Readouts for Multiplex Selections

Latest Update: 1-Nov-1997 , (minor revision 12-Mar-1999) , Up to: Church Lab page

See also: E.coli Gene Knockout Registry

pKO3 Genome engineering manual

The objective is to measure the relative growth (or death) rates of a variety of mutant strains in a pool under various environmental conditions for all members of the pool. This multiplexing guarantees identical growth conditions and by decreasing the effort per strain-environmental-condition combination it increases the number of conditions tested and hence the likelihood of finding the best match between gene-response and environment. Each strain in a pool is tagged by either an engineered insertion/deletion/point mutant or random insertions. The ratios of each strain can be readout out by hybridization and/or size tagging.

Two methods with similar goals have been used for yeast, Genetic footprinting (size tags) and Barcoding (hybridization tags). The former requires roughly one primer and sequencing reaction per gene-condition. The latter requires an engineered tag for each mutant and an Affymetrix chip scanner. Our size tagging method is likely to be compatible with 400 or more gene-environment combinations. Similarly hybridization methods can use random insertions and hybridization DNA targets arrayed on membranes or slides allowing increased experimental flexibility.

Size tagging example:

A 33 base-pair tag replacing the deleted ORF is used as a universal priming site. A second, specific "size tagged" primer is designed to prime at a precise distance from the C-terminus of the deleted gene in question. When used in pairs the universal and specific tags will generate a fragment of known length which will be unique for each mutant. Amplification using the universal primer, the specific size tags, and a mixed population of mutants as a template will result in a collection of bands which will quantitatively reflect the populations of the various mutants in a mixed culture.

Cllick here for an image from a high-throughput version for 48 strains (bands, y axis) 64 conditions (lanes, x axis) in a ABI 377 gel run.