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Laboratory : Unit 4: purify amplicon ligate to plasmid transform; agarose gel (pages 84-86) Lecture : topoisomerase-mediated ligation; lacZ & alpha complementation In-Class Writing : edit sequences; Unit 3 (56-61) Read : Day, Chapter 27; manual 71-100, 213 - PowerPoint PPT Presentation
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Laboratory: Unit 4: purify amplicon ligate to plasmid transform; agarose gel (pages 84-86)
Lecture: topoisomerase-mediated ligation;lacZ & alpha complementation
In-Class Writing: edit sequences; Unit 3 (56-61)
Read: Day, Chapter 27; manual 71-100, 213
Hand In: editorial (pages xlii, section E & 96)
Due Next Class: draft of lab report 3 (pp. 109-115)
CCCTTGGGA
AGGGTTCCC
P
P
topoisomerase I
topoisomerase I
5’3’
3’5’
Topoisomerase-Bound Plasmid DNA
untemplatedA
AA
CCCTTGGGA
5’3’
AGGGTTCCC
3’5’
PCR Product
topoisomerase I
topoisomerase I
Alpha complementation"blue-white" screening
β-galactosidase (LacZ) = tetramer
α-peptide (aa 1-92) LacZ tetramers
ω-peptide (rest of LacZ) enzyme
wild-type -galactosidase tetramer
amino acids 11-41 of LacZ no tetramers no β-galactosidase
ω-peptide monomers inactive
α + ω peptides active LacZ tetramers
NH2- -COOH
NH2- -COOH
wild-type -galactosidase (LacZ)
LacZM15
Alpha complementation: Used in many vectors
Distinguish E. coli with: empty vector from vector + foreign DNAvector + foreign DNA
Plasmid has: lac promoter/operator restriction sites for cloning 5' end of lacZ gene LacZ α-peptide
Plasmid (NO insert) LacZ αE. coli LacZ ω Lac+ (blue colonies)
Plasmid + foreign DNA NO LacZ αE. coli LacZ ω Lac- (white colonies)
pCR4
80lacZM15
active LacZ tetramer
E.coli LacZ ωplasmid LacZ
Medium: IPTG = isopropyl D-thiogalactoside (inactivates LacI Lac+ phenotype)
X-gal = colorless =5-bromo-4-chloro-3-indolyl-β-D-galactoside
Cl
Br
NH
OHO
O
OH
HOCH2
OH
X-Gal
E.coli LacZ ωplasmid LacZ
LacZ cleaves β-D-galactoside bond indole ring released 2 indole rings indigo blue colonies
Cl
Br
NH
O
Cl
BrNH
O
indigo
E.coli LacZ ωplasmid + insert NO LacZ
NO LacZ X-Gal not cleaved remains colorless white colonies
L agar + ampicillin+ X-gal + IPTG
Lac+
Lac-