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 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