The goal for this experiment is to assess whether calf thymus DNA is sufficient in vitro to yield a disparity between \(D_{ap}\) and \(D_{phys}\). I will do this by comparing IDAs coated in 1% agarose +/- 1mg/mL ctDNA. If this experiment is successful it will help us to determine whether the disparity we observe for the biofilm is due to physical diffusion only (influenced by the heterogeneity of the biofilm) or truly self-exchange. This in vitro system should recapitulate the biofilm, but will be far more homogeneous.
First, I anticipate that we will observe a difference in the decays +/- DNA. Hopefully, enough of the PYO will bind to the DNA that diffusion is slowed. If no difference is observed between the two conditions then I think we will have to reassess how to approach the in vitro system.
Second, the DNA may yield a disparity between Dap and Dphys - this would support the self-exchange model. Alternatively, the DNA may look different than no DNA, but not yield a difference. This would support the diffusion only model, where the geometry of the biofilm is causing an artifactual \(D_{phys}\)
2% agarose will be prepared in PBS 50. Then the molten 2% agar will be combined 1:1 with either ctDNA (2mg/mL) or just PBS 50. 50uL droplets will be deposited on the IDA and solidified for 15min.
Then IDAs will be immersed in a 75uM PYO solution. The influx of PYO will be monitored by SWV only every ~10min to avoid unnecessary buildup on the electrode and incubated for at least 30min to insure PYO equilibrates into the gel. Then the IDA will be directly transferred into fresh medium and the macro will be started to acquire the SWV / GC data. Based on past data I expect this transfer acquisition to take at least 1 hour.
library(htmltools)
base_path="https://scott-saunders.github.io/labwork/IDA/08_13_19_agarose_ctDNA/"
files <- dir(path=".", pattern = "*\\.html$",include.dirs = T,recursive = T)
for(link in files){
print(
tags$a(href = paste(base_path, link, sep=''), link)
)
writeLines("")
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