plasmids and vectors - Instructor Supplement to pGlo Bacterial Transformation

 People believed that “safe” strains of bacteria, viruses and vectors could be made in a few weeks NIH formed the Recombinant DNA Advisory Committee RAC  It took 1 year 1976 before t

Plasmids and Vectors

Instructor Supplement to pGlo Bacterial Transformation

A more detailed look at plasmids

Origin of

Replication

Promotor Site

Antibiotic

Resistance

Cloning into a Plasmid

 People believed that “safe” strains of bacteria, viruses and vectors could be made in a few weeks

 NIH formed the Recombinant DNA

Advisory Committee (RAC)

 It took 1 year (1976) before the first

“safe” (EK2 category) line of E coli

was released

 That year, RAC released a set of

guidelines requiring the use of safe bacteria

Asilomar Conference

NIH Guidelines

 Self Regulation in Science Milestone

 Contents

 Specified handling and construction processes

 Microorganisms containing recombinant DNA were prohibited outside of the laboratory

 Vectors that sexually move to “unsafe” bacteria was prohibited

The First “Safe” Bacterium

 Released in 1976 by Roy Curtiss III at the University of Alabama

 E coli χ 1776

 Required diaminopimelic acid (DAP)

 Fragile cell walls (low salt, detergent

In the 1970’s and 1980’s

pSC101 had limited functionality

smaller plasmids

 Increased efficiency of transformation

 Easier to restriction map

 Higher copy numbers

The Cadillac of Cloning Vectors

untransformed bacteria) and loss of resistance to interrupted antibiotic

resistance gene (selects for recombinant

molecule)

pBR322 4,361 bp

EcoRI

Tet R Amp R

APstI

BamHI

Screening bacteria by replica plating

Next Major Advance in Plasmid(ology)

 The inclusion of

polylinkers into plasmid vectors

 Polylinker is a tandem

array of restriction endonuclease sites in a very short expanse of DNA

 For example, pUC18’s

polylinker

 Sites for 13 RE’s

 Region spans the equivalent of 20 amino acids or 60 nucleotides

Source: Bio-Rad Laboratories

The Polylinker Advantage

 Unique sites (usually)

 Insert excision facilitated

 Restriction endonuclease mapping and Subcloning made easier

Another Major Advance: Blue-White Screening

•Small size

•Origin of replication

•Multiple cloning site (MCS)

•Selectable marker genes

•Some are expression vectors and have sequences that allow RNA polymerase to transcribe genes

•DNA sequencing primers

Features of many modern Plasmids

The Major Limitation of Cloning in Plasmids

 Upper limit for clone DNA size is 12 kb

 Requires the preparation of “competent” host cells

 Inefficient for generating genomic libraries as overlapping regions needed to place in proper sequence

 Preference for smaller clones to be transformed

 If it is an expression vector there are often

limitations regarding eukaryotic protein expression

Bacteriophage lambda (λ)

o A virus that infects bacteria

o In 1971 Alan Campbell showed that the central third

of the genome was not required for lytic growth People

started to replace it

Lambda genome is approximately 49

kb in length.

Only 30 kb is required for lytic growth.

Thus, one could clone 19 kb of

“foreign” DNA.

Packaging efficiency 78%- 100% of the lambda genome.

A complete animation of the lytic cycle:

http://www.blackwellpublishing.com/trun/artwork/Animations/Lambda/lambda.html

Circularized lambda

ori

Not Quite Bacteriophage lambda

 Eliminate the

non-essential parts of lambda

 Can now insert

large pieces of DNA (~ 20 kb)

COS

Tail Replication

ori

Lambda was great:

 Larger insert size

 Introducing phage DNA into E.coli by phage infection

is much more efficient than transforming E.coli with

plasmid DNA

 Have to work with

plaques

But:

 Hybrid vectors: plasmids that

contain bacteriophage lambda

cos sites

 DNA (~ 33-48 kb) cloned into

restriction site, the cosmid

packaged into viral particles

and these phages used to infect

E.coli

 Cosmid can replicate in

bacterial cell, so infected cells

grow into normal colonies

 Insert DNA limited by the

amount of DNA that can fit into

cos TetR

Other Vectors

 BACs (Bacterial artificial chromosomes)

 Large low copy number plasmids (have ori and selectable marker)

 Can be electroporated into E coli

 Useful for sequencing genomes, because insert size

100 - 300kb

 YAC (Yeast Artificial Chromosome)

 Can be grown in E.coli and Yeast

 Miniature chromosome (contains ori, selectable markers, two telomeres, and a centromere

 Can accept 200 kb -1000 kb; useful for sequencing

 Ti plasmids; to introduce genes into plants

 Expression vectors

How do you identify and clone a gene

Genomic Library

cDNA library

What can you do with a library?

 Can be used to complement a mutant (this is more common for research in bacteria).

 Can use it in a colony hybridization.

Screening libraries

by colony hybridization

Polymerase Chain Reaction

(PCR)

Agarose gel electrophoresis

Restriction Mapping