BioMolecules : Genetic Engineering

Human Cells with a level of assessment for Laws. Just give the analysis of DNA, RNA and others.

Genetic Engineering
1. Classical breeding practices focus on 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 modification of traits may involve:

      I. introduction of new traits into an organism
      II. enhancement of a present trait by increasing the expression of the desired gene
      III. enhancement of a present trait by disrupting the inhibition of the desired genes’ expression.
3. A general outline of recombinant DNA may be given as follows:

      I. cutting or cleavage of DNA by restriction enzymes (REs)
      II. selection of an appropriate vector or vehicle which would propagate the recombinant DNA (  eg. circular plasmid in bacteria with a foreign gene of interest)
      III. ligation (join together) of the gene of interest (eg. from animal) with the vector ( cut bacterial plasmid)
      IV. transfer of the recombinant plasmid into a host cell (that would carry out replication to make huge copies of the recombined plasmid)
      V. selection process to screen which cells actually contain the gene of interest
      VI. sequencing of the gene to find out the primary structure of the protein

4. Ways in which these plasmids may be introduced into host organisms.

Biolistics. In this technique, a “gene gun” is used to fire DNA-coated pellets on plant 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.

Plasmid insertion by Heat Shock Treatment. Heat Shock Treatment is a 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 (usually with CaCl2) is said to make the cells “competent” for accepting the plasmid DNA. After the cells are made competent, they are incubated with the desired plasmid at about 4°C for about 30min. 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°C for 1 minute then back to 4°C for 2 minutes.

The rapid rise and drop of temperature is believed to increase and decrease the pore sizes in the membrane. The plasmid DNA near the membrane surface are taken into the cells by this process. The cells that took up the plasmids acquire new traits and are said to be “transformed”.

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.

5. Some methods to screen recombinant cells are as follows:

Selection of plasmid DNA containing cells
A selection marker within the inserted plasmid DNA sequence allows the selection of “transformants”. Usually, an antibiotic resistance gene (e.g. AMP ampicillin resistance gene) is included in the plasmid DNA. This allows only “transformed