Notebooks:

library(htmltools)

base_path="https://scott-saunders.github.io/labwork/IDA/01_17_19_psoralen_nonequil_2/"

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("")
}

Analysis/01_17_19_psoralen_nonequil_2_Dap_analysis.html

Analysis/01_17_19_psoralen_nonequil_2_Dm_analysis.html

Processing/01_17_19_psoralen_nonequil_2_Dap_processing.html

README.html

Purpose

This is the second replicate of the psoralen experiment using the non-equilibrium measurements. As discussed previously, the purpose is to test the effect of DNA CT in the biofilm echem signal, since psoralen + 365nm light introduces interstand crosslinks into DNA. The first replicate seemed to work ok, although the control sample showed some weird behavior that may have been caused by an uncharacteristically thin biofilm. I made no major changes, the purpose of this experiment was to repeat in the hopes that it would clarify the first replicate data as well.

Protocol

Essentially the same protocol was used as 1/08/19:

  1. Biofilms were grown in reactors with minimal succinate medium for 4 days, at ~30 degrees C, as usual.
  2. Before any echem, one biofilm was submerged in a 10uM trioxsalen solution in a petri dish (from 2mM EtOH stock) and exposed to 365nm light from a handheld lamp placed directly above it. The other biofilm was submerged in MM succ with an ethanol control.
  3. After an hour of treatment, the biofilms were briefly left in the petri dishes, but the UVA light was turned off.
  4. I then used a non-equilibrium type protocol, similar to the one first attempted on 12_10_18.
  5. To measure \(D_{ap}\) I performed the following echem:
  1. To measure \(D_{m}\) I performed the following echem:

*SWV slow was run several times for the psoralen biofilms because of high background current.

Note that I had two potentiostats running. There were three chambers total, 1 soak and 2 transfers, so only one biofilm could soak at one time, but two transfers could be monitored simultaneously. I kept the biofilms staggered, first control, then psoralen, but I made sure that much of the transfer time overlapped to try to make it efficient.

Data

In total 5 transfers were performed for each biofilm. For the control biofilm the first 3 were data acquisitions to try to measure \(D_{ap}\) with the paired GC/SWV measurements taken as the biofilm equilibrated. The last two were the attempts to measure \(D_m\). For the psoralen biofilm the first 3 transfers were taken using the SWV fast settings as with the control and the 1/8/19 experiment, however, there was a high background current that looked like it would be difficult to quantify. Therefore, for the final two transfers I repeated the \(D_{ap}\) estimate, but I used a short SWV slow (~20sec), which made the PYO peak more easily identifiable. This changed scan rate will change the slope of the titration and should be corrected accordingly in the analysis.

In addition to the “transfer” measurements there are the corresponding “soak” measurements taken before each transfer (numbered in the same way). Hopefully the rest of the file naming is obvious.

Imaging

Immediately following the final data acquisition, the IDA/biofilms were sliced off the epoxy holder and mounted on slides with 2uM TOTO-1 and 10uM Syto60 to stain for eDNA/all DNA. There was clearly biomass on both electrodes, although this time the control biofilm actually had much more biomass coming off the electrode surface. It is unclear if this difference in biomass was due to psoralen treatment or not.

Tilescan/zstacks were taken of the entire IDA working surface at 10x. The folder should be 01_17_19_psoralen-2 and the filenames are:

  • control_biofilm_dPHZstar_10x_tile_zstack_syto60_toto1.czi
  • psoralen_biofilm_dPHZstar_10x_tile_zstack_syto60_toto1.czi