What are the examples of unlinked genes

Introducing human genes into bacteria

There are many different uses for genetic engineering methods. For example, if you put the gene for a certain protein in bacteria and then these bacteria are propagated in the laboratory, they will produce large quantities of this protein. That way you can get enzymes or drugs that couldn't be made any other way.

Recombining pieces of DNA: DNA recombination

Not only do human genes consist of DNA, but also bacterial plasmids. Different pieces of DNA can be combined with each other. Recombination means: inserting an additional gene into the gene ring of a bacterium (a plasmid).

First, the biologists have to prepare the following DNA:
A) The gene to be introduced into the plasmid is isolated from a cell (for example from a human cell).
B) The plasmid is isolated from bacteria.

Both pieces of DNA must now be cut so that the gene to be examined can be inserted into the plasmid of the bacterium. To do this, DNA scissors (restriction enzymes) are added:
A) Human DNA is cut in many places. Fragments of different sizes are created. The piece of DNA containing the desired gene is separated from the other pieces of DNA using gel electrophoresis (as described in the article Cutting DNA into pieces).
B) The plasmid is cut only once. This opens the gene ring.

The human DNA and plasmid must be cut with the same scissors so that the ends match. One speaks of "sticky ends". The ends are linked with one another with the help of ligases. The result is a recombinant gene ring: a plasmid from a bacterium with a human gene.

In addition to this traditional technique, there are now also newer methods of inserting genes into plasmids. These modern methods often have one decisive advantage: They are faster and less time-consuming. It can e.g. For example, certain fragments that have been amplified using a polymerase chain reaction (PCR) can be inserted directly into a plasmid without having to be additionally treated with restriction enzymes. To do this, the PCR must be set up in such a way that the gene pieces get the correct ends during this reaction.

Bringing the plasmid into the bacterium: transformation

In order for the bacterium to be able to use the new gene, which is now integrated into the plasmid, the gene ring must be brought back into the bacterium. Bacteria and plasmids are brought together in a tube with a nutrient solution.

In order for the bacteria to take up the plasmid, they must be treated with heat. The tube is therefore kept in warm water at 42 ° C for a short time. The bacteria suffer a shock from the heat: small holes form in their cell walls and / or cell membranes through which the plasmids can slip. If you take the tube out of the warm water, the holes close and the plasmids remain in the bacteria. After the shock, the tube is often placed on ice in warm water so that the holes close as quickly as possible.

selection

Not all bacteria take up a plasmid, but only about one in 10,000. Then how can those with a plasmid be distinguished from those without?

A common approach is to also insert a so-called antibiotic resistance gene into the plasmid. Using this gene, the bacterium produces a protein with which it can protect itself against a certain antibiotic. However, the antibiotic is toxic to bacteria without this gene.

To distinguish between bacteria with and without a plasmid, a small amount of the antibiotic is mixed into the nutrient solution for the bacteria. This mixture is mixed with agar as a gelling agent and poured into a plastic dish, where it solidifies into a gel. The bacteria are distributed on the nutrient agar and then placed in a 37 ° C incubator for several hours. The bacteria with the plasmid, i.e. those with the antibiotic resistance gene, multiply strongly, so that over time you can see small clusters (so-called colonies) with the naked eye. The bacteria without a plasmid die.

Obtain human protein

With a thin pen or needle, the biologist can now remove bacteria from a colony and place them in a nutrient solution. The bacteria continue to multiply there. They all have the recombinant plasmid with the human gene. The bacteria use this gene to produce the corresponding human protein. This can then be cleaned and used again.

This post integrates content from the former website gene-abc.ch, which was taken over by SimplyScience in 2016. The Gene ABC was an initiative of the Swiss National Science Foundation (SNSF) and also comprised a series of YouTube videos.