Bacterial Transformation and Plasmid Recovery

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Exercise 5




      Bac terial
 Transformation and
  Plasmid Recovery




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INTRODUCTION

When Frederick Griffith observed the conversion of Pneumococcus colonies
from `rough' to `smooth', he did not realize that the change was due to the
uptake of DNA from the dead `smooth' cells by the live `rough' cells. Even
Avery and his co-workers, when they showed that the transforming agent
was DNA, did not foresee the significance of this technique. This process,
transformation, has become one of the key techniques in genetic
engineering. Transformation can be defined as the uptake of exogenous, or
foreign, DNA by a recipient cell and the insertion of that DNA into the
recipient cell's genome, resulting in a new and heritable trait.

Bacterial cells must be in a particular physiological state before they can be
transformed. This state is referred to as competency. Competency can occur
naturally in certain species of Haemophilus and Bacillus when the levels of
nutrients and oxygen are low. Competent Haemophilus express a membrane-
associated transport complex, which binds and transfers certain DNA
molecules from the medium into the cell where they are incorporated and
their genes are expressed. In nature, the source of the external DNA is cells
that have died and released their DNA.

Much of the current research and experimentation in molecular biology
involves the transformation of E. coli. However, this organism does not
enter a stage of natural competency. E. coli can be artificially induced to
enter competency by treating the cells with the chloride salts of the metal
cations calcium, magnesium and rubidium. In addition, sudden cycles of
heat and cold help to bring about competency. The metal ions and
temperature changes affect the structure and permeability of the cell wall
and membrane so that DNA molecules can pass through. Competent E. coli
cells are fragile and must be treated carefully.

The number of cells transformed per 1 microgram of DNA is called the
transformation efficiency. In practice, much smaller amounts of DNA are
used (5 to 100 nanograms) since too much DNA inhibits the transformation
process. In research laboratories, transformation efficiencies generally range
from 1 x 104 to 1 x 107 cells per microgram of DNA. There are special
procedures that can produce cells having transformation efficiencies
approaching 1010. Transformation is never 100% efficient. Approximately 1
in every 10/000 cells successfully incorporates the DNA in preparations
having average competency. However, there is such a large number of cells

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in a sample (typically 1 x 109) that only a small fraction needs to be
transformed to obtain colonies on a plate. These ideas can be demonstrated
by plating the same volume of recovered cells on selective and non-selective
agar media. The nonselective media will have many more growing cells. In
this case, all the untransformed cells also survive. The bacterial agar plates
will be covered heavily with untransformed cells forming a "lawn", in
contrast to individual colonies. Because such a small percentage of even
competent cells ever take up DNA, one of the problems facing geneticists is
how to identify and keep the rare transformed cells, while getting rid of the
rest.

The most common solution to this problem is to use plasmids. A plasmid is
an extrachromosomal, circular piece of DNA that usually has very specific,
and useful, properties. Plasmids naturally exist as supercoiled molecules.
The two strands of DNA in the supercoiled molecule are wound and folded
around each other in a way that produces a condensed, entangled structure
when compared to relaxed (non-supercoiled) DNA. Competent E. coli are
sensitive to the conformation of the DNA they will accept. Supercoiled
DNA gives the highest transformation efficiencies. Most plasmids used
nowadays have been genetically engineered to contain some very useful
features:




The origin of replication tells the bacterial cell that this piece of DNA should
be copied. In most plasmids this causes the cell to make dozens or even
hundreds of copies of the plasmid. This type of plasmid is called a `high-
copy-number' plasmid.

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