Download lacZ

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)
Topoisomerase-Bound Plasmid DNA
topoisomerase I
5’
3’
CCCTT
GGGA
P
P
AGGG
TTCCC
topoisomerase I
3’
5’
untemplated
A
topoisomerase I
5’
3’
CCCTT
GGGA A
PCR Product
A AGGG
TTCCC
3’
5’
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








D amino acids 11-41 of LacZ 
no tetramers  no β-galactosidase
ω-peptide monomers inactive
α + ω peptides  active LacZ tetramers
wild-type -galactosidase (LacZ)
NH2-


-COOH
LacZDM15
NH2-


-COOH
Alpha complementation:
Used in many vectors
Distinguish E. coli with:
empty vector from
vector + foreign DNA
Plasmid has:
lac promoter/operator
restriction sites for cloning
5' end of lacZ gene  LacZ α-peptide
Plasmid (NO insert)  LacZ α
E. coli  LacZ ω
 Lac+ (blue colonies)
Plasmid + foreign DNA NO LacZ α
E. coli  LacZ ω
 Lac- (white colonies)
pCR4

active LacZ tetramer

80lacZDM15
D

D
E.coli  LacZ ω
plasmid  LacZ 
Medium:
IPTG = isopropyl -D-thiogalactoside
(inactivates LacI  Lac+ phenotype)
X-gal = colorless =
5-bromo-4-chloro-3-indolylβ-D-galactoside
X-Gal
HOCH2
O
HO
Cl
O
Br
OH
OH
N
H
E.coli  LacZ ω
plasmid  LacZ 
LacZ cleaves β-D-galactoside bond
 indole ring released
 2 indole rings  indigo
 blue colonies
indigo
Cl
O
N
H
Br
Br
O
N
H
Cl
E.coli  LacZ ω
plasmid + insert  NO LacZ 
NO LacZ  X-Gal not cleaved
 remains colorless
 white colonies
L agar
+ ampicillin
+ X-gal
+ IPTG
Lac+
Lac-