Bacterial Plasmids

Bacterial Plasmids

What 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:

An origin of replication
A selectable marker gene (e.g. resistance to ampicillin)
A cloning site (a place to insert foreign DNAs)

Origins of replication: 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.

Selectable markers 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 population.

Cloning sites 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.

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 coil.

You can click on the image below to see an electron micrograph of a supercoiled circular DNA.

Supercoiled DNA (Bock lab)

Relaxation
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 DNA" form.

Another electron micrograph: This one is of relaxed DNA

Relaxed DNA (Bock lab)

What is 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.

Bacteriophage lambda (Bock lab)

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.

Orientation One important concept is that depending on the cloning strategy employed, a gene could be inserted into the plasmid in either of two orientations:

Left to right orientation

Right to left orientation

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.

How do we clone a plasmid?

How do we isolate a plasmid we want?

We introduce the reclosed (ligated) products into E. coli, a process called "transformation", and select for bacteria resistant to a drug (such as amipicillin, for example).
We screen the individual bacterial colonies to find one that contains a plasmid of the correct structure.

Transformation is natural. Bacteria naturally take up DNA from their environment, and we call that process transformation.

Efficiency of transformation in the lab.
When we are transforming DNA in the laboratory (i.e. for experimental purposes), we have several ways of making the uptake of DNA by E. coli cells more efficient.

One method is to starve the cells in ice-cold calcium chloride solution, add a sample of ligated DNA, and "heat-shock" the cells at 42 degrees C for a short period of time - about 45 seconds. The fluidity of the membrane increases to the point where DNA is taken up by the cell.
A second method is to deliver an electric shock to the cell, releasing a charged capacitor with a field strength in the sample of approximately 1200 volts per millimeter. The DNA is swept into the cells as the membranes are temporarily breached. This process is called electroporation.

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)

	A plasmid is an extra-chromosomal element, often a circular DNA. The plasmids we will use in this class typically have three important elements: An origin of replication A selectable marker gene (e.g. resistance to ampicillin) A cloning site (a place to insert foreign DNAs)
Origins of replication:	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.
*Selectable markers*	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 population.
Cloning sites	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.

How do we isolate a plasmid we want?	We introduce the reclosed (ligated) products into E. coli, a process called "transformation", and select for bacteria resistant to a drug (such as amipicillin, for example). We screen the individual bacterial colonies to find one that contains a plasmid of the correct structure.
Transformation is natural.	Bacteria naturally take up DNA from their environment, and we call that process transformation.
Efficiency of transformation in the lab.	When we are transforming DNA in the laboratory (i.e. for experimental purposes), we have several ways of making the uptake of DNA by E. coli cells more efficient. One method is to starve the cells in ice-cold calcium chloride solution, add a sample of ligated DNA, and "heat-shock" the cells at 42 degrees C for a short period of time - about 45 seconds. The fluidity of the membrane increases to the point where DNA is taken up by the cell. A second method is to deliver an electric shock to the cell, releasing a charged capacitor with a field strength in the sample of approximately 1200 volts per millimeter. The DNA is swept into the cells as the membranes are temporarily breached. This process is called electroporation.
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)