Source: Amoeba sisters

Genetic Engineering
- Changing an organism’s genotype

1. The specific DNA is inserted into the Plasmid

Plasmid
- Is like an extra set of genes in addition to the bacterial chromosome that bacteria can
use
- Tend to be in a circular shape
- Common in bacteria (can also find in yeast)

2. To get specific DNA into the plasmid, you have to make space for that. For that, you can
use RESTRICTION ENZYMES. After that, LIGASE comes in to seal the specific DNA
into the plasmid. The plasmid with the specific DNA is now called the RECOMBINANT
DNA.

Restriction Enzymes
- Enzymes that cut in specific spots like tiny scissors
- Can cut a specific spot in the plasmid so you can add in that human insulin gene
(example of specific DNA).
- Are actually part of the natural defense systems bacteria have against bacteriophages.

Ligase
- Used to seal the specific DNA into the plasmid.

Recombinant DNA
- Is made up of DNA from different sources

3. In order to encourage a bacterium to pick up the plasmid in transformation (recombinant

DNA), certain chemicals and temperature changes may be used.
4. Once it picks up that plasmid, when the bacterium reproduces by splitting (Binary
fission), the resulting cells will both inherit the plasmid and then their daughter cells
will inherit it. In this way, the plasmid continues to be produced over and over. The
bacteria can use the human insulin gene to produce human insulin and the insulin can
be purified in a lab setting to be used for humans.

Note:
● You can consider the bacteria to be transgenic: any organism or microorganism that
has genetic material from other organisms is considered transgenic.
● The plasmid is the vector in this situation. Vector: thought as the vehicle for getting the
recombinant DNA into the organism.
● Plasmid is a common vector but aren't the only vectors in genetic engineering where
viruses are another example.
 ● If a virus’ own genetic material is removed and a gene of interest instead is placed
inside, the virus can the be permitted to attach to target cells to deliver that gene of
interest. When it attaches to a target cell, it inserts the gene of interest in which this is
another type of delivery system.
● Sometimes if the plasmid or viral vector is just not ideal for delivering DNA into a cell,
you have other options such as microinjection.

But what if you had a way to customize the exact place you want to cut in DNA?

CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)

- This gene editing tool allows for the editing of DNA using a special kind of nuclease
called Cas9 (a nuclease).
- The CRISPR-Cas9 is also part of the natural defense system bacteria have against
bacteriophages.
- But in CRISPR, by using a specific guide RNA that can be designed in the lab, Cas9
can be guided with the specific guide RNA to cut a points around a specific target gene.
By doing that, one can do gene editing by removing a selected target gene.

Source: Fuse School

Genetic Engineering
- Manipulation or changing of the DNA of an organism
- Involves removing a gene from one organism which is called the donor and transferring it
to another organism which is called the recipient organism.

Recipient
- Is called a transgenic or genetically modified organism

Two basic purposes genetic engineering:

1. Require large volumes of a particular protein to be made where scientists use transgenic
microorganisms to produce large volumes of this protein.
2. To give organism A the gene of organism B to give it some advantage that Organism A
has naturally.
Source: PPT ni madam (Lesson-2-Genetic…)

Modifying Techniques
1. Classical Breeding
- Focus on controlled pollination of plants and the mating of organisms with
desirable qualities.
2. Genetic Engineering
- Involves the use of molecular techniques to modify the traits of a target organism.
- The process of artificial manipulation, modification and recombination of DNA or
other nucleic acid molecules in order to modify an organism or population of
organisms.
- Genetic information is transferred via vector. A vector can be a bacterium,
through its circular DNA called a plasmid, or a virus. A specific target genetic
segment is spliced into a bacterial plasmid and allowed to be replicated.

Modification of traits may involve:

1. Introduction of new traits into an organism
2. Enhancement of a present trait by increasing the expression of the
desired gene
3. Enhancement of a present trait by disrupting the inhibition of the desired
genes’ expression.
Other application of recombinant DNA technology
● Molecular biology - Gene Mapping
● Identification of Genetic Disorder
● Gene Therapy
● DNA Fingerprinting (forensics, paternity and identification of a person)
● Vaccines
● Pharma Products

General Steps of Recombinant DNA Technology

1. Isolating of DNA
2. Cutting or cleavage of DNA by restriction enzymes (REs)
3. Selection of an appropriate vector or vehicle which would propagate the recombinant
DNA.
4. Ligation of the gene of interest with the vector
5. Amplifying the recombinant DNA:
- Biolistics
- Plasmid insertion by Heat Shock Treatment
- Electroporation
6. Selection process to screen which cells contain the gene of interest
- Selection of plasmid DNA containing cells
- Selection of transformed cells with the desired gene
- PCR detection of plasmid DNA
7. Sequencing of the gene to find out the primary structure of the protein