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Transposable Mutagenesis for an E. coli Methionine or Histidine Synthesis Gene

Transposable Mutagenesis for an E. coli Methionine or Histidine Synthesis Gene. Jon Kissel Mary Maschek. Transposon mutagenesis. Mutating prototrophic bacteria to become auxotrophic using a transposon Making plasmid library from bacterial genome Sequencing plasmid with interrupted gene.

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Transposable Mutagenesis for an E. coli Methionine or Histidine Synthesis Gene

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  1. Transposable Mutagenesis for an E. coli Methionine or Histidine Synthesis Gene Jon Kissel Mary Maschek

  2. Transposon mutagenesis • Mutating prototrophic bacteria to become auxotrophic using a transposon • Making plasmid library from bacterial genome • Sequencing plasmid with interrupted gene

  3. Transposons • Mobile DNA elements • Randomly insert into genome • Can contain resistance genes

  4. Types of transposons • Size • Number of genes

  5. Bacteriophage Mu • Lysogenically inserts into bacterial genome • Inserts at multiple locations • Can recombine out of genome and form a plasmid library

  6. Plasmid Libraries • Contain bacterial genome • Individual genes are located on each plasmid • Can be used to transform wild-type bacteria

  7. Inserting the Transposon • EZ::TN™ <R6Kγori/KAN-2>Tnp Transposome™ Kit • Electroporation

  8. EZ::TN™ <R6Kγori/KAN-2>Tnp Transposon Epicentre EZ::TN™ <R6Kγori/KAN-2>Tnp Transposome™ Kit protocol

  9. Finding the Auxotroph • Plate cells on minimal medium + met/his • Replica plate on minimal medium • Select for colonies that don’t grow

  10. Making the Library • DNA purification • Restriction digest • End-It DNA Repair Kit • DNA ligation

  11. Finding Resistance • Mix library with wild-type E. coli • Plate on kanamycin • Select for kanamycin-resistance

  12. Plasmid Purification • Good ole Biology 107 • Run on gel just to be sure

  13. Sequencing and BLAST • Send purified plasmid to IUPUI • Primers come with transposome kit that tell sequencers where to begin • BLAST returned sequence

  14. Epicentre EZ::TN™ <R6Kγori/KAN-2>Tnp Transposome™ Kit

  15. Timeline • September 13-20 – make plates, LB broth • September 20-27 – begin work with transposome and obtain auxotroph • September 27-October 15 – ligate genomic DNA, plasmid library ready • October 15-22 – isolate kanamycin-resistant colony • October 22-29 – isolate and purify plasmid • October 29-November 5 – enter sequence into BLAST • November 5 to December 8 – work on manuscript

  16. Budget • EZ::TN™ <R6Kγori/KAN-2>Tnp Transposome™ Kit $375? • End-It DNA Repair kit $70 • Sequencing at IUPUI $17

  17. Grade Agreement • Obtaining auxotroph 15 points • Purifying DNA 5 points • Digesting DNA 5 points • Assembly of plasmid library 10 points • Obtaining kanamycin-resistant colony 10 points • BLAST results give methionine or histidine synthesis gene 5 points

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