Laboratory: Unit 4: purify amplicon ligate to plasmid transform; agarose gel (pages 84-86)...

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-