Psoralen treatment and toxicity - Non equilibrium #3

Purpose
Protocol
Data
- Notes
- Imaging

Notebooks:

library(htmltools)

base_path="https://scott-saunders.github.io/labwork/IDA/01_23_19_psoralen_nonequil_3/"

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_21_19_analysis_psoralen_toxicity.html

Analysis/01_23_19_psoralen_nonequil_3_Dap_analysis.html

Processing/01_23_19_processing_psoralen_nonequil_3.html

README.html

Purpose

This is the third replicate of the psoralen nonequilibrium experiment.

Protocol

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

Biofilms were grown in reactors with minimal succinate medium for 4 days, at ~30 degrees C, as usual.
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.
After an hour of treatment, the biofilms were briefly left in the petri dishes, but the UVA light was turned off.
I then used a non-equilibrium type protocol, similar to the one first attempted on 12_10_18.
To measure \(D_{ap}\) I performed the following echem:

Soak only working electrode part of IDA (100mL liquid in reactor) in 75uM PYO in MM succ medium for at least 10 min. Take SWV fast scan in soak reactor. During soak, make sure that transfer reactor has 180mL fresh MM succ medium and that it is stirring with N2 bubbling to make anaerobic and equilibrate temperature.
Hook up counter and reference in transfer reactor. Remove the biofilm from the soak reactor, dip once in a MM succ wash, and then transfer to other reactor. Connect working electrodes and check open circuit potential (OCP). Take first SWV fast scan. Try to time the seconds between immersing the biofilm in the transfer reactor and starting the first scan.
Run a 1 segment GC ( +0.2 to -0.4 V only ~2min), and then immediately run an SWV fast* to pair the data. Repeat for ~30min when the signals start to level off.

To measure \(D_{m}\) I performed the following echem:

The first step was the exact same as the first bullet point for \(D_{ap}\).
Prepare a SWV fast repetitive run in the software (56 sec gap, repeat 60 times).
Hook up counter and reference in transfer reactor. Remove the biofilm from the soak reactor, dip once in a MM succ wash, and then transfer to other reactor. Connect working electrodes and check open circuit potential (OCP). Start the repetitive run. Try to time the seconds between immersing the biofilm in the transfer reactor and starting the first scan.

*SWV slow used during run 4 for both biofilms.

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

Before running this echem experiment, I was also able to test the toxicity of trioxsalen in the plate reader. Therefore in the data folder there is a separate folder for the plate reader psoralen toxicity data. There is a separate analysis notebook for this experiment. This notebook contains the protocol etc.

For the echem the files are separed into the control biofilm and the psoralen treated biofilm. In total 5 runs were taken for each biofilm. The first 4 runs were paired SWV, GCs scans for measuring \(D_{ap}\), and the last run was an SWV repetitive run for measuring \(D_m\). For runs 1 and 2, I used the 760b potentiostat with the psoralen biofilm, and the 760e potentiostat with the control biofilm. For runs 3 and 4, I used the 760b with the control and 760e with the psoralen.

For run 4, I used SWV slow on both the control and psoralen biofilms (to enable comparison), because the SWVfast signal was not working well with the psoralen biofilm.

In general for the \(D_{ap}\) runs I took 15 paired scans as the biofilm equilibrated.

Notes

Before the psoralen #2 run with the 760b potentiostat, the open circuit potential was > -1V and it was unclear why. I tried replacing the ref electrode etc, but eventually hooking up the potentiostat to the other IDA and then moving back to the psoralen IDA fixed the problem. Later on this happened again, and I fixed the problem by restarting the 760b potentiostat, therefore I believe it was a potentiostat issue.

In the GC data you can see “wiggliness.” At first I thought this may be a potentiostat specific issue, which is why I switched potentiostats/biofilms halfway through. I also tried orienting the biofilms slightly differently in the reactor. I do not know if any of this truly made a difference.

Imaging

Zstack tilescans were of both IDAs were taken at the BIF. It seemed that the control biofilm was significantly larger/more complex coming off the electrode, although both biofilms clearly had cells and DNA on the IDA band surface. The filenames are:

01_23_19_psoralen_biofilm_dPHZstar_10x_tile_zstack_syto60_toto1.czi
01_23_19_control_biofilm_dPHZstar_10x_tile_zstack_syto60_toto1.czi