the method used to transform the linear substrate into the recombining cell. Selection/counterselection technique In some cases, one desires a deletion with no marker left behind, to make a gene fusion, or to make a point mutant in a gene. This can be done with two rounds of recombination. In the first stage of recombineering, a selection marker on a cassette is introduced to replace the region to be modified. In the second stage, a second counterselection marker (e.g. sacB) on the cassette is selected against following introduction of a target fragment containing the desired modification. Alternatively, the target fragment could be flanked by loxP or FRT sites, which could be removed later simply by the expression of the Cre or FLP recombinases, respectively. A novel selection marker "mFabI" was also developed to increase recombineering efficiency. Recombineering with ssDNA Recombineering with ssDNA provided a breakthrough both in the efficiency of the reaction and the ease of making point mutations. This technique was further enhanced by the discovery that by avoiding the methyl-directed mismatch repair system, the frequency of obtaining recombinants can be increased to over 107/108 viable cells. This frequency is high enough that alterations can now be made without selection. With optimized protocols, over 50% of the cells that survive electroporation contain the desired change. Recombineering with ssDNA only requires the Red Beta protein; Exo, Gamma and the host recombination proteins are not required. As proteins homologous to Beta and RecT are found in many bacteria and bacteriophages (>100 as of February 2010), recombineering is likely to work in many different bacteria. Thus, recombineering with ssDNA is expanding the genetic tools available for research in a variety of organisms. To date, recombineering has been performed in E. coli, S. enterica, Y. pseudotuberculosis, S. cerevisiae and M. tuberculosis. Red-Independent recombination In the year 2010, it has been demonstrated that ssDNA recombination can occur in the absence of known recombination functions. Recombinants were found at up to 104/108 viable cells. This Red-independent activity has been demonstrated in P. syringae, E. coli, S. enterica serovar typhimurium and S. flexneria. Applications and benefits of recombineering The biggest advantage of recombineering is that it obviates the need for conveniently positioned restriction sites, whereas in conventional genetic engineering, DNA modification is often compromised by the availability of unique restriction sites. In engineering large constructs of >100 kb, such as the Bacterial Artificial Chromosomes (BACs), or