CH391L/S12/CounterSelection
Counterselectable Genetic Markers
Introduction
In contrast to selection markers, counter-selection markers serve to eliminate unwanted elements. These markers are often toxic or otherwise inhibitory to replication under certain conditions. Selective conditions often involve exposure to a specific substrates or shift in growth conditions. These elements are often incorporated into genetic modification schemes in order to select for rare recombination events that require the removal of the marker or to selectively eliminate plasmids or cells from a given population.
Application: Allelic Exchange
The introduction of specific mutations in a genetic sequence is a powerful way to learn about gene function or to engineer an organism for a desired application. A common way to introduce specific mutations into a target sequence is through allelic exchange. In a typical allelic exchange experiment, the chromosomal sequence to be mutated is synthesized and cloned onto a vector. This sequence is either highly homologous (except for the introduced mutations) to the chromosomal version or else is flanked by homologous sequences specifying the desired insertion site. Upon introduction to the cell, the cell’s homologous recombination machinery will recognize the sites of homology between the vector and the chromosome and at some frequency will stimulate the integration of the vector at the site of homology. This event is often selected for by the presence of a selectable marker present on the vector. Following this selection, it is often desirable to remove the vector to produce a “seemless” insertion. For this purpose, a counterselectable marker is included on the vector. Upon induction of the counterselectable condition, only those cells that have excised the vector sequence through a second recombination event will survive. Since this recombination event is usually rare the counterselection step is essential to find the desired mutants.
Parts
tetAR
The tetAR genes endow the cell with resistance to the antibiotic tetracycline by altering the cell membrane making it impermeable to the drug. However, this alteration makes the cell hypersensitive to lipophilic chelating agents such as fusaric or quinalic acids. This enables selection of cells that have lost the tetAR genes by exposure to fusaric acid.
sacB
rpsL
The rpsL gene encodes the S12 protein component of the 30S ribosome. Certain mutations of the gene cause resistance to the antibiotic streptomycin which targets the 30S ribosome. Resistance to streptomycin resistance is recessive meaning that an additional wild-type copy of the gene will lead to streptomycin sensitivity. Therefore, the wild-type rpsL gene can be used as a counterselective marker in a strain that already possesses a mutant allele. Selection on streptomycin will only permit those cells that have lost the wild-type gene to survive.
ccdB
ccdB is the toxin component of the toxin-antitoxin system of the F plasmid. ccdB is a DNA gyrase inhibitor which causes cell death when the ccdA antitoxin is not present. There is a known mutation of the DNA gyrase gene (gyrA462) that confers resistance to the ccdB toxin. This allows plasmid vectors that contain ccdB to be propagated without associated toxicity in the absence of ccdA. Cloning vectors that contain the ccdB gene can be used to select against vectors that fail to accept a desired insert when transformed into a wild-type gyrA strain. This cloning scheme virtually eliminates background. The Invitrogen "Gateway" cloning system takes advantage of this method [1].
