We will now discuss how genes are transferred into a variety of different organisms, from microbes to maize and mammals. There are a variety of terms that are used in this area of research, so a little glossary is given below.
I will first discuss the methods and problems of transformation of E. coli. Many of the points raised in this discussion are equally relevant to the transformation of other organisms - fungi, plants, animals.
When talking about the discovery of DNA as the genetic material and the experiments of Griffith, it was clear that bacteria were able to take up DNA. That is how the non-pathogenic bacteria became pathogenic: they took up DNA, including the gene(s) for pathogenesis, from the heat-killed pathogenic bacteria. In 1970 it was shown that E. coli could be transformed and take up DNA. Refinements in these methods of transformation are one of the principle reasons why E. coli has become the workhorse of recombinant DNA technology.
What do you need for transformation of an organism?
The host must be competent for transformation, that is, able to take up DNA. The bacteria used by Griffith were naturally competent to take up DNA. E. coli, however, is not so obliging. For E. coli, a variety of treatments are used to make cells competent to take up DNA. One of these is treating the bacteria with ice cold calcium chloride. It is not fully understood what this treatment does to the bacteria, but it is assumed that the physical barriers to DNA getting into the bacteria, the cell wall and membrane, are made more permeable thereby allowing DNA to enter the cell.
The competent E. coli cells and the DNA are mixed together in a tube. After several minutes, the bacteria are briefly heated, then placed on an agar medium and allowed to grow. Are all of the bacteria transformed? No. For E. coli, the highest transformation frequency that can be obtained is about 1%. In other words, at best only 1 bacterium out of every 100 will be transformed. In almost all transformation systems, the frequency of transformation, the fraction of cells that are transformed and take up DNA, is low. (For E. coli, a typical transformation would use about 0.1 micrograms of DNA. On average only 1 molecule out of every 10,000 successfully transforms the host.) So a major experimental problem is how to identify the relatively rare transformed cells among the mass of non-transformed bacteria.
How can the rare transformed bacteria be identified? The transformants must be distinguishable from the others. The DNA that the bacteria take up should carry a gene that gives the transformed bacteria a distinct property or phenotype that can distinguish them from the non-transformed bacteria. In transformation of bacteria, the DNA used for transformation carries a gene for resistance to an antibiotic, such as ampicillin or tetracycline. Bacteria that have been transformed with the DNA will be able to grow in the presence of the antibiotic, while untransformed bacteria (the vast majority) will not grow.
After mixing the bacteria and DNA, the bacteria are placed on medium that contains the antibiotic. Only transformed bacteria will grow on medium containing the antibiotic. This antibiotic resistance gene is also known as a selectable marker because it allows for direct selection of transformed bacteria. Selectable marker genes are essential for transformation of virtually all organisms because the frequency of transformation is very low. Transformation with vectors that carry a selectable marker allows the transformed organisms to be simply identified.
So transformation of bacteria can be summarized as:
The DNA used to transform the bacteria needs to carry a selectable marker, such as a gene for resistance to an antibiotic, and an origin of replication. If the DNA introduced into the bacterium is not replicated each time the bacterium divides, then the DNA transferred into the cell will not be present in all of the progeny and will rapidly be lost. So these two components are required in the DNA used for transformation. But the purpose of transforming E. coli is not simply to introduce and copy these pieces of DNA, but to move other more interesting genes into the host organism. These essential components are assembled into vectors that can be used to carry other pieces of DNA into the bacterium. In the same way that vectors carry infectious diseases between organisms, transformation vectors carry DNA into the host.
Most transformation vectors are plasmids, circular DNA molecules that carry these essential components and can also have other DNA added to them.
See overhead of simple bacterial plasmid vector.
How are other pieces of DNA added to a plasmid vector? The vector must contain a site where the circular DNA molecule can be cut with a restriction enzyme to open the molecule. Other DNA fragments can then be inserted into the vector using DNA ligase to join the DNA molecules and create a new circular plasmid DNA. These recombinant DNA molecules are formed in a test tube and are then transferred into E. coli by transformation.
See overhead of DNA ligation
Note that plasmid vectors are normally quite small DNA molecules, many are only 3,000 base pairs in size. There are a number of reasons for this: it is easier to transform E. coli with small DNA molecules; as the size of a DNA molecule increases, the likelihood of there being a single site for any restriction enzyme in the vector declines. If the vector is cut into 2 fragments, it is quite unlikely that it will be joined together in the right way by DNA ligation.
Vectors for transformation of E. coli are now quite advanced and have some added features that make cloning of DNA fragments more straightforward. For example, when you ligate together vector DNA and other pieces of DNA, you can create a number of different combinations. As a researcher, you are only interested in those that recombine the vector with other DNA. But the vector can also simply ligate back to itself and produce a DNA molecule that will transform the bacteria and give antibiotic resistant colonies.
See overhead of products of ligation
How can we distinguish between the bacteria that have been transformed with recombinant plasmids and just religated vector plasmids that do not carry any new piece of DNA? In many vectors, the vector contains a second gene (the "blue gene"), and when this gene is expressed the bacteria are blue rather than the usual white when grown on a special indicator medium. The site for cloning fragments of DNA is in the middle of this "blue gene". When a DNA fragment is inserted at this site, the blue gene is disrupted and inactivated. The bacteria that take up this recombinant DNA molecule will still be resistant to the antibiotic, but will now be white rather than blue. So the blue colonies on the plate contain just the rejoined vector, while the white colonies contain recombinant DNA plasmids.
Overhead of blue plasmid vector.
These plasmids have been manipulated so that one plasmid can serve many different functions. Many sites for restriction enzymes have been introduced into the vector so that DNA fragments with many different types of "sticky ends", produced by different restiction enzymes, can be cloned in one vector. The vector can also be used to allow the DNA sequence of the cloned DNA fragment to be determined quite easily. RNA can be transcribed from the DNA that has been cloned into the vector, which can be useful in a number of research methods.
See overhead of multi-functional plasmid vector.
This gives you examples of how E. coli is transformed, how DNA can be transferred into this bacterium. Similar methods have been developed for other prokaryotes. What methods are used on eukaryotes? First I will talk about baker's yeast, Saccharomyces cerevisiae. This is a eukaryote, but can grow on agar plates very much like E. coli.
The first vectors that were developed for yeast transformation were based on a plasmid that was found in this yeast. This plasmid contains an origin of replication, which is critical if this DNA is to be replicated and maintained in the yeast. (What would happen to a plasmid in a cell if it was not replicated?) The origin of replication serves as the starting point to assemble yeast transformation vectors.
The selectable marker that is used in yeast transformation is normally a gene involved in synthesizing an essential compound such as an amino acid like leucine. The host is a yeast strain that carries a mutation in the pathway to make this essential compound, in this case leucine.
Overhead of yeast vector, host, and transformation scheme.
The yeast has a cell wall made of cellulose. This must be removed before yeast can be transformed. This is done using a treatment with enzymes. The yeast cells, now without their cell walls, are mixed with the vector DNA, again with another chemical such as calcium chloride or polyethyleneglycol, and DNA is taken up by the yeast. The yeast is then placed on an agar medium that contains no leucine. To grow, the yeast must make their own leucine, and only those cells that contain the vector have all of the genes for the biochemical pathway to synthesise leucine. Yeast cells that grow on medium lacking leucine are transformed.
Today I have described vectors that replicate on their own, not as part of the host chromosome. Next time I will talk about how transformed microbes can be used to produce proteins of commercial value. In future lectures I will describe vectors used for transformation of plants and animals that rely on integration of DNA into a chromosome of the host.