GENETIC ENGINEERING OF MICROORGANISMS and PLANTS

Recombinant DNA technology or genetic engineering is a method that allows the combination
of genes in a test tube to form hybrid DNA. It allows the transfer of a specific gene (from the same or
different organism) to produce a new trait.

Basic Steps in genetic engineering:

1. extraction of DNA from source organism, isolating and cloning the gene from the DNA

2. design and preparation of the “gene construct” or “hybrid DNA” or “recombinant DNA”

3. delivery of gene construct to the new host organism

4. checking the presence and expression of gene in new host organism

To design the gene construct, one must consider the different parts of a gene :(figure 8)

1. the promoter region, the so called structural gene part

As the term connotes, the promoter region contains elements or sequences which drive the
reading of the gene, instructing when and where the gene will be expressed. For example, the promoter
can direct the expression in all parts of the organism or in specific tissues or organs, at all times or only
at night or at daytime or at specific stage of the development of the organisms. The structural gene part
provides the information for the trait itself (ex. a specific protein).

2. the terminator sequence – marks the end of the transcription of the gene.

Pic fig. 8
A. Genetic engineering of microorganisms

The steps involved in the process of genetic engineering of microorganism are shown in Fig.9 to
illustrate the introduction of human insulin gene into bacteria:
 Figure 9. Cloning and the introduction of the human insulin gene in bacteria.

Step 1. The gene of interest, in this case, human insulin is isolated from the human DNA and cloned.
(cloning of DNA or a gene means making multiple copies).

Step 2. The human insulin gene is inserted into a vector, a plasmid at specific sites cut by enzymes
called “restriction enzyme”. A plasmid is a genetic element {DNA} which can carry a DNA fragment into a
recipient cell like a bacterium. The plasmid contains the promoter elements and terminator sequence
that will work for the host organism, in this case, bacterial cells.

Step 3. The recombinant plasmid is introduced into bacterial cells by physical means. The bacterial cells
are subject to heat and then put on ice. This will result into formation of pores in the cellular membrane
that allow the DNA to get inside the cell. This process is called “transfection or transformation”. The
recombinant plasmid is not incorporated into the chromosome of the bacteria but it is multiplied into
many copies in the cell.
Step 4. This results into the production of many copies of the inserted gene ( the human insulin gene)
and into the production of human insulin by the bacterial cells. This is called “cloning of the insulin” and
this is how insulin is produced by bacterial cells

The same process is basically followed for the cloning of other gene in microorganisms to produce
various products.

NOTE: THIS IS DONE IN LABORATORY BY GENETIC ENGINEERS

B. Genetic Engineering in Plants

Steps in genetic engineering or recombinant DNA technology of plants are the following:

1. The gene or genes needed are isolated and cloned.

2. The gene is inserted in an appropriate vector to form the gene construct. A vector is like a vehicle
or carrier and has the necessary regulatory elements such as the promoter and terminator which can
make the gene work. The promoter will tell when and where the gene will be expressed and how many
copies of protein will be produced. The terminator will command the end of expression or reading of the
gene. A selection gene marker is also usually in the gene construct that help in determining which of the
bombarded tissues have incorporated the gene construct in their DNA.

3. The gene construct is delivered to the plant cell by either of two methods:

1. By coating the gene on tungsten or gold particles and delivering these particles into plant
tissues by using a particle bombardment device. This device is attached to a gas tank containing helium
gas which forces the DNA-coated particles into the tissues with pressure.

2. By inserting the gene construct into Agrobacterium tumefaciens which is then used to infect a
plant and eventually transfer the gene construct and its other genes to the plant genome. The
Agrobacterium is commonly found in nature causing galls or swelling on plants due to infection.

4. Determine which among the plants cells have integrated the introduced gene. The selection
marker gene will do this. For example, if the selection marker gene is an antibiotic resistance gene
marker, cells which have this gene will be able to survive in a medium containing antibiotic. These cells
are termed transformed or transgenic. (Another type of selection marker gene is the green fluorescent
protein or GFP marker, cells that integrate this in their DNA will produce this protein which gives off
green fluorescence when beamed under a UV light)

The transformed or transgenic plant tissues are allowed to grow and regenerate completely.

The breeder will screen the resulting plants for the desired trait and evaluate as well as their
agronomic or horticultural traits. The breeder will select lines which have the desired traits and stable.
The molecular biologist/biochemist will determine the presence of the inserted gene and other
biochemical characteristics of the transgenic plants.

Fig 11

Steps in Genetic Engineering of Plants

QUIZ

Fill-in the blanks:

1. This process allows a pre-designed recombination of genes in an organism to produce a new

trait.__________________ or ____________.
2. The first step in genetic engineering is the _______________ of the gene of interest.
3. A vector is a ______________ which is a genetic element or DNA which can carry a DNA
fragment such as a gene into a recipient cell like a bacterium.
4. There are several enzymes which can cut DNA at specific sites. These enzymes are called
____________ enzymes.
5. When a gene of interest is inserted into a vector, the resulting combination is called a
recombinant plasmid or _________________.
6. The introduction of a recombinant plasmid into a bacteria ( or other types of organism ) is called
________________or _____________.
 7. The gene construct can be introduced to a plant tissue by either of two methods:
_________________________or __________________.
8. The term _____________ means that the gene of interest has been transferred and integrated
into the genome of the host plant.
9. The gene for green fluorescent protein ( GFP ) is an example of _______________ and is used in
the selection of transformed of plants.
10. Conventional plant breeding techniques are used in the selection and evaluation of transgenic
plants. True or FALSE.

Activity:

1. If you are a genetic engineer what plant and its trait you want to improve? Why? (15 pts}