laboratory : purify plasmid & restriction lecture : restriction mapping
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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. - PowerPoint PPT PresentationTRANSCRIPT
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 SalI
cut with SalI
cut with SalI
5’…G/TCGA C…3’3’…C AGCT/G…5’
5’ TCGAAGCT 5’
lux operon
5’ TCGAAGCT 5’
pBR322
Mix SalI restriction fragments.Add DNA ligase and ATP.
DNA ligase will join the moleculesat their annealed cohesive ends.
TCGAAGCT 5’
lux operon5’ TCGAAGCT
pBR322
DNA ligase + ATP
DNA ligase + ATP
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).
promoter luciferase gene
Reporter Gene
plant promoter luciferase gene
RNAPol transcription
lux mRNA
luciferase protein
veins bright capillaries veins dark
Transgenic tobacco leaves expressing luciferase
Image courtesy of Wayne M. Barnes
wound
plant promoter luciferase gene
RNAPol
WI TrFactor
wound-inducibletranscription factor
transcription
lux mRNA
luciferase protein
unwounded wounded
unwounded/woundedunwounded/wounded
unwoundedwounded
Transgenic tobacco leaves expressing luciferase
WoundInducible
Veins Bright
Veins Dark
Capillaries
Image courtesy of Wayne M. Barnes