Bacterial Transformation & Selection

Transfer of plasmid DNA into bacteria. How bacteria are selected. Protein production and purification.

Key points:

• Bacteria can take up foreign DNA in a process called transformation.
• Transformation is a key step in DNA cloning. It occurs after restriction digest and ligation and transfers newly made plasmids to bacteria.
• After transformation, bacteria are selected on antibiotic plates. Bacteria with a plasmid are antibiotic-resistant, and each one will form a colony.
• Colonies with the right plasmid can be grown to make large cultures of identical bacteria, which are used to produce plasmid or make protein.

The big picture: DNA cloning

Transformation and selection of bacteria are key steps in DNA cloning. DNA cloning is the process of making many copies of a specific piece of DNA, such as a gene. The copies are often made in bacteria.

In a typical cloning experiment, researchers first insert a piece of DNA, such as a gene, into a circular piece of DNA called a plasmid. This step uses restriction enzymes and DNA ligase and is called a ligation.

After a ligation, the next step is to transfer the DNA into bacteria in a process called transformation. Then, we can use antibiotic selection and DNA analysis methods to identify bacteria that contain the plasmid we’re looking for.

Steps of bacterial transformation and selection

Here is a typical procedure for transforming and selecting bacteria:

1. Specially prepared bacteria are mixed with DNA (e.g., from a ligation).
2. The bacteria are given a heat shock, which "encourages" them to take up a plasmid. Most bacteria do not take up a plasmid, but some do.
3. Plasmids used in cloning contain an antibiotic resistance gene. Thus, all of the bacteria are placed on an antibiotic plate to select for ones that took up a plasmid.
4. Bacteria without a plasmid die. Each bacterium with a plasmid gives rise to a cluster of identical, plasmid-containing bacteria called a colony. A typical colony looks like a small, whitish dot the size of a pinhead.
5. Several colonies are checked to identify one with the right plasmid.
6. A colony containing the right plasmid is grown in bulk and used for plasmid or protein production.

1. Specially prepared bacteria are mixed with DNA (e.g., from a ligation).
2. The bacteria are given a heat shock, which causes some of them to take up a plasmid.
   

   
   

   The basic answer is that a heat shock makes the bacterial membrane more permeable to DNA molecules, such as plasmids. It appears that the heat shock causes the formation of pores in the bacterial membrane, through which the DNA molecules can pass.
3. Plasmids used in cloning contain an antibiotic resistance gene. Thus, all of the bacteria are placed on an antibiotic plate to select for ones that took up a plasmid.

Diagram of a plasmid. The plasmid contains an antibiotic resistance gene, a promoter to drive gene expression in bacteria, and the target gene inserted during the ligation.

4. Bacteria without a plasmid die. Each bacterium with a plasmid gives rise to a cluster of identical, plasmid-containing bacteria called a colony.
5. Several colonies are checked to identify one with the right plasmid (e.g., by PCR or restriction digest).

6. A colony containing the right plasmid is grown in bulk and used for plasmid or protein production.

Why do we need to check colonies?

The bacteria that make colonies should all contain a plasmid (which provides antibiotic resistance). However, it’s not necessarily the case that all of the plasmid-containing colonies will have the same plasmid.

How does that work? When we cut and paste DNA, it's often possible for side products to form, in addition to the plasmid we intend to build. For instance, when we try to insert a gene into a plasmid using a particular restriction enzyme, we may get some cases where the plasmid closes back up (without taking in the gene), and other cases where the gene goes in backwards.

Left: gene goes into plasmid forwards (pointing in the same direction as the promoter sequence). This is the desired plasmid from the ligation.

Middle: plasmid closes back up without taking in the gene. This is not a useful plasmid.

Right: gene goes into plasmid backwards (pointing back towards the promoter sequence). This is not a useful plasmid if we want to express the gene in bacteria.

[More explanation]

Let's say we are trying to insert a gene into a plasmid so it can be expressed in bacteria. In order to do so, we must "paste" the gene into the plasmid next to the promoter, pointing in the forward direction:

Starting materials:

• Target gene digested at both ends with a particular restriction enzyme.
• Plasmid cut with the same restriction enzyme at a site following a promoter for bacterial expression. The promoter "points" towards the right, meaning that it will drive transcription of the DNA sequence that lies to the right.

