Transformation is a parasexal method of introducing new genes into an
organism. These transgenes may give the organism new functions. Examples
of a new function may be antibiotic resistance, new metabolic capabilities,
or a change in growth and shape. Often the gain of function allows
selection of the organisms that have gained this function against a large
background of those that have not. Transgenic organisms may be selected by
the ability to grow on a new food source, by the ability to withstand a
metabolic poison (antibiotic), or by a change in rate of growth or in
morphology, such as change in color or shape that may be seen by eye. Here
we will give E. coli new traits by transformation with a
plasmid DNA carrying a functional gene. One trait is ampicillin resistance
and the other trait is a new color due to the presence of the pigment,
carotenoid. The molecular nature of each new function and their phenotypes
are discussed below.
Ampicillin resistance is conferred to the bacterium by transforming it
with a plasmid carrying the gene for lactamase. Lactamase is an enzyme
that cleaves and destroys chemical structures with lactam rings, such as penicillin
and ampicillin. These antibiotics stop bacterial growth by interfering
with the synthesis of the bacterial cell wall. The lactamase must be secreted
by the bacterium where it destroys the antibiotic in the surrounding
medium. This allows the bacterium to synthesize a cell wall and thus grow.
Here the phenotype conferred by the lactamase gene is growth in the
presence of ampicillin.
These genes are carried on a plasmid named pMPDS3. It has
the vector backbone pBluescript. This bacterium is
light yellow and produces zeta-carotene. Thus, transformants will be able to grow on
ampicillin-containing plates.
The protocol below describes simply how to put a plasmid into E.
coli and select for transformants. The genetic engineering has been
done for you.
