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Streptomyces integrating vector e.g. pSET152

Streptomyces integrating vector e.g. pSET152. ori E. coli. Res. Streptomyces chromosome. int. PP = att P. BB = att B. transformation or conjugation. B P. P B. Do we always obtain a single copy of the vector integrated into BB?.

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Streptomyces integrating vector e.g. pSET152

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  1. Streptomyces integrating vector e.g. pSET152 ori E. coli Res Streptomyces chromosome int PP =attP BB=attB transformation or conjugation BP PB Do we always obtain a single copy of the vector integrated into BB?

  2. E. coli plasmids from rec- strains are mostly dimers. 1 2 ori E. coli BP PP PB Res int PP How can we find out?

  3. Some streptomycetes have several inefficientbb bb bb How can we find out? bP bP Pb Pb

  4. Sequence the unknown flanking regions R Restriction digest R R R R R R bP bP Pb Pb

  5. R R bP Pb ori E. coli LM-PCR Ligation-mediated PCR Res int PP b R b

  6. Summary Integrating vectors often for tandem inserts Some streptomycetes lack a perfect BB, and insertion occurs into multiple, secondary bbs This can be important if experiments need to be reproducible.

  7. Complementation: testing whether a mutation of a particular gene causes the observed phenotype. E.g. does a gene knockout have a polar effect on downstream genesin an operon? mRNA

  8. Mutant phenotype Wild-type phenotype ? integrated or on plasmid Can we be sure that the downstream gene is not involved ?

  9. homogenotization homo-geno-tization up to 5% !! and How can we prove that complementation was correct?

  10. homogenotization homo-geno-tization up to 5% !! and How can we prove that complementation was correct? Is there another possible gene arrangement?

  11. Integrating plasmids, dimers and bbs Homogenotization

  12. Kleinia a species of Mangrove

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