By John N.Abelson, Melvin I.Simon
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Extra info for Advanced Bacterial Genetics
This provides a simple screen on arabinose‐utilization plates for the presence or absence of the Tn10dTc‐araCþ derivative. Plan of the Experiment Isolation of Mud Insertions in and Tn10‐araCþ Near Genes of Interest Mud: The random mutagenesis of Salmonella by MudJ transposition is described in Chapter 7. MudJ transposition is selected by selecting for MudJ‐encoded KmR. When the selection for MudJ insertions is done on rich medium, about 4% of the insertions are in auxotrophic genes. , 1980). For some pathways such as the his biosynthetic genes, all the genes are in a single nine‐gene operon; consequently, the chromosomal location of a his::MudJ insertion is known immediately.
Because the duplication harbors two tandem copies of this large chromosomal segment, cells grown in the absence of tetracycline lose the duplication at a high frequency, and are visualized as sectored colonies on arabinose‐indicator plates because segregation events result in the loss of the Tn10dTc‐araCþ element at the joint point. When the duplicated strain is plated in triphenyl tetrazolium chloride‐arabinose (TTC‐ Ara) medium, the resulting colonies produce white (Araþ) and red (AraÀ) sectors as well as red‐only (AraÀ) colonies.
Insertions defective in carbon or nitrogen source utilization must be screened for according to the specific carbon or nitrogen source. Tn10dTc‐araCþ: Plasmid pDH10 was transformed into Salmonella strain SL4213 (a restriction‐deficient Salmonella strain). , 1984). Transposition recombinants were selected for by selecting for Tn10dTc‐araCþ‐encoded TcR. Tens of thousands of TcR transposition recombinant colonies were pooled, and a P22‐transducing lysate prepared on the randomized pool of insertion mutants.
Advanced Bacterial Genetics by John N.Abelson, Melvin I.Simon