Expression Plasmids:

A focus on the Ti plasmid of Agrobacterium tumefaciens

 

What is a transgenic organism?

A transgenic organism contains genes foreign to its own genome (Griffiths 1999).  Such organisms can be used for research or for specialized commercial applications.  There are multiple ways to construct transgenic organisms.  Expression plasmids are widely used tools, especially in the genetic modification of plants.

 

What is an expression plasmid?

An expression plasmid is a circular piece of DNA that contains an origin of replication, the appropriate regulatory regions located 5’ to the site in which the gene of interest is inserted, and a criterion for selection.  The origin of replication is important to assure the replication of the plasmid within the host bacterium (Darnell 1990).  Containing the correct regulatory regions, such as promoters, allows for the transcription and translation of the inserted gene, ultimately resulting in the expression of the gene.  Having a mechanism for selection enables for the discrimination between self-ligated (which do not contain the gene of interest) and recombinant plasmids (which do contain the gene of interest).  Selectable bacterial genes, such as antibiotic resistance, are often used for the selection process. Expression is finally achieved when the plasmid is reinserted into bacteria, which have replication, transcription, and translation machinery (Griffiths 1999).

 

Why use expression plasmids with plant transformation?

One property of plants that facilitates genetic modification with expression plasmids is that the growing cells of a dissected or excised plant will regenerate in culture to form callus tissue.  Therefore, with the addition of plant growth hormones, development from the callus will occur, resulting in a new, whole, fertile plant.  Introducing new genes to the callus enables the new genes to be contained in all the newly developing cells (Darnell 1990).

 

Which plasmid is used? (illustration)

The bacteria Agrobacterium tumefaciens is a naturally occurring pathogen found in soil.  A. tumefaciens has the unique ability to transfer its DNA into a plant’s genome.  A. tumefaciens infection, normally occurring at the site of a wound in the plant, causes the formation of crown gall tumors (Hall 1 Sept 2002).  A large (200 kb) circular DNA plasmid is responsible for the production of the tumor and the transforming abilities.  This tumor inducing (Ti) plasmid transfers and inserts a region of the plasmid, called T-DNA, into the host chromosome.  The T-DNA transfer genes are located outside the T-DNA on the Ti plasmid.  The T-DNA region codes for the production of the tumor and the synthesis of opines (Griffiths 1999). 

 

How is the desired plasmid made?

Modifications are made to the plasmid in order to enable a variety of inserts.  The Ti plasmids are too large for easy manipulation and have few unique restriction sites to be readily made smaller.  Therefore, an intermediate plasmid that contains the insert of interest and other recombination segments, is integrated into the Ti plasmid.  This integration forms a cointegrate plasmid that is eventually introduced into a plant cell via transformation.  To begin, the Ti plasmid that will be the recipient of the intermediate vector is attenuated (right-hand (R) region of T-DNA is deleted).  The left-hand (L) region remains as the cross over site with the intermediate vector.  The gene of interest is inserted into the intermediate vector, which has a variety of restriction sites, making insertion of any cDNA possible.  The intermediate vector also contains the selectable bacterial genes (spcR) and (kanR) for spectinomycin and kanamycin resistance, respectively.  The vector also contains two segments of T-DNA; the nopaline-synthesis gene along with the right-hand T-DNA border, and a left-hand border region that facilitates recombination with a homologous part of region L in the disarmed Ti plasmid.  The integrate plasmid is then introduced into A. tumefaciens, which contain the disarmed Ti plasmid, by conjugation with E. coli.  The recombinant plasmids cointegrate are selected for by plating the A. tumefaciens on spectinomycin (Griffiths 1999).

 

 

How do you make the plant transgenic? (illustration)

Incubate the wounded plant with the genetically altered A. tumefaciens:  The plasmids are duplicated in the culture.  The A. tumefaciens transfers the new gene into one of the plant cell chromosomes.  As the plant cell divides subsequent cells also receive the new gene.  New shoots will develop from the callus.  Newly formed shoots are separated from the original wounded plant.  The transgenic plant develops into a full-grown plant in which all the cells contain the insert of interest (Hall 1 Sept 2002, Industry 3 Sept 2002).

 

Links

Bibliography

 

 

This page has been created for Bio 361, an undergraduate GMO course at Davidson College.

© Copyright 2002 Department of Biology, Davidson College, Davidson, NC 28035
Send comments, questions, and suggestions to: mosiegenthaler@davidson.edu