Genetic engineering

- direct maniputlation of genes “for practical purposes”

- the applications of genetic engineering include the “manufacture of hundreds of useful products”
(research)
- w/ the use of “biochemical and mechanical tools” of DNA technology, scientists can make
recombinant DNA in VITRO
- it can introduce the DNA into cultured cells that replicate the DNA and may express its genes,
yielding a desired product

MODIFICATION OF TRAITS
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

MODIFYING TECHNIQUE

- CLASSICAL BREEDING-
> focus on the “mating of organisms” with desirable qualities

Examples:
- controlled pollination of plants
- mating of animals w/ desired traits

-RECOMBINANT DNA TECHNOLOGY-

> “Molecular bilogy techniques” are being employed in the insertion and expression of proteins in
different organisms for various purposes

GENERAL OUTLINE OF RNA

1. Cutting or cleavage of DNA by restriction enzymes (REs)
2. Selection of an appropriate vectore which would propagate the rDNA
3. Ligation of the gene of interest with the vector
4. Transfer of the recombinant plasmid into a host cell
5. Selcetion process to screen which cells actually contain the gene of interest
6. Sequencing of the gene to find out the primary structure of the protein

3 WAYS IN WHICH PLASMID MAY BE INTRODUCED INTO HOST ORGANISMS

 1. Plasmid- a small circular double-stranded DNA molecule found in bacteria and some
microscopic organisms
- Carry only few genes and exist independently of chromosomes, the primary structures
that contain DNA in cells
- Able to self- replicate and can be picked up from the environment and transferred
between bacteria
- They are used by their host organism to cope with stress-related conditions

 2. Biolistics- “gene gun” technique is used to fire DNA-coated pellets on plants tissues
- cells that survive the “bombardment”, and are able to take up the expression plasmid coated
pellets and acquire the ability to express the designed protein

GENE GUN HAVING DESIRED DNA --- DNA COATED GOLD PARTICLES --- TARGET CELLS ---
 3. Heat schock Treatment- process used to “transfer plasmid DNA into bacteria”
- the target cells are “pre-treated” before the procedure to increase the pore sizes of their
plasma membranes
- this pretreatment is said to “make the cells competent “ for accepting the plasmid DNA
- after the cells are made competent, they are “incubated” with desired plasmid at about 4
degree celcius for about 30 mins
- the plasmids concentrate near the cells during this time
- afterwards, a “Heat Shock” is done on the plasmid- cell solution by incubating it at 42 degree celcius
for 1 minute then back to 4 degree celcius for 2 minutes
- the rapid “rise and drop” of temperature increase and decreases the pore sizes in the membrane
- the plasmid DNA near the membrane sureface are taken into the cells by this process
- the cells that took up the plasmids acquire new traits and are said to be “transformed”

HEAT-SHOCK TRANSFO. OF E.COLI-PROTOCOL

VECTOR W/ THE DESIRED INSERT --- VECTOR ADDED TO THE E.COLI COMPETENT CELLS --- GROWTH
OF E.COLI COLONIES HARBORING VECTOR ON LB PLATE W/ A SELECTION MARKER

- ELECTROPORATION-
> this technique follows a similar methodology as “heat shock treatment”, but, the expansion of the
membrane pores is done through an electric “shock”
> this method is commonly used for insertion of genes into mammalian cells

ELECTROPORATION
GENETICALLY MODIFIED ORGANISMS (GMOs)

ACTIVITY

CLASSICAL BREEDING- B AND D

BOTH (SIMILAR) - A AND F
MODERN GENETIC ENGINEERING- C AND E