Counter selection

Flanking counter-selectable markers Eliminates vector backbone Reduces transformation efficiency 23,24... 

Schweizer HP. 1992. Allelic exchange in Pseudomonas aeruginosa using novel ColEl-type vectors and a family of cassettes containing a portable oriT and the counter-selectable Bacillus subtilis sacB marker. Mol Microbiol 6 1195-204. 

If an aptamer is desired that binds one molecule with high specificity, but should not bind a second, very similar molecule, this can be achieved by using this second molecule immobilized to the matrix during the preselection [10]. This process is referred to as counter selection. 

In addition, NR is a very convenient genetic marker, that has the potential to be selected or counter-selected in many organisms, whether they be plants (Caboche Rouze, 1990 Nussaume et al., 1991) or microorganisms (Unkles et al., 1989). NR-deficient (NR-) mutants will not grow on nitrate, but unlike wild-type organisms will not be killed by chlorate, a nitrate analogue that is toxic when reduced by NR. 

This strategy has also been applied for the selection of active //-lactamases from a library of mutants also containing penicillin-binding proteins. For this purpose, the protocol had to be modified to circumvent a difficulty of selections with suicide substrates in mechanisms involving a covalent intermediate. If inhibition arises from a covalent intermediate (Y in Scheme 5.2, an acyl-enzyme in the case of serine //-lactamases), enzymes whose rate of release of this intermediate (hydrolysis of the acyl-enzyme) is slow will be efficiently selected as the efficiency of inhibition depends on the ratio of rate constants k4/k3 (Scheme 5.2). To prevent the selection of enzymes with inadequate turnover, a counter-selection step was included in the protocol the library of mutants was incubated with substrate in order to block them as covalent intermediates before adding the biotinylated inhibitor. The library could be enriched from 6 ppm to 25 % active //-lactamases in four rounds of selection [62]. 

Figure 1 The classic yeast two-hybrid method and derivatives, (a) Schematic diagram of the yeast two-hybrid approach, describing an interaction between protein X and protein Y. Protein X is fused to a transcription factor DNA-binding domain (the "bait" construct), and protein Y is fused to a transcription factor activation domain (the "prey" construct), (b) High-throughput applications of the yeast two-hybrid method use mating of haploid strains carrying bait and prey, respectively. Hybrids can be mated in arrayed formats (as shown) or as libraries, (c) The reverse two-hybrid method uses a counter-selectable marker to indicate loss of protein interaction because of disruption by an inhibitor protein/small molecule ("/" illustrated in the diagram) or mutation(s) in proteins X and/or Y.

Meanwhile prepare the Ni-NTA beads (the partition matrix). We have a stock of protein bound beads, which serves as our selection matrix, and we prepare the unbound beads, our counter selection matrix, before each experiment by transferring 20 pL of the beads into 500pL of selection buffer. We then wash them three times with selection buffer by repeated centrifugation at a low speed at room temperature in a mini centrifuge and gently mixing the beads with fresh buffer each time. 

Finally, add 100 pL of binding buffer to the beads and mix the annealed DNA library with the beads for counter-selection step. Incubate the tube on a gentle rocker-shaker for 15 min. 

Centrifuge the counter selection tube at maximum speed and transfer the supernatant to the tube containing the protein bound beads. Incubate the beads for another 15 min on rocker-shaker. 

After initial few rounds of counter-selection, it is trivial to counter-select in each round. After observing a signal on native gel-shift, the selection can even be switched onto a gel-based selection, thereby obviating the need for a bead-based selection. The gel-shifted band, which has the sequences bound to the protein, is cut out, and after elution, DNA sequences can be PCR amplified for the next round. The use of Taq polymerase for PCR can introduce a few mutations to the selected DNA species. This happens due to the low fidelity, error-prone properties of the DNA... 

Casanova Most of the Mendehan mutations discovered in the field of primary immunodeficiencies have been recessive. It could take quite some time before they would be counter selected by natural selection. The few but increasingly recognized dominant mutations that I did not discuss today can be counter-selected in populations at a higher rate, but there are still de novo mutations. There are even a few multiplex kindreds, with patients in multiple generations. In most cases the children have died before bearing their own children. Theoretically what you say is possible, but I don t have examples in mind of infectious disease-predisposing human mutations for which we know a factor that has acted as a positive selection factor. On the other hand, we know deleterious mutations that have been selected because they protect from some infectious diseases, such as the sickle cell trait and malaria. 

Zhang, X.Z. et al (2006) mazF, a novel counter-selectable marker for unmarked chromosomal manipulation in Bacillus subtilis. Nucleic Acids Res., 34 (9), e71. 

Heap, J.T., Ehsaan, M., Cooksley, C.M., Ng, Y.-K. et al (2012) Integration of DNA into bacterial chromosomes from plasmids without a counter-selection marker. Nucleic Acids Res., 40, e59. 

Begemann, M.B., Zess, E.K., Walters, E.M. et al. (2013) An organic acid based counter selection system for cyanobacteria. PLoS One, 8 (10), e76594. 

An earlier example of a coenzyme acting as a receptor is the porphyrin molecule of cytochrome oxidase. The lethal action of hydrogen cyanide, counter-selective for mammals, follows directly from the binding of this poison to the free valence of the chelated iron in the porphyrin. Many bacteria, lacking this enzyme, are not affected. 

Other ansamycins are found in Nature, e.g. streptovaricin (from another Streptomyces) which acts in the same way as rifamycin but is not so selective amanitin (from the fungus Amanita phalloides) which is counter-selective, inhibiting eukaryotic but not prokaryotic DNA-dependent RNA polymerase and maytansine (from the bark of an African flowering plant) which has anti-cancer properties but is not established in the clinic. 

---