Exconjugants were selected on MS agar [ 35] containing apramycin, and the products of double crossovers identified by screening for kanamycin sensitivity.
Selection for single crossovers and screening for the double crossovers was performed as described [60], [69].
Then, pLQ258 was introduced into strain 5008 by intergeneric conjugation, and single-crossover exconjugants were screened by thiostrepton resistance (TsrR).
Single crossovers of ΔptmR1/ΔptmU4 were selected by screening for apramycin resistance on ISP4 medium.
After another round of passaging the single-crossover exconjugants in solid ISP4 medium, the ΔptmR1/ΔptmU4 mutant SB12040, a result of double-crossover homologous recombination, was selected for by screening for an apramycin-resistant and kanamycin-sensitive phenotype.
Reflecting Holby City origins as a spin-off from Casualty and the closely related premises of the two programmes, the BBC has screened occasional crossover mini-dramas entitled Casualty@Holby City, featuring a number of characters from each of the two casts.
This, too, was a crossover moment - in the early 1970s, Hollywood was still largely segregated, with the urban Detroit movie houses screening only the latest "blaxploitation" hits such as Shaft, Super Fly and Hit Man.
Nevertheless, follow-up researchers suggested that this method is of low reproducibility and laborious to screen for double-crossover integration events [34], [38].
In C. thermocellum, a replicating allelic exchange vector was also adopted to delete targeted genes because the current transformation efficiency does not meet the requirement for genetic manipulation; therefore this method should be laborious to screen for double-crossover integration events [37].
The frequencies of the second recombination event differed between the four repeats (Fig. 2B), but on average, the frequency between days 3 and 9 remained between 4% and 13% of the total clones screened; therefore, double-crossover clones were readily isolated by replica plating 25 colonies.
