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Synthetic AHL Concentration Inductions of LasR, LuxR, and TraR
The experiments performed to characterize the Las, Lux, and Tra receivers included the synthetic AHLs of 3-oxo-C12 (Las), 3-oxo-C6 (Lux), 3-oxo-C14 (Sin), Coumaroyl (Rpa), C4 (Rhl), and 3-oxo-C8 (Tra). The following graphs show the maximum (DGFP/OD)/DT across a concentration range of 1E-14M to 1E-4M. Each data point was done in triplicate then averaged. These points determine where the GFP is changing the fastest, and represents the maximum rate of change at a specific synthetic AHL concentration.
The graphs below depict the GFP production from the F2620 receiver to be higher at a higher concentration of the synthetic AHL of Las. The second graph, depicts the same nature however at a higher concentration of the synthetic AHL of Lux. It is interesting to note that the Las sender, at a lower concentration, promotes a higher GFP expression than the Lux. Our group hypothesized that it would be opposite considering the LuxI sender comes from the same system as F2620. These results show differently.
As Las receiver and Tra receiver are also induced by the synthetic AHLs, a similar trend emerges. There is little to no GFP expression for the lower concentration, between 1E-14 through 1E-9, 1E-8, or 1E-7 in some cases. Then a dramatic increase in GFP expression as the AHL concentration increases. This indicates that the system shows significant GFP expression at higher concentrations.
Another interesting result was the with the synthetic AHL Rpa. As depicted in the graph below, it shows an almost steady state of GFP production, independent of the concentration of the AHL signal. This trend can also be seen in multiple systems such as the induction of LasR with a Rhl AHL, LuxR with Rhl AHL, and LuxR with a Rpa AHL. This might be a useful finding if researchers wanted to use a smaller amount of signal due to limiting resources of RpaI or RhlI sender. However, it did not show an orthogonal pathway since it did in fact promote GFP expression.
The trend outlined above is also expressed in the below graphs. From concentrations 1E-14M through 1E-5M there is a steady production of GFP that is independent of the AHL signal concentration. However, as the receiver is induced by the 1E-4M signal, an outlier of GFP expression is produced. This is an unusual result as the 1E-4M concentration in the LasR and Lux AHL system is expected to be around 2-6 arbitrary units. This unexpected point is also present in the TraR and Rhl AHL system. The max GFP expression of 1E-4M is an outlier as the trend indicates orthogonality between the receiver TraR and RhlI sender. Within our experiments, we implied that orthogonality is defined as not expressing GFP or having no communication within the systems. This might be an important find for synthetic biologist and genetic engineering to use this orthogonal quorum sensing circuit within their genetic circuit to be able to, with confidence, produce results of a protein or phenotypic expression. This orthogonality does not apply to the system of LasR and Lux synthetic AHL, however, as there was still induction and GFP was shown to express. As stated above, this system could be beneficial to researchers who have limited resources of the Lux sender and wish to express the same amount of GFP that would occur in higher concentrations.
The graph of LasR and Las AHL displayed below is a combination of different sender concentrations of LasI in respect to the GFP expression of LasR. It is interesting to note that the corresponding sender and receiver system, Las, does not express as highly in GFP expression as other experiments with different senders. Our group hypothesized that this sender/receiver system would still promote a higher GFP expression, even at a more diluted concentration of synthetic AHL. This trend can also be seen in the graph of LasR and Sin AHL. The 1E-7M expresses a higher GFP of around 20 arbitrary units than the 1E-4M, which is an unexpected result. This is beneficial to researchers looking for maximum GFP expression using the Las receiver and a small supply of sender.
A notable conclusion can be drawn from the below graph of the Tra receiver and Lux AHL system. It is seen from 1E-14 to 1E-7 that there is little to no GFP expression, while the 1E-4M concentration reached 6 arbitrary units with high error. This type of fluctuation also occurs in the LuxR with Sin AHL, although it is more varied throughout the concentration range. Both graphs appear to have an upward trend despite the fluctuations as concentration increases.
