8/3/2019 23_phage

1/16

Cloning Vectors

plasmids

viruses/phage lambda () filamentous/M13

(ssDNA, display) combinations

Recombinant DNA

Major Limitation of Plasmids transformation is inefficient

105-109 colonies per g DNAfor heat shock 109 -1010 colonies per g DNA

for electroporation 1 g of a 5 kb plasmid = 2 x1011 copies

efficiency with size (10-20 kb maximum)

8/3/2019 23_phage

2/16

large phage that infects E. coli

phage attaches to bacteria and

injects DNA complex genetics and life

cycle with two phases:

lytic lysogeny

Bacteriophage

8/3/2019 23_phage

3/16

8/3/2019 23_phage

4/16

as a Cloning Vector infectious can be assembled in vitro foreign DNA can be incorporated into

the genome non-essential genes removed

phage assembly can occur with 40-52kb of DNA (wild-type 50kb)

13 kb stuffer fragment

(lysogeny genes) discarded

accommodates 11-20 kbforeign DNA

Insertion Vector Replacement Vector

accommodates up to 7-

10 kb foreign DNA

(depending on vector)

8/3/2019 23_phage

5/16

1 Prepare foreign DNA

2 Prepare vector DNA

3 Mix vector, foreign

DNA and ligase

4 In vitro packaging

5 Infect host E. coli

6 Screen plaques7 Plaque purification

8 Subclone fragment

into plasmid

Cloning in Target DNA

digest gDNA

make cDNA

Vector DNA

purchase pre-cut anddephosphorylated arms add adapters if needed

EcoRI BamHI

AATTCGAACCCCTTCG GATCCNNNNNN----GCTTGGGGAAGCCTAG GNNNNNN----

8/3/2019 23_phage

6/16

1 Prepare foreign DNA

2 Prepare vector DNA

3 Mix vector, foreign

DNA and ligase

4 In vitro packaging

5 Infect host E. coli

6 Screen plaques7 Plaque purification

8 Subclone fragment

into plasmid

COSLLLLLLLLG AATTCFFFFFFFG AATTCRRRRRRRRR

||||||||| ||||||||| ||||||||||

LLLLLLLLCTTAA GFFFFFFFCTTAA GRRRRRRRRRCOS

Cloning in

8/3/2019 23_phage

7/16

8/3/2019 23_phage

8/16

rDNA + packagingextract phage particlesself assemble

In Vitro

Packaging

8/3/2019 23_phage

9/16

1 Prepare foreign DNA2 Prepare vector DNA

3 Mix vector, foreign

DNA and ligase4 In vitro packaging

5 Infect host E. coli

6 Screen plaques7 Plaque purification

8 Subclone fragment

into plasmid

Library: mixture of phage with

different DNA inserts

Cloning in

8/3/2019 23_phage

10/16

Cloning in 1 Prepare foreign DNA

2 Prepare vector DNA

3 Ligation reaction4 In vitro packaging

5 Infect host E. coli

(plate on lawn)

6 Screen plaques

7 Plaque purification

8 Subclone fragment

into plasmid

Plaque: clear zone on bacterial

'lawn' cause by lytic phage

8/3/2019 23_phage

11/16

Plaque Lift

8/3/2019 23_phage

12/16

Cloning in 1 Prepare foreign DNA

2 Prepare vector DNA

3 Ligation reaction

4 In vitro packaging

5 Infect host E. coli(plate on lawn)

6 Screen plaques

7 Plaque purification8 Subclone fragment

into plasmid

amplify cloned phage

purify phage DNA

excise insert ligate into plasmid

8/3/2019 23_phage

13/16

Identifying Recombinants

based on interruption of a gene eg., lacZ gene = -galactosidase intact -galactosidase produces

blue color in presence of X-gal

-complementation or blue-white screening

8/3/2019 23_phage

14/16

mixed synthetic oligonucleotidesbased on partial protein sequence

antibodies

Probes Based on Protein

Codons for Hypothetical HexapeptideMet Trp Glu Leu Ile Ala Gly

ATG TGG GAA (87) CTT (11) ATT (40) GCT (42) GGT (47)GAG 13 CTA (7) ATA (53) GCA (43) GGA 46

CTG (2) ATC (7) GCG (4) GGG (3)CTC (2) GCC 11 GGC (3)

TTA (64)TTG (13)

A-T-G-T-G-G-G-A-R-Y-T-N-A-T-H-G-C-N-G-G-N

1x1x1x1x1x1x1x1x2x2x1x4x1x1x3x1x1x4x1x1x4 = 768

8/3/2019 23_phage

15/16

Screening Expression Libraries

prepare library in expression vector

cDNA better than gDNA

(introns and reading frames) screen with protein based probe

antibody/western blot

ligand binding or other activity

gt11

8/3/2019 23_phage

16/16