• Table of Contents

Purpose

<aside> 🛰️ Quantify salt tolerance of first library winners in IC50 assay compared to negative control (mStayGold) and positive control (Dr-IrrE)

</aside>

Background

In 1st Selection of Functional Library we got out 8 stocks that ‘won’ selection, coming from functional genomics libraries made from H. elongata gDNA. Then in @Transform AarI library winners into K-12 & isolate clones we transformed them into K-12, picked individual clones and isolated 7 unique inserts across 18 total stocks:

Now it’s time to actually compare them vs. IrrE with an IC50 assay and see how good they are!

Experiment Design

The overall experiment looks much like a normal @Endpoint IC50 Assay Protocol (v2), but with a few tweaks below.

1. Day 1, start overnights in a 96-well plate, shaking at 1000 rpm. Plate layout below.
2. Day 2, measure OD of overnights but don’t OD normalize, instead, dilute 1:25 into seeding plates. Setup salt plates with the gradient below using the Opentrons script (resulting in a 1:750 final dilution into salt plates)
3. Day 3, measure OD of all salt plates, that’s your data.

Details/experimental justifications:

Planning on using a salt concentration range from 0, 0.5, 1, 1.5-5.5%

Also using the 1:25 dilution demonstrated in @IC50 assay optimization: do we need OD normalization? Though we should still measure & record general ODs in the plate reader, just to be able to reference back to if anything went weird.

Due to a change in the deck layout of the OT flex, using a new script (tested in @Perchlorate tolerance of PydG and ClpP in sodium perchlorate + testing new assay setup script (PER-005))

The plan is to run 8 total plates, using mStaygold as a negative control, These are our controls:

Plus all of the new strains. We can run three strain replicates on each plate (in addition to the mStaygold negative control), so each plate looks like this: