Team:Aberdeen Scotland/WetLab/quorumsensing/results

Results and Discussion
Aims and background for testing our Quorum Sensing construct BBa_K182200, and the AND gate BBa_K182101 

In our first series of experiments, we wanted to test whether our quorum sensing construct BBa_K182200 (expressing LuxI and LuxR and hereafter referred to as pIR) is functional, and is capable of inducing a pLux promoter. As a test of BBa_K182200 (pIR) function the reporter construct BBa_J37032 was used. BBa_J37032 consists of a pLux promoter and the gene for the green fluorescent protein GFP. For this purpose SCS1  E.coli  cells were transformed with 1) pIR alone, 2) BBa_J37032 alone, and 3) pIR and J37032 together. The pIR transformant represents the negative control. Without any GFP gene inside the cells they should not fluoresce. The BBa_J37032 transformant should also not fluoresce as the promoter needs to be activated by quorum sensing and this single J37032 transformant does not express the required LuxI/LuxR combination. The double transformant should show low fluorescence at low cell density and high fluorescence at high cell density as quorum sensing is triggered. Micrographs were taken from over night cultures, i.e. at high cell density, under a light microscope (Figure 1; left hand-side) and a fluorescence microscope (Figure 1: right hand-side) from the 3 different SCS1  E.coli  cell transformants. No fluorescence was exhibited by the pIR-only transformants. However, the single J37032 transformants showed as great a fluorescence as the double transformants.

Figure 1 Those results suggest that reporter construct BBa_J37032 is not responsive to quorum sensing as it does not need LuxI/LuxR to produce GFP. In other words, the pLux promoter in J37032 seems to be extremely leaky. This BBa_J37032 construct was therefore revealed by these experiments as an unsuitable reporter of LuxI/LuxR quorum sensing.

In a further series of experiments, the pIR construct BBa_K182200 was re-tested with the pLux responsive promoter construct BBa_K182101 created by the Aberdeen Team. BBa_K182101 (herafter referred to as pLG) represents an AND-Gate promoter controlling the expression of GFP; it requires two inputs to initiate the transcription of GFP, firstly IPTG to release LacO repression and secondly quorum sensing, in the form of LuxR-homoserine-lactone 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 lacIq which over-produces LacI. This is needed to sufficiently suppress the Lac operator in the hybrid pLux-LacO promoter in the pLG construct in the starting cultures. It was expected that a high fluorescence would only occur in the pTrc99A / pIR / pLG triple transformed cells, and only in the presence of IPTG and when the cells are at a high density to trigger quorum sensing. The pTrc99A / pIR double transformant lacks the gene for GFP and hence should show no fluorescence at all; this acts as a negative control. The pTrc99A / pLG double transformant lacks quorum sensing ability, since it expresses neither LuxI or LuxR. It thereby should not show fluorescence at any cell density, whether ITPG is added or not. GFP fluorescence was then assessed in the three transformant types. The graph below (Figure 2) shows the negative control (pTrc99A / pIR double transformant). All quantitative fluorescence measurements in these experiments were taken with the help of a fluorimeter at an emmision wavelength of 514.5nm, and an excitation wavelength of 490nm. As this pTrc99/pIR negative control expresses no GFP these fluorescence values were defined as the background fluorescence reading of the cells and the mean of those values were subtracted from all of the subsequent measurements before the graphs were plotted. As expected, under the fluorescence microscope no fluorescence was observed for the pTrc99A/pIR double transformant since it is not transformed with a GFP gene.

Figure 2

The second control was the SCS1 strain transformed with pTrc99A and pLG. It is expected that when grown with or without IPTG no fluorescence should be detectable. The results in Figure 3 show hardly any fluorescence for the culture grown without IPTG, but a high fluorescence when grown with IPTG. Measurements of fluorescence were taken after the cells reached an optical density of 0.200 at 600nm. This was chosen because it is suggested that quorum sensing only switches on at a much higher density than this. Those results indicate that the Lac operator works as expected and is sufficiently repressed by LacI as no fluorescence occurs without IPTG. However, since the pTrc99A/pLG double transformant lacks a quorum sensing mechanism (it contains no LuxI or LuxR gene), the fluorescence occuring when IPTG was added cannot be due to quorum sensing. This unexpected result led us to conclude that the pLux promoter is extremely leaky and requires only the presence of IPTG for its induction.

Figure 3

Figure 4 shows the fluorescence of the triple transformed cells (pTrc99A/pLG/pIR) over a range of cell density, grown with and without IPTG. Similar results were obtained to those of the pLG/pTrc99A transformants were observed. There was very little fluorescence present in the absence of IPTG, however, high levels of fluorescence when IPTG is added. In this case this is actually expected at high cell density, as both inputs of the AND-gate - IPTG and quorum sensing - are provided. Therefore one interpretation of this experiment might be that, quorum sensing appears to be switched on at low cell density as at the first measuring time of 240 minutes the optical density of the cultures were only 0.2 at a wavelength of 600nm. However, when the results from the double transformants pLG/pTrc99A are taken into account (Figure 3 above), it is clearly apparent that this fluorescence is due to the pLux-LacO hybrid promoter exhibiting a high degree of leakiness, and being independent of LuxR-homoserine-lactone for its activation.

Figure 4

In a further experiment, two cultures were grown without IPTG up to an optical denisty of 1.0 at 600nm wavelength. This was carried out for the transformants with 1) pLG and pTrc99A and 2) pIR, pLG and pTrc99A (Figure 5). At this cell density it is predicted that some quorum sensing should occur in the second culture, but not in the first culture as no pIR construct is present. Figure 5 below shows the first culture. It suggests that a high fluorescence GFP expression is induced in a relatively short time, i.e. 30 minutes. This leads to the conclusion that as in the above experiments the AND-gate can be triggered by one input (IPTG addition) alone and does not need quorum sensing. Figure 5

The second culture contains the pIR, pLG and pTrc99A triple transformant, inducing the quorum sensing construct pIR, and it is therefore expected that as soon as IPTG is added the AND-gate is switched on and GFP production is triggered. The time delay until the fluorescence develops represents the time needed to release the repression from the Lac operator. This was shown (see Figure 6). However, if compared with the control, double transformant (pLG and pTrc99A: Figure 5), then no significant difference in GFP expression was evident. Figure 6

In conclusion, it can be summarised that the quorum sensing construct BBa_K182200 (pIR in the experiments above) could not be tested properly as both lux promoter constructs with which it was paired appeared not to work as expected and showed no responsiveness to LuxR/LuxI. However, it was clear from the experiments conducted that the Lac operator sequence in the AND-gate Lux-Lac hybrid promoter, constructed as part of this work, functioned very well and tightly repressed transcription from this promoter. However, the second input of the AND-gate construct did not function as expected. The hybrid promoter is therefore leaky in the presence of IPTG, and in contrast to expectation, is activated independent of the presence of LuxR-homoserine-lactone.