In the last four Instructions, you have been learning about genetic engineering.

In this Instruction, we thought you ought to know about the unsung heroes of the process- bacteria.

We've mentioned bacteria briefly before, but now we'd like to talk about them in depth.

But first we need to explain about vectors.

Vectors
 

In biology, a vector is something that carries a thing between species. A mosquito is a disease vector because it carries the malaria parasite into human beings.

In genetic engineering, a vector is a length of DNA that carries a gene into a host cell during cloning - which is the first step in genetic engineering.

There are many different kinds of vectors, but the most common are plasmids -- short circular bits of DNA found naturally in the cytoplasm in the cells of bacteria.
 

Plasmids
http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/R/RecombinantDNA.html 

As we said, plasmids are short circular bits of DNA found naturally in the cells of bacteria. Most plasmids contain 3-5 genes, and there are usually about 10 copies of a plasmid in a bacterial cell.

Plasmids are copied separately from the main bacterial DNA during cell division, so the plasmid genes are passed on to all of the daughter cells. And since plasmids are used naturally for gene exchange, bacterial cells will take them up readily.

Plasmids were discovered in research on the reproduction of the human intestinal bacteria Escherichia coli (E. coli).

Two enzymes are required to introduce foreign DNA into a DNA vector (like a plasmid).

The first (restriction) enzyme is a bacterial enzyme that cleaves the DNA. The second enzyme - DNA ligase - then seals the foreign DNA fragments into the gap created by the restriction enzyme. The treated cells take up the plasmids and the bacteria and plasmids reproduce.

Eventually, there are many copies of the plasmid and many copies of the foreign gene (the gene that has been introduced). When this process is complete, the end result is the desired rDNA (recombinant DNA) molecule.

One of the most commonly used plasmids is the R-plasmid (also called pBR322).

Transformation
http://en.wikipedia.org/wiki/Gene_transfer

The process of gene transfer- which we've described above-takes place when vectors containing the desired genes are transferred into living cells so they can be replicated or expressed.

The cells receiving the vector are called host cells - and once they have successfully incorporated the vector, they are said to have been transformed. This process is called transformation.

There are several different methods by which vectors can be introduced into host cells.

These methods include:

1. Mechanical means (like heat shock or the use of high-voltage electricity)
    
2. The use of bacteria (in which the gene to be inserted is hooked on to the bacterial DNA)
    
3. The use of viruses

Gene Transfer by Virus

Although, as we said, bacteria are the most commonly used vectors, viruses are frequently used, too.

When the virus method is used, a vector is incorporated into a virus and the virus is then used to infect cells -carrying the foreign gene along with its own genetic material.

Viruses must first be genetically engineered (usually through the use of the appropriate proteins) to make them safe so that they can't reproduce or make toxins. Interferon is used to increase human resistance to viral infections. And a protein like the insecticidal protein produced by Bacillus thuringiensis is not harmful to humans because we metabolize it.

Here are the three classes of viruses that are most commonly used for gene transfer:

1. Bacteriophages (or phages) - viruses that infect bacteria.
    
2. Adenoviruses - human viruses that causes respiratory diseases including the common cold. Their genetic
   material is double-standed DNA, so they are ideal for delivering genes to living patients in gene therapy.
    
3. Retroviruses - a group of human viruses that include HIV. They are enclosed in a lipid membrane and their genetic material is double-stranded RNA, which is copied onto DNA for incorporation into the host cell chromosome.
    

Polymerase Chain Reaction (PCR)
http://en.wikipedia.org/wiki/Polymerase_chain_reaction

As we have told you, genes can be cloned by cloning the bacterial cells that contain them. But this requires a lot of DNA.

There is another method called PCR (Polymerase Chain Reaction) that can clone DNA samples as small as a single molecule. It is a fairly recent technique and earned its developer, Kary Mullis, a Nobel Prize in 1993. Some scientists feel that PCR has revolutionized biotechnology.

Instead of using bacteria to clone DNA fragments, PCR uses DNA fragments that are copied millions of times by the direct use of the enzyme DNA Polymerase.

But no matter which cloning method is used, the success of gene insertion is measured by marker genes.

Marker Genes
http://en.wikipedia.org/wiki/Marker_gene

Marker genes are used to identify those cells that have successfully taken up a vector and have become transformed.

The most common way to find them is with the use of bacterial host cells.

For example, one common marker (used in the R-plasmid) is a gene for resistance to the antibiotic tetracycline. When cells are grown on a medium that contains tetracycline, all the untransformed cells (and all the cells that have taken up non-plasmid DNA) will die.

Only the transformed cells - the cells that contain the marker genes - will survive. These cells can then be selected to be grown and cloned to complete the genetic engineering process.

One of the most useful procedures for identifying specific genes is the Southern blot method.

The Southern Blot Method
http://www.bio.davidson.edu/COURSES/GENOMICS/method/Southernblot.html 

The Southern blot has nothing to do with North, South, East or West - it's a procedure named after a man named Edward M. Southern, who developed it at Edinburg University in the 1970's.

The Southern Blot Method is a sophisticated way to locate a particular sequence of DNA within a complex mixture.

It can even be used to locate one particular gene within an entire genome.

This diagram shows the basic steps involved in a Southern blot.



Experiments for Home and Classroom

For experiments and activities relating to this Instruction, refer to the activities and experiments suggested at the end of the previous Instruction, Instruction 5-4: DNA Technology and Recombinant DNA.

A particularly relevant activity would be the first -- "Gel Electrophoresis" -- which allows students to actually hunt for and analyze DNA segments to be used in genetic engineering.


 

for Students, Parents and Teachers

Now let's do Practice Exercise 5-5 (top).

Next Page:  Problems (top)