxI; producing more AHL, or LuxR; that can bind AHL and activate the target gene, or both under control of the inducible promoter. All three systems create a positive feedback loop, which results in higher sensitivity towards AHL. But on the other hand they also create a bad signal-to-noise-ratio.

Experiments

In our experiments we use bacteria supplied with a plasmid containing the reporter part. Apart from varying the composition and order of these parts we also test all devices with different reporters, namely GFP, RFP, mCherry, YFP and pigments.

We induce the bacteria with different concentrations of AHL ranging from 0.1 nM to 2000 nM and measure the kinetics of the development of the reporter using a photospectrometric plate reader. We expect that the initial signal is the same for all bacteria since the reporter is not being expressed, also because the bacteria and the medium used only show some background fluorescence. After approximately 20-30 minutes we expect to see the first developments of the fluorescent signal due to transcriptional activation by the LuxR-AHL-complex. The fluorescence is expected to further increase until either the maximum rate of induction; according to the AHL concentration applied, or the maximal amount of the reporter protein that a cell can bear, is reached. For very low concentrations we do not anticipate any signal but the background fluorescence.