For competitive fitness assays, one must be able to distinguish two E. coli subpopulations in a mixed culture. One way this can be accomplished is by mutating genes required for sugar fermentation to create a genetic marker (Ara, Mal, Lac, etc.). Plating a – version of these strains will produce red colonies and plating the + version will produce pinkish-white colonies on tetrazolium indicator agar containing a rich media base plus the sugar of interest (TA, TM, etc).
This procedure has been used to create defined Ara+ and Ara– versions of Escherichia coli B (REL606) and Mal- derivatives of Escherichia coli K12 containing the malF in-frame deletion from the Keio collection strain with its KanR marker removed by FLP recombination.
For a more detailed description of the Ara genetic marker, see also: Ara Marker.
Strain | Plasmid | Marker | Resistance |
---|---|---|---|
JEB205 | pJEB11 | →Ara– | AmpR+KanR |
JEB207 | pJEB12 | →Ara+ | AmpR+KanR |
JEB600 | pJEB15 | →Mal– | AmpR+KanR |
JEB208 | pACBSR | for gene-gorging | CamR |
Expected results are 0-10 colonies with the marker change per 200 colonies of the other color.
Competitive fitness assays should be used to test for non-neutral mutations that sometimes occur during strain construction by this method. Testing three independent isolates usually assures one success.
Barrick Lab > ProtocolList > ProtocolsGeneGorgingMarker