What we want to get is:

• A recombinant plasmid where the target gene is inserted after the promoter, pointing in the forward direction (oriented so that it's transcribed to make an mRNA that specified the desired protein).

Suppose we cut our gene and plasmid with the same enzyme and join the fragments together with DNA ligase. In some cases, the plasmid DNA and the gene DNA will combine in the right way and form the plasmid we're looking for. In other cases, though, the plasmid may simply close back up (without taking in the gene), or the gene may go into the plasmid backwards. A backwards gene cannot be expressed in bacteria to make a protein.

Left: gene goes into plasmid forwards (pointing in the same direction as the promoter sequence). This is the desired plasmid from the ligation.

Middle: plasmid closes back up without taking in the gene. This is not a useful plasmid.

Right: gene goes into plasmid backwards (pointing back towards the promoter sequence). This is not a useful plasmid if we want to express the gene in bacteria.

A ligation involves many fragments of DNA (billions of copies of the plasmid, and billions of copies of the gene). Thus, in every ligation, we will get some number of "good" plasmids and some number of "bad" ones. Each colony starts from a single bacterium with a single plasmid, so all the bacteria in a colony with have the same plasmid (either "good" or "bad").

See the article on restriction enzymes and DNA ligase for a more concrete example of how and why these different ligation products can form.

Why does it matter if a gene goes into a plasmid backwards? In some cases, it doesn't. However, if we want to express the gene in bacteria to make a protein, the gene must point in the right direction relative to the promoter, or control sequence that drives gene expression. If the gene were backwards, the wrong strand of DNA would be transcribed and no protein would be made.

Because of these possibilities, it's important to collect plasmid DNA from each colony and check to see if it matches the plasmid we were trying to build. Restriction digests, PCR, and DNA sequencing are commonly used to analyze plasmid DNA from bacterial colonies.

Protein production in bacteria

Suppose that we identify a colony with a "good" plasmid. What happens next? What's the point of all that transforming, selecting, and analyzing?

Possibility 1: Bacteria = plasmid factories

In some cases, bacteria are simply used as "plasmid factories," making lots of plasmid DNA. The plasmid DNA might be used in further DNA cloning steps (e.g., to build more complex plasmids) or in various types of experiments.

In some cases, plasmids are directly used for practical purposes. For instance, plasmids were used to deliver a human gene to lung tissue in a recent gene therapy clinical trial for patients with the genetic disorder cystic fibrosis$^1$start superscript, 1, end superscript.

Possibility 2: Bacteria = protein factories

In other cases, bacteria may be used as protein factories. If a plasmid contains the right control sequences, bacteria can be induced to express the gene it contains when a chemical signal is added. Expression of the gene leads to production of mRNA, which is translated into protein. The bacteria can then be lysed (split open) to release the protein.

A chosen colony is grown up into a large culture. The bacteria in the large culture are induced to express the target gene through addition of a chemical signal to the culture medium. Inside each bacterium, the target gene is transcribed into mRNA, and the mRNA is translated into protein. The protein encoded by the target gene accumulates inside the bacteria.

Bacteria contain many proteins and macromolecules. Because of this, the newly made protein needs to be purified (separated from the other proteins and macromolecules) before it can be used. There are a variety of different techniques used for protein purification.

Cells that have produced protein are burst open (lysed), releasing the protein and the other cell contents. The molecules extracted from the cells are applied to a column that contains antibodies specific for the target protein. Thus, the protein is trapped in the column while other molecules from the bacteria flow through. In the final step, after all the non-target proteins have been washed away, the target proteins are released from the antibodies in the column, and the pure protein is collected for use.

In one technique called affinity chromatography, a mixture of molecules extracted from the lysed bacteria is poured through a column, or a cylinder packed with beads. The beads are coated with an antibody, an immune system protein that binds specifically to a target molecule.

The antibody in the column is designed to bind to our protein of interest, and not to any other molecules in the mixture. Thus, the protein of interest is trapped in the column, while the other molecules are washed away. In the final step, the protein of interest is released from the column and collected for use.

[Source: Khan Academy]