1. 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 , 213
Hand In: editorial (pages xlii, section E & 96)
Due Next Class: draft of lab report 3 (pp )

4. untemplated A

8. Alpha complementation "blue-white" screening
β-galactosidase (LacZ) = tetramer
α-peptide (aa 1-92)  LacZ tetramers
ω-peptide (rest of LacZ)  enzyme

10. no tetramers  no β-galactosidase ω-peptide monomers inactive
D amino acids of LacZ 
no tetramers  no β-galactosidase
ω-peptide monomers inactive
α + ω peptides  active LacZ tetramers

12. Alpha complementation:
Used in many vectors
Distinguish E. coli with:
empty vector from
vector + foreign DNA

13. Plasmid has:
lac promoter/operator
restriction sites for cloning
5' end of lacZ gene  LacZ α-peptide

15. Plasmid (NO insert)  LacZ α
E. coli  LacZ ω
 Lac+ (blue colonies)
Plasmid + foreign DNA NO LacZ α
 Lac- (white colonies)

18. E.coli  LacZ ω plasmid  LacZ a Medium:
IPTG = isopropyl b-D-thiogalactoside
(inactivates LacI  Lac+ phenotype)
X-gal = colorless =
5-bromo-4-chloro-3-indolyl-
β-D-galactoside

19. X-Gal

20. E.coli  LacZ ω
plasmid  LacZ a
LacZ cleaves β-D-galactoside bond
 indole ring released
 2 indole rings  indigo
 blue colonies

21. indigo

22. E.coli  LacZ ω
plasmid + insert  NO LacZ a
NO LacZ  X-Gal not cleaved
 remains colorless
 white colonies