<aside> 🛰️ We have the exciting new salt-selected cassettes from the H. elongata genome from 1st Selection of Functional Library . But are they better than WT? What about IrrE? I hope they are!
</aside>
Also, we want to try a slightly different plate setup, where we put every strain in every plate in different replicates, so plate 1 has all of the replicate 1’s, etc. Basically, making 4 copies of the same plate, each with 1 replicate of all 4 strains being tested. This should only require us to refactor our seeding plate, nothing else.
We just did our first selection experiment, trying to make E. coli more tolerant of high salt with a functional genomics library. @c.00186.pTwist_BsT-mStayGold_Barcode-GG-Helongata Unfortunately that library contained smaller inserts, because it got further fragmented by the AarI enzyme. Details in First time: fragment genomes
Then we selected it in 1st Selection of Functional Library with 5 days of selection in 4% salt, and got down to probably clonal libraries.
Additionally, we’re pretty sure our ability to calculate IC50s is pretty good, Salt IC50 normalization
One complicating factor is that the cloning strain used here is more salt resistant than the K12 E. coli we’ve been using before. So we’re adjusting our salt concentration up, to span 1.4-6% salt.
So now it’s time to put those all together and measure the IC50 values of our selected functional genomics isolates!
Use the following strains, starting 16 cultures overnight - 4 each of the IrrE & mStaygold, 1 each of each replicate of the library. 4 total plates.
| Strain
start? | | --- | --- | --- | --- | | s.Ec.C.00184 | c.00154.pTwist_BsT-Dr-irrE | Dr-IrrE | 4 | | s.Ec.C.00403 | c.00185.pTwist_BsT-mStayGold_Barcode-GG | mStaygold | 1 | | s.Sl.S.00404 | c.00185.pTwist_BsT-mStayGold_Barcode-GG | mStaygold | 1 | | s.Sl.S.00405 | c.00185.pTwist_BsT-mStayGold_Barcode-GG | mStaygold | 1 | | s.Sl.S.00406 | c.00185.pTwist_BsT-mStayGold_Barcode-GG | mStaygold | 1 | | s.Sl.S.00505 | c.00186.pTwist_BsT-mStayGold_Barcode-GG-MetagenomicLib | AarI-1-1 | 1 | | s.Sl.S.00506 | c.00186.pTwist_BsT-mStayGold_Barcode-GG-MetagenomicLib | AarI-1-2 | 1 | | s.Sl.S.00507 | c.00186.pTwist_BsT-mStayGold_Barcode-GG-MetagenomicLib | AarI-1-3 | 1 | | s.Sl.S.00508 | c.00186.pTwist_BsT-mStayGold_Barcode-GG-MetagenomicLib | AarI-1-4 | 1 | | s.Sl.S.00509 | c.00186.pTwist_BsT-mStayGold_Barcode-GG-MetagenomicLib | AarI-2-1 | 1 | | s.Sl.S.00510 | c.00186.pTwist_BsT-mStayGold_Barcode-GG-MetagenomicLib | AarI-2-2 | 1 | | s.Sl.S.00511 | c.00186.pTwist_BsT-mStayGold_Barcode-GG-MetagenomicLib | AarI-2-3 | 1 | | s.Sl.S.00512 | c.00186.pTwist_BsT-mStayGold_Barcode-GG-MetagenomicLib | AarI-2-4 | 1 |
Set up the starter culture like THIS:
and seed into the plates using the normal script, though with adjusted salt concentrations:
There should be 4 plates.