Table of Contents

Purpose

We successfully created a library containing H. elongata metagenomic inserts and now we want to select it for salt tolerance genes. Ideally, this will allow us to identify salt-tolerance genes that are even more effective than IrrE. We also want to hash out the experimental procedure for selection and identify a selection workflow that works.

Experimental design considerations

Stocks of libraries that will be selected:

Strain Construct Library size Metagenomic origin
s.Ec.C.00348 c.00185.pTwist_BsT-mStayGold_Barcode-GG 1.1 million transformants None, just barcode.
s.Ec.C.00349 c.00186.pTwist_BsT-mStayGold_Barcode-GG-MetagenomicLib 5.9mil transformants. NEB 10-Beta E-Comp Cells. 6/13 F1 H. elongata fragmented genome inserted into linearized barcoded library.
s.Ec.C.00350 c.00186.pTwist_BsT-mStayGold_Barcode-GG-MetagenomicLib 8.7mil transformants. NEB 10-Beta E-Comp Cells. 6/13 H1 H. elongata fragmented genome inserted into linearized barcoded library.

These libraries are still in the 10beta cloning strain, which is good enough for now. Eventually we’d like them to be in K-12.

The general experimental design here is:

  1. Recover cells from glycerol stock in 0% salt.
  2. Passage into a salty culture, and do a daily passage into another salty culture
  3. Take glycerol stocks & pellets at every timepoint.

The watchword here is avoiding bottlenecking.

First we’ll need to figure out how much glycerol stock to take to get a good library size. Our target is in the 50-100 million CFU range.

Second, let’s do 3 mL cultures and transfer 100 uL from each culture to the next, which should be about 250 million cells, but it still allows ~8 generations in each culture. Over 6 growths that’s 48 generations, which should be enough to see 1% fitness advantages.

This is a new experiment. It’s going to take some fiddling to figure out.

Experiment - Take 1 (had to restart because 5% is too salty!)

Experimental design: