In V. fischeri, the PL−luxpromoter of the lux operon provides weak LuxR constitutive ex-
pression. However, the binding of LuxR to lux boxes along the operon may reverse the transcriptional state of this promoter, and it may exhibit leaky opposite direction activ- ity. Since LuxR is expressed from the complementary DNA strand, we placed a double- terminator directly downstream of its promoter sequence to reduce background noise observed in genetic circuits. In continuous plate reader assays, we tested the induction of the PR−luxpromoter by detecting mCherry (replacing the native LuxICDABEG poly-
cistronic mRNA). In Figure 4.16, we observe a strong variation between induction pro- files of individual experiments and AHL detection could only be reported for 100nM + inducer concentrations. Although individual profiles showed variable reponse over different assays, we demonstrated that we could detect environmental levels of AHL in E. coli for this basic synthetic circuit using red fluorescence.
As we detailed for sender genetic circuits, different fluorophores were tested for the induction of bacterial response. We have shown previously that a significant signal was detected with red fluorescence, and attempted to improve this system by using the sfGFP instead of mCherry. As shown in Figure 4.17, we detected a stronger flu- orescent response when using sfGFP as a reporter. Besides showing brighter signal, we also observed, for sfGFP constructs, a more homogeneous signal within bacterial populations. In induced conditions, mCherry constructs produced an average 85x in- duction varying from 50x - 500x, whereas sfGFP devices were very centered around a 200x induction level. These results show that - based on discrete time points - the sfGFP was brighter and provided a better signal robustness for the detection of AHL.
Reporter devices presented so far were based on the constitutive PL−lux promoter
to drive low expression of LuxR. To improve the AHL detection threshold we obtained with previous constructs, we upregulated the expression of LuxR and controlled its appropriate degradation via an ssrA-tag. We tested a range of promoters varying from low to very high constitutive expression and obtained best fluorescence induction pro- files for relatively high LuxR levels. Figure 4.18 displays the differential response that was observed between the use of the native PL−lux and J23104 to drive LuxR expres-
sion. With higher levels of environmental LuxR proteins, we observed fluorescence induction for a few nanomolars of AHL, making the system 100 times more sensitive than with the use of the native V. fischeri promoter. The bacterial fluorescent response
FIGURE4.16: Plate reader assays results for the induction of receiver ge-
netic circuits by AHL over red channel channel fluorescence. In all graphs, the y-axis represents variation of fluorescence intensity in a range of in- ducer concentrations over time (x-axis). On the left, fluorescence pro- files of individual assays performed on different days are presented, while their average are displayed on the right. The top right corner graph dis- plays the mean of all fluorescence measurements while the bottom right
4.3. Single cell and population scale in vivo characterisation 109
FIGURE4.17: Flow cytometry results for the induction of receiver quorum
circuits. Colored peaks represent the count of individual cells (y-axis) for certain fluorescence levels (FL1, x-axis). Samples were analysed in the absence of inducer (no AHL, top panel) and in the presence of 1mM AHL
FIGURE 4.18: Effect of modifying LuxR regulation over receiver circuits fluorescence induction. For different AHL concentrations, both graphs show the induction of a green fluorescence signal (sfGFP, y-axis) over time (x-axis). In (A) and (C), LuxR is regulated via V. fischeri PL−lux promoter
and in (B) via the strong E. coli J23104 promoter. In (C), LuxI was cotran- scribed with the fluorophore.
was reduced for stronger constitutive promoters, probably due to a LuxR crowding effect, where overnumerous LuxR molecules compete for AHL and binding to PR−lux.
For low synthetic LuxR expression, we also observed reduced signals due to a short- age of LuxR proteins compared to the available inducer. Driven by the expression of the J23104 promoter, we obtained robust and identical induction profiles for several inducer dilutions. One induction rate in particular produced a better response than all higher AHL concentrations, which is the closest to the theoretical PR−lux promoter
KD. Moreover, while it would take about 2h for cells to peak in fluorescence in the
native PL−lux context, J23104 regulation allowed a quasi instant peak of fluorescence
after starting time-lapse experiments. Here, we showed how adjusting cofactor pro- moter strength provided a greater control over the inducible PR−lux in E. coli. Based
on the optimisation of the response fluorophore and LuxR regulatory mechanism, we engineered receiver genetic circuits that coordinate a robust emission of fluorescence when sensing environmental AHL.
In Figure 4.18, we can observe a reduction of the overall fluorescence profile in the amplifier circuit (C) as opposed to the native V. fischeri context (A). One would natu- rally expect the opposite situation, where a rise in fluorescence levels should be created by an autoinduction loop. However, there are a few possible explanations: cells may be undertaking a certain metabolic load that, over a certain induction threshold, becomes toxic to bacteria, or the lux promoter may also display some properties such that higher inducer concentrations become less active at starting transcription (not uncommon for
4.3. Single cell and population scale in vivo characterisation 111
FIGURE 4.19: AHL sensor induction by sender genetic devices super- natant. On the left, receiver cultures were grown after plating 50µl of uninduced sender circuit supernatant. On the right, the sensor was im-
aged after induction by 50µl of induced sender circuit supernatant.
inducible promoters, as exemplified in Figure 4.13). Finally, it is also worth keeping in mind that the response detected via these experiments is solely based on fluorescence, which is only a reporter for the activity of the promoter and an approximation of both transcription and translation.