Plasmids and Vectors

Instructor Supplement to pGlo Bacterial Transformation

A more detailed look at plasmids

Origin of Replication

Multiple Cloning Site

Promotor Site

Antibiotic Resistance Gene

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 Subsequent modifications

1986 expanded to include animals and plants, and 4 biosafety levels

1994 officially relinquished control of GMO plants in the environment to EPA and APHIS

The First “Safe” Bacterium

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

E. coli 1776 Required diaminopimelic acid (DAP) Fragile cell walls (low salt, detergent

sensitive) Difficult to work with Slow grower Poor receptor for transformation

In the 1970’s and 1980’s

The first cloning vectors such as pSC101 had limited functionality

The next trend was to develop smaller plasmids

Advantages Increased efficiency of

transformation Easier to restriction map Higher copy numbers

The Cadillac of Cloning Vectors

pBR322 Clone fragment in one

antibiotic gene Select for other antibiotic

resistance Screen for presence of

one resistance gene (selects against untransformed bacteria) and loss of resistance to interrupted antibiotic resistance gene (selects for recombinant molecule)

pBR322

4,361 bp

EcoRI

TetR

AmpR

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 bacteriao In 1971 Alan Campbell showed that the central third of the genome was not required for lytic growth. People started to replace it with E. coli DNA

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

Bacteriophage lambda

Protein capsule of lambda has a tight constraint on the amount of DNA that will fit inside it (~ 55kb)

By the early 1970’s we knew that a good portion of lambda was not required

“Junk” DNA

COS site: Cohesive “sticky” ends

Lysis

Lysogeny

Head

Tail

Replication

Circularized lambda

ori

Not Quite Bacteriophage lambda

Eliminate the non-essential parts of lambda

Can now insert large pieces of DNA (~ 20 kb)

COSLysis Head

Tail

Replicationori

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 phage capsule

Somewhat unstable, difficult to maintain

cos

TetR

EcoRI

21.5 kb

ori

Cos site is the only requirement for packaging into phage particle

Cosmids

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 of interest?

Screen A DNA library: Genomic cDNA

Use Polymerase Chain Reaction (PCR) to clone gene of interest

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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 librariesby colony hybridization

Polymerase Chain Reaction (PCR)

Agarose gel electrophoresis

Restriction Mapping