pLux-Lac Hybrid with GFP Reporter and Double Terminator
Hybrid Promoter with LacO site and pLux-Lac sites for Quorum Sensing reporting via GFP.
As it also responds to LacI, this part acts as an AND logic gate, relying upon the inputs of AHL and IPTG (or some other form of LacI repression) in order to produce the GFP output
Comprised of parts BBa_I751502 (as developed by Tokyo Tech) upstream and BBa_E0840 downstream in a pSB3T5 vector.
BBa_K182101 (hereafter 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. The BBa_K182101 BioBrick was tested with another construct, BBa_K182200 (hereafter referred to as pIR) designed to generate a quorum sensing output signal.
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 1) 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.
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 2 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 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.
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 4). 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.
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 5). However, if compared with the control, double transformant (pLG and pTrc99A: Figure 5), then no significant difference in GFP expression was evident.
In conclusion, it was clear from the experiments conducted that the Lac operator sequence in the AND-gate Lux-Lac hybrid promoter BBa_K182101, 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 independently of the presence of LuxR-homoserine-lactone. It was not clear from the experiments conducted whether further induction of BBa_K182101 by LuxR/homoserine lactone would have generated further promoter activity over and above its observed ‘leaky’ nature. However, BBa_K182101 clearly represents a lacI-regulated promoter activity, with potential to be regulated additionally by LuxR.
Sequence and Features
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- 1000INCOMPATIBLE WITH RFCIllegal BsaI.rc site found at 747