Part:BBa_K223047:Experience
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Applications of BBa_K223047
This summer, the team built three of the four device subparts, namely the SoxR/SoxS sensor, the retinoic acid generator and the 5-methyl tryptophan mutant trp sensor. All the sensors were built with GFP downstream of the sensor itself. As a result, only the sensing capability, that is the ability of sensor to recognize inflammation or immunosuppressive signal molecules, was tested. Of these three device subparts, the SoxR/SoxS sensor was characterized on both a high and low copy plasmid and found to be functional. In the section below are the graphs relating OD to time and GFP to time for the sensor on both the high and low copy vector strains.
Note on Assays
In order to characterize our device, we needed to use an inducer which imposed superoxide stress within the cell. In vivo, this stress would take the form of nitric oxide, a downstream product related to increasing levels of Th-17 cells. However, in vitro, we needed an inducer which was more suitable for us to test. Thus, we used [http://en.wikipedia.org/wiki/Paraquat paraquat], which causes oxidative stress in a similar manner as nitric oxide.
Low Copy SoxR/SoxS Sensor
Experimentation done on the low copy vector indicated that our SoxR/SoxS sensor was not only capable of inducible GFP (or another downstream protein) production but also that the downstream production levels can be modulated through varying concentrations of inducer paraquat. In this case, our group demonstrated that increasing the concentration of superoxide inducer paraquat increased the production of GFP as expected. Additionally, it was noted that at higher concentrations of the inducer, the rate of change of fluorescence increased as well.
However, it is interesting to note that the induction chemical paraquat, which can be toxic to E. coli cells at large concentrations, begins to have a negative effect on the cells at higher concentrations. Specifically, it seems that at 80uM concentration of paraquat, there is a marked decrease in growth rate with time. This is shown by the OD levels, which seem to begin stabilizing around an OD600 of 1.1 three hours after induction.
While growth inhibition seems to occur at 60 and 80 uM concentrations of paraquat, the overall stability of the low copy system and its ability to sense a large range of inducer concentrations over a long period of time suggests that it is ideal sensor system for our anti-inflammatory device. In particular, by virtue of the low copy system's ability to induce GFP production over a longer time and larger range range of concentrations, we would like to use it in the diagnosis and treatment of stable post-operative Crohn's patients who need long-term, stable retinoic acid therapy.
High Copy SoxR/SoxS Sensor
The SoxR/SoxS sensor was similarly tested on a high copy vector. Both of these assays lead to our characterization of this system with respect to its production of downstream GFP through induction by superoxide stress (in this case paraquat). As shown by the graphs, our results from the high copy varied sufficiently from the low copy vector. In the case of the high copy, one should first note that there is a greater growth inhibition occurring at lower inducer concentrations. As compared to the low copy in which growth inhibition seems to occur at 60 and 80 uM, the high copy showed growth inhibition occurring around 30uM. The second factor to take note of is the greater standard deviation occurring in the OD levels. Both of these factors are most likely due to the nature of the high copy vector itself. Since the cells are more metabolically active and have a higher rate division, they are under greater stress. When coupled to the stress of the inducer, this leads to larger growth inhibition and discrepancies in the OD with varying inducer concentrations.
The differences between the high and low copy results allow us to tailor our device to meet individual patient needs. The high copy vector will have the greatest application in treating high risk, post-operative patients of Crohn's Disease. In this stage of the disease, the patient's will require a burst of high dosage retinoic acid localized to a specific section of the small intestine. This would result in a short-term, more intensive treatment. However, this system is not sustainable; eventually the treatment would switch to the low copy vector as more of a monitor device.
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