Team:Aberdeen Scotland/WetLab/quorumsensing/results

Results and Discussion

Aims and background for testing our Quorum Sensing construct

In our first experimental set-up, we wanted to test whether our Quorum Sensing construct (further referred to as pIR) works on its own, and induces a pLux promotor to enhance trancription. The testing construct , which was used, consists of a pLux promoter and the gene for the green fluorescence protein. Therefore, it is expected that without Quorum Sensing construct no fluorescence occur.

For this purpose SCS1 E.coli cells were transformed with 1) pIR alone, 2) alone, and 3) pIR and J37032 together. The first one is the negative control. Without any GFP gene inside the cells they should not fluorescence. The second set of cells should show no fluorescence as the promoter needs to be activated by Quorum Sensing or only to a small degree due to leakiness of the promoter. The third set should show low fluorescence at low cell density and high fluorescence at high cell density.

Pictures were taking from over night cultures, i.e. at high cell density, under a light microscope (left hand-side) and a fluorescence microscope (right hand-side) from the 3 different SCS1 E.coli cell transformances. No fluorescence were shown on the pIR only transformance. However, the J37032 transformance shows as high a fluorescent as the double transformance.

Those results suggest that is not responsive to Quorum Sensing as it does not need LuxI/LuxR to produce GFP. Or as an alternative the pLux promoter could be extremely leaky. Hence, the pIR construct was further tested with the pLux responsive promoter construct this team created.

Apart from testing the pIR construct, the AND-gate, i.e. part (further referred to as pLG) was also tested in this experimental set-up. needs two inputs to initiate the transcription of GFP, firstly IPTG to release LacO repression and secondly Quorum Sensing for triggering the pLux promoter.

SCS1 cells were transformed with either 1) pTrc99A and pIR, 2) pTrc99A and pLG or 3) pTrc99A, pIR and pLG. The plasmid pTrc99A contains the gene lacI^q which over-produces LacI. This is needed to sufficiently suppress the Lac operator in the pLG construct in the starting cultures. It is expected that a high fluorescence occurs only in the third triple transformed cells and only when IPTG is added and the cells are at a high density. The first one is lacking the gene for GFP and hence should show no fluorescence at all. Whereas, the second one lacks Qourum Sensing producing part and thereby should not show fluorescence at any cell density and whether ITPG is added or not. However, adding IPTG releases the LacO repression and hence some GFP production might result from a leaky promoter.

Below graph and pictures show the negative control. All measurements in this experiments were taken with the help of a fluorimeter at a wavelength of 514.5nm. As this strain of transformances should not have any fluorescence this first set of values were said to be the background reading of the cells and the mean of those values were subtracted from all of the following measurements before the graphs were plotted. Also, under the fluorescence microscope no fluorescence were observed as expected.

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