How to optimize the population fitness step during a SATAY experiment depending on what we want to know from it?

[Wteunisse]

Depending on what you want to measure, different decisions might be made during the growth phase of the SATAY experiment.

• Werner suggested that in synthetic media, WT cells grow slower relative to bem1del cells and other less fit cells. Therefore, this could be used to increase the resolution between bem1del cells and lethal transposon insertions (the resolution in the less fit cell regime). On the other hand, this will decrease the resolution and relative fitness comparison between WT cells and cells with a positive mutation or cells with neutral mutations.

• On a side note, growth in a flask might be different from growth in the biotek. As in a flask there are more cells there is a higher chance for a mutation to occur and sweep the population. Take this in mind while designing the experiment and deciding on the growth time.

[leilaicruz]

This is how looks like a typical reseed step in SATAY, where you grow the cells in a 3L flask of SC-ADE media.

The source of the figure is the documented protocol from the group of ETH who set up this technique for yeast : https://sites.google.com/site/satayusers/complete-protocol/liquid-library-generation-w303

[Wteunisse]

Ah that is interesting, thanks Leila!

[Wteunisse]

In comment to the idea of using synthetic media; Enzo reminded me that SATAY experiments are performed in synthetic media anyway, so we should note that in the analysis of the relative fitness, the difference between WT and negative transposon insertions is influenced by this effect.