Nov 24, 2020
Cerulenin experiment
This protocol is a draft, published without a DOI.
- Elizabeth Fozo1
- 1In-house protocol
Protocol Citation: Elizabeth Fozo 2020. Cerulenin experiment. protocols.io https://protocols.io/view/cerulenin-experiment-bpzump6w
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: November 24, 2020
Last Modified: November 24, 2020
Protocol Integer ID: 44820
Keywords: cerulenin experiment rescue from cerulenin inhibition, cerulenin inhibition, cerulenin experiment rescue
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Abstract
Rescue from Cerulenin Inhibition
Based on Saito, et al., 2018
Troubleshooting
Before start
What you can expect: cerulenin addition with solvent control should inhibit growth completely forE. faecalis. However, you will see a few rounds of division, and then the cells “level-off” at an OD600 nm 0.1-0.3.
If the fatty acid can rescue from cerulenin, you should reach a similar OD600 nm value at the 24-hour mark as the control tube (cells with no antibiotic). Note that in the case of the specific fatty acid or fatty acid combination, the cells may grow more slowly out of cerulenin inhibition (which is why the 24-hour recording is critical).
Note, as of August 2019, no evidence for suppressors arising from this assay.
Steps
Grow strain of interest overnight in 5 ml BHI
The next day, set up the following tubes:
Tube with no cerulenin
Tube with cerulenin + solvent control (for fatty acid)
Tube with cerulenin + fatty acid of interest
Note
For current studies, use 5mg/ml of cerulenin (note, can dissolve stock in 10 mg/ml ethanol) and 5ug/ml of fatty acid interested in testing.
Perform in glass tubes with either 5 or 10 mL total volume
In am, dilute the strain to be an OD600 nm of 0.01 into each of the above tubes. This is time “0”
Record the OD600 nm at time 2, 4, 6, and 24 hours
Graph the results, making sure to add temporal separation in the graphs for the 24 hour time point.