This week you will first prepare competent cells of
, and then you will transform the
ligation reactions you performed last week into these competent cells.
In the transformation
process, each of the transformed bacterial cells will take up one circular DNA molecule
In this way, we can analyze the phenotype that the individual plasmids confer on the
bacteria, and find the plasmids that have the characteristics expected for the desired clones (in
this case they will be: Ampicillin resistant, Chloramphenicol resistant, and white on X-gal).
Since each transformed bacterial cell will divide to produce a colony, and each cell in the colony
will carry an identical copy of the plasmid, the transformation process also provides a method to
produce many copies (enough to analyze) of each of the plasmids in the ligation mixture.
Potential clones of the desired type will be initially chosen based on the phenotype they confer
upon the bacteria on plates.
Next week you will isolate the plasmid DNA from promising clones
so that we can analyze it in more detail (at the level of the genotype).
Transformation is a form of genetic exchange in which
is taken up
by a bacterial cell
Some bacteria (ie -
) are capable of taking up free DNA from the
environment naturally - Natural Transformation.
This is not true of
requires that the cells be first chemically treated to make them competent – Chemically-
However, since we know so much about
, and so many useful
strains have been developed,
is by far the organism of choice for performing plasmid
Unlike natural transformation systems,
can only be transformed by circular DNA
molecules that carry an origin of replication.
In other words, the DNA that is taken up by
must be able to replicate independently of the bacterial chromosome to be maintained in the cell.
can take up linear DNA, however, linear DNA will be degraded in the cell and the
nucleotides used to make more
DNA - this process is called “eating” not transformation.
Competent Cells of
We understand an enormous amount about the bacterium
However, the mechanism by which this bacterium becomes “competent” (able to take up DNA
from solution) for transformation, and the mechanism it subsequently uses to take up plasmid
DNA from its environment (transformation) are only beginning to be understood.
cells are made competent by harvesting cells in early logarithmic growth, then keeping the
cells very cold, and treating them with divalent cations, usually including calcium chloride.
our experiment, we will treat the cells with FSB (frozen storage buffer) which contains Calcium
Chloride as well as Potassium Chloride, Potassium Acetate and Glycerol.
The two potassium