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Laboratory : purify plasmid & restriction Lecture : restriction mapping

Laboratory : purify plasmid & restriction Lecture : restriction mapping In-Class Writing : rewrite sentences (pages 60-61) Hand In : flow chart 1 Read : Appl. Environ. Microbiol. 63: 4920-8, 1997. cut with Sal I. 5’…G/TCGA C…3’ 3’…C AGCT/G…5’. cut with Sal I. cut with Sal I.

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Laboratory : purify plasmid & restriction Lecture : restriction mapping

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  1. Laboratory: purify plasmid & restriction Lecture: restriction mapping In-ClassWriting: rewrite sentences (pages 60-61) HandIn: flow chart 1 Read: Appl. Environ. Microbiol. 63: 4920-8, 1997

  2. cut with SalI 5’…G/TCGA C…3’ 3’…C AGCT/G…5’ cut with SalI cut with SalI

  3. pBR322 5’ TCGA AGCT 5’ Mix SalI restriction fragments. Add DNA ligase and ATP. lux operon 5’ TCGA AGCT 5’

  4. DNA ligase will join the molecules at their annealed cohesive ends. DNA ligase + ATP pBR322 lux operon 5’ TCGA TCGA AGCT 5’ AGCT DNA ligase + ATP

  5. Transform ligated DNA into E. coli. Select ampicillin-resistant colonies. Only circular molecules survive; RecBCD nuclease destroys linear DNA. Transformants contain vector or vector + lux(either orientation).

  6. promoter luciferase gene Reporter Gene

  7. plant promoter luciferase gene transcription RNAPol lux mRNA luciferase protein

  8. Transgenic tobacco leaves expressing luciferase Image courtesy of Wayne M. Barnes veins bright capillaries veins dark

  9. WI TrFactor luciferase protein wound-inducible transcription factor wound plant promoter luciferase gene transcription RNAPol lux mRNA

  10. Transgenic tobacco leaves expressing luciferase Image courtesy of Wayne M. Barnes Veins Dark unwounded wounded Wound Inducible unwounded/wounded unwounded/wounded unwounded wounded Capillaries Veins Bright

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