• Ability to replicate independently (so that a lot of copies could be generated)

• A recognition sequence for a restriction enzyme (so that we can introduce our DNA of interest)

• Reporter genes (to confirm we have successfully introduced the vector into the host cell)

• Small size in comparison with host’s chromosomes (for easy manipulation)

Characterstics of a vector

Recombinant DNA Technology

• Vectors– Generally plasmids or viruses

• Plasmids– Small circular DNA molecule– Introduced into bacteria by transformation

• Small size in comparison with host’s chromosomes (for easy manipulation)

• Ability to replicate independently (so that a lot of copies could be generated)

• A recognition sequence for a restriction enzyme (so that we can introduce our DNA of interest)

• Reporter genes (to confirm we have successfully introduced the vector into the host cell)

Characterstics of a vector

•Transform E.coli with plasmid

–Treat with CaCl2

–Makes walls more permeable to small DNA molecules–Can also be introduced by electroporation

• Two challenges remain: First, how can you make sure that all the

bacteria that is growing contain a plasmid?Second, how can you identify which of

the bacteria contains the recombinant plasmid.

• Bacterial plasmids carry two reporter genes to overcome these challenges.

• First problem is solved with the help of antibiotic (ampicillin) resistance on the plasmid vector.

Recombinant DNA Technology

Recombinant DNA Technology

Second challenge is solved with the help of lacZ gene.

Recombinant DNA Technology

Lac Z gene• Genetic indicator system

– From lac operon– Codes for enzyme beta-galactosidase– Cleaves beta-galactoside bonds

• Cleaves a synthetic beta-galactoside– 5-bromo-4-chloro-3-indoyl-beta-D-galactoside

(X-gal)– Galactose with a blue dye attached by a beta-

galactoside bond

Recombinant DNA Technology

X-gal (galactose + blue dye) is colorless

• If the beta-galactoside bond in X-gal is cleaved after taken up by the bacteria:– The dye is released from X-gal– Results in blue colonies of bacteria

• Why?– The lac Z is not interrupted – Beta-galactosidase is produced – X-gal is cleaved releasing the dye– The colonies are blue

Recombinant DNA Technology

X-gal (galactose + blue dye) is colorless

• If the lac Z gene is interrupted with a foreign DNA– The gene is inactivated (the beta-galactosidase is

inactive)– The dye is not released– The colonies are white

• Final confirmation is obtained by retrieving the plasmid DNA from E. coli cells and performing restriction digestion to examine cloned DNA

Viewing DNA Fragments

• Gels:– Are porous– Agarose (a polysaccharide from red algae)

• Use electrophoresis to separate molecules on the basis of:– Size and electrical charge

Eco RI Eco RI

+

Cut vector

Insert DNA

Size stds

DNA of interest can also be isolated by PCR amplification

• http://bcs.whfreeman.com/mga2e/pages/bcs-main.asp?s=003&n=84&i=168&v=category&o=|26|132|131.1|37|14|60|72|84|&ns=209&uid=0&rau=0

DNA of interest can also be isolated by PCR amplification