is a plasmid? A plasmid is an extra-chromosomal element, often a circular
DNA. The plasmids we will use in this class typically have three important elements:
Since a plasmid is (by definition) an extrachromosomal element, it cannot make use
of any origin of DNA replication in a chromosome. That is, DNA synthesis within
(i.e. copying of) a plasmid depends on its having an origin of DNA synthesis of its
own. Obviously, if a plasmid couldn't be copied, it would be rapidly diluted out
in a population of dividing cells because it couldn't be passed on to daughter cells.
A selectable marker is not actually a required element of a plasmid, but it makes
it possible for us to maintain stocks of cells that contain the plasmid uniformly.
Sometimes, carrying a plasmid puts a cell at a selective disadvantage compared
to its plasmid-free neighbors, so the cells with plasmids grow more slowly. Cells
that happen to "kick out" their plasmid during division may be "rewarded"
by having a higher rate of growth, and so these plasmid-free (sometimes referred
to as "cured") cells may take over a population. If
a plasmid contains a gene that the cell needs to survive (for example, a gene encoding
an enzyme that destroys an antibiotic), then cells that happen to kick out a plasmid
are "punished" (by subsequent death) rather than "rewarded" (as
in the previous scenario). That selective pressure helps to maintain a plasmid in
A cloning site is not required at all, but it sure is nice to have! What I mean by
"cloning site" is a place where the DNA can be digested by specific restriction
enzymes - a point of entry or analysis for genetic engineering work. This is a matter
we will be discussing in great detail at a later point. For now, think of the following
example: Suppose you are really thirsty and you buy a can of soda. Does it occur
to you that one end of the can (the "top") is designed so that you can
open it easily? If you bought a can of soda with two bottom ends and no top, you
would have a hard time drinking it! It's the same way with plasmids. You can have
a plasmid with lots of terrific features, but you might lack an easy way of "getting
it open" with restriction enzymes.
A plasmid is an extra-chromosomal element, often a circular DNA. The plasmids we will use in this class typically have three important elements:
|Coiling in a plasmid||
You probably remember that double-stranded DNA has the form of a "double helix" which looks a bit like a telephone handset cord (except that the telephone cord is a single helix). You may also recall that the double helix is right-handed (for an expose on the difference, take a look at the Left Handed DNA Hall of Fame Site.)
You've probably also noticed how knotted up a telephone cord can get, if your roommate twists the handset around a few times before hanging up. Those knots are a higher order structure that lead to "coiled coils."
DNA has the
same problem, though your roommate isn't to blame this time! Aside from the double-helical
structure that we all know and love, DNA can take on a higher order coiling that
twists one double helix around another. We call this "superhelical coiling"
or simply "supercoiling." In a linear molecule these twists can
unravel by themselves, provided the ends are not prevented from rotating. In a circular
molecule with no free ends, the superhelical twists are "locked in" and
the molecule cannot relax. This coiling is not the same as the right-handed double
helix coil with which you are all familiar. The supercoiled molecule is a coiled
What's needed to
get supercoiled circular DNA to relax? A few weeks of pampering at a spa perhaps?
No! If one of the two strands is broken so that it has free 5' and 3' ends, the supercoils
can relax even though the overall structure of the molecule remains a circle. The
free ends of the broken strand rotate around the phosphate backbone of the intact
strand (the one that wasn't broken). This loss of superhelical stress puts the plasmid
into a "relaxed
a vector? Plasmids are sometimes
called "vectors", because they can take DNA from one organism
to the next. Not all vectors are plasmids, however. We commonly use engineered viruses,
for example bacteriophage lambda, which can carry large pieces
of foreign DNA. Left to right orientation
Right to left orientation
Why do we use the word "vector," which we've been trying to forget ever
since we took Physics 100? The word has a connotation of taking something from one
place to another. A mosquito is said to be a "vector" for malarial parasites,
and a velocity "vector" in physics indicates a direction in which an object
is travelling. In molecular biology, a "vector" is a piece of DNA that
may be introduced into a cell, usually after we've played around with it a bit in
a test tube.
One important concept is that depending on the cloning strategy employed, a gene
could be inserted into the plasmid in either of two orientations:
Perhaps we don't care which orientation we obtain as our final product, but we should note that there is a fundamental difference between the two. The arrow in the diagram shows the direction of transcription/translation of the "red gene" coding sequence, and the two orientations differ with respect to the outside markers Amp and ori.
Plasmids are sometimes called "vectors", because they can take DNA from one organism to the next. Not all vectors are plasmids, however. We commonly use engineered viruses, for example bacteriophage lambda, which can carry large pieces of foreign DNA.
Left to right orientation
Right to left orientation
How do we clone a plasmid?
|How do we isolate a plasmid we want?||
|Transformation is natural.||Bacteria naturally take up DNA from their environment, and we call that process transformation.|
|Efficiency of transformation in the lab.||
|Selection||After transformation, we challenge the bacteria with an antibiotic
(such as ampicillin). If the E. coli have taken up and expressed an ampicillin resistance
gene on a plasmid, they will live - otherwise they will die. This process is called
selection, because we are
selecting which bacteria may survive.
Transformation is a rare event, so most bacteria in an experiment are killed by the antibiotic. If a bacterium takes up a piece of DNA that cannot be maintained in a cell (e.g. if it lacks an origin of DNA replication) that cell also will not survive. It's a tough world!
|Screening||At this stage we have a bacteriological plate (agar medium
containing ampicillin) with bacterial colonies on it. Each colony contains a different
plasmid type, because each was grown up from a single transformed cell. What we do
now is to isolate DNA from each colony (or a small growth of cells propagated from
the colony), and analyze the structure of the plasmid with restriction enzymes or
by DNA sequencing. We can use gel electrophoresis to identify the sizes of restriction
fragments that are released from the plasmid and to check the purity of the preparation.
If you are unfamiliar with the principles of gel electrophoresis, you may be helped by this explanation (in which fish and DNA are one and the same)