From this point of view, we can dramatically reduce costs by pooling multiple mutant strains in two distinct ways: 1. library-pooling: mixing equal amounts of DNA from multiple mutant strains into a single library preparation.
Since phenotype sequencing requires sequencing complete genomes of multiple mutant strains, and is potentially expensive, we wish to optimize the information yield per cost.
Multiple mutant strains were generated using standard genetic and molecular methodologies.
One of the best producer strains is E. coli JCL206, a multiple mutant strain (ΔadhE, ΔfrdBC, Δfnr, ΔldhA, Δpta, ΔpflB) derived from E. coli K12 strain BW25113, bearing two plasmids, pSA65 and pSA69, that produces about 7 g l−1 of isobutanol in flasks (Atsumi et al. 2008, 2010) when cultured in similar conditions to that used in this work.
We believe the rodent model in this experiment could be applied to various situations using multiple mutant mouse strains and the Cre/loxP technology, as well as for in vivo administration of potential reagents to investigate colon cancer mechanisms.
However, if we can obtain multiple independent mutant strains from our phenotype screen, the statistics of independent selection events will quickly help distinguish the true target genes.
For such organisms, phenotype sequencing can open up a fast path for directly identifying a phenotype's genetic causes, for any phenotype where a good screen exists for generating multiple independent mutant strains.
Overall, these data demonstrate that PKA substrates have increased levels of phosphorylation in multiple kinetochore mutant strains, suggesting that PKA activity is elevated in these strains.
To test this postulate, we studied imaginal disc tissues isolated from multiple Top2 mutant strains, which revealed that imaginal discs and brains were larger in strains carrying hypomorphic alleles than in strains carrying null alleles.
Both multiple-eyespot mutant strains, mlt1 and mlt2, are characterized by supernumerary, nonasymmetrically localized eyespots.
For YopK pulldown of proteins in Hela cell lysates a 10-µl portion of anti-FLAG beads was preincubated with 200 µl of culture supernatants from Y. pseudotuberculosis multiple yop mutant strain (YPIII pIB29MEKBD) expressing or not expressing YopK-FLAG (grown as described above).
