Team:Lubbock TTU/Future Outlooks
Future Outlooks & Developments
Plate reader Experiments with 8 Constructs
Because we encountered issues with the construction of our L1 transcription units, we were unable to conduct plate reader experiments with the 8 constructs. In addition to measuring fluorescence of the eight constructs at different concentrations of chitin induction, we would like to design a similar well plate induced with different concentrations of Bd zoosporangia. Dr. Rodriguez sent us samples of heat-killed Bd that we have conducted serial dilutions of to induce our constructs with. Because we are inducing our biosensor with chitin oligosaccharides, attempting to induce it with the chitin naturally-found in Bd zoosporangia cell walls may be problematic in that it cannot enter through the chitoporins in the membrane. Thus, in the future, we will explore adding a chitinase to the biosensor to degrade chitin polysaccharides.
Modeling
Once we determine the chitin concentration ranges that exist in zoosporangia, we can tune our biosensor to respond to these ranges and determine the dynamic range of the biosensor when used in the field. Furthermore, because the parameters of V. cholerae’s chitin utilization program have not been published, the parameters used in the model are sourced from protein interactions that we determined can be comparable to our system (see "Determining Parameters" table in the Modelingpage). However, once we determine the induced fluorescence values of our system, these parameters can be adjusted to best fit the data (performing sensitivity analysis). Given more time, we can experimentally estimate these parameters.
Testing ChiSPY On Swabs Collected With Dr. Barnes
We want to put our biosensor to the test with the swabs we collected with Dr. Barnes. Once we know which section of the frog has a higher probability of housing zoosporangia, we can compare the accuracy of our swabbing protocol to qPCR detection of chytridiomycosis.
Measuring chitin concentration in different Bd strains
We choose to use chitin as our biomarker as it is independent of DNA and thus circumvents the copy number variation issue associated with traditional detection methods. However, there is no data on whether chitin concentration in zoosporangia cell walls is actually consistent across different Bd strains. To put our assumption to the test, we would like to measure the chitin concentration in the zoosporangia of different Bd strains.