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Making a mutant

Making a mutant. Goal: to create 3 strains of mutants in Yersinia pseudotuberculosis from previously generated strains of Yersinia pestis . Strains: Y. pestis Δ pmrA , Y. pestis Δ phoP , Y. pestis Δ pmrA Δ phoP. NWRCE.

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Making a mutant

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  1. Making a mutant

  2. Goal: to create 3 strains of mutants in Yersiniapseudotuberculosis from previously generated strains of Yersiniapestis. • Strains: Y. pestisΔpmrA, Y. pestisΔphoP, Y. pestisΔpmrAΔphoP

  3. NWRCE

  4. >y0677|basR|Yersinia pestis KIM|500 nt upstream|500 nt downstream|:c760025-761687 GTCTTCAGAACCATTGGTCATCAGCGTTTTGGGGTTCCGGGTACTTGGCGCTCGGGTGCCGATGCCGTTC GCCAAATTTTACGCCAAAAAGCAGGCGTTGATCTTGGTAACAGCATTGTGGTGGATGGATCTGGCCTATC GCGCCATAATCTTATTTCACCAGCAACAATGATGCAGGTTTTGCAATATATTGCTCAAAATGATCAAGAG CTTAATTTTATATCAATGCTGCCTCTAGCAGGATATGACGGTACTCTACGTTATCGTGGTGGCCTGCATG AAGCGGGGGTCAACGGTAAAGTTTCAGCGAAGACGGGTGCACTGCAAGGGGTTTACAATCTTGCCGGTTT CATTACTACCGCTAGTGGGCAACGTATGGCATTTGTACAATTTTTATCCGGCTATGCCGTTCCACCTGAA GACCAAAAAAATCGCCGAGCCCCCTTGGTTCGATTTGAGAGCCGCCTCTATAAAGATATTTATCAGAATA ACTGAGAGCTATGAAATTACTGATTGTTGAAGATGATGAACTGCTACAACGCGGTATAGCCATGGCGCTGACCAGTGAAGGCTATGTCTGCGACTGCGCAGCTACCGCCGCCGAGGCTCACAGTTTGCTGCAAACCAGTC AGTACAGCATGATTATTCTTGATCTCGGCCTGCCGGATCAGGATGGTACTCTGTTATTACGTCAATGGCG TCGCCAACATGTCACCCTGCCAGTACTCATCCTCACCGCACGTGATGCGCTTGAAGACAGGGTTGACGGG CTGGATGCCGGTGCTGACGATTATCTTGTCAAACCTTTTGCCTTGGCTGAATTACTGGCCCGGGTACGTG CTCTCATCCGCCGTTATCAGGGGCAGAGTGACAACCTGGTACAGCAAGATGACCTGAGCCTTAACTTGTC GACACAACAAGTCTGTTTACAAGGGCAGCCATTAGAGATAACCCCTAAAGAGTTTGCGATCCTCTCACGT CTAATCATGCGAGCTGGGCAAACAGTTAACCGCGAGTTGTTACAACAGGATCTATATACCTGGAATGACG ACCTTAGCTCTAATACGCTGGAAGTTCATATTCATAATTTGCGACGCAAACTGGGTAAAGATCGTATCCG AACCGTTCGAGGTATTGGTTATCGATTGGAAGCCTTATCATGATCAGCATGCGCCGTCGTTTGCTGTTAA TGCTGGCGCTGATTTTACTGGTGACCCAGTTGATCAGCGCATTCTGGCTATGGCATGAAAGTCAGGAACA AATAAGTTTTCTGGTCGATGAAACTCTTAGCGCAAAAGCCCGTAATGAGCAGGTTGATAAAGAAATAGCT GAGGCGATTGCCTCCCTGCTTGCACCGTCGCTAATCATGATGACAATCACTCTGCTGCTGTCTTTCTGGG CAATCAGTTGGATTATTCGTCCACTTGATCAATTACAACAAAAGTTGGCAGAACGCTCCGCAGATAACCT TTCCCCCTTGGTTGTTAACAGTGAAATGCAAGAAATTGTCTCTGTTACCTCAACACTCAATCAATTGTTA TTGCGACTTTCTAATACCATTCAGCAAGAACGGTTGTTTACCGCCGATGCCGCCCATGAACTGCGTACAC CACTCGCAGGAATACGCCTCCACCTAGAATTAATGGAAAAGCAGGGAATTGCC • Sequence Massager

  5. pCVD442 is R6K oriV (pirdependent). Must integrate into the genome to be replicated in a pir-strain Take PCR fragment, digest with enzymes (Xba1) and add CIP (optional) Ligate at 16°C overnight Transform by electroporation or heat shock

  6. pCVD442 is in B083 (pir+ host strain) • Plasmid is not self-transferrable • Need helper strain that has tra genes (B001) • Alternatively, place vector and gene into a pir+ host with tragenes. • Perform tri (bi) parental mating.

  7. Selection series: 1st round – BHI+Irg+cb, why? • 2nd round – LB + 10% sucrose, why? • 3rd round – Screen on LB and LB + cb, why?

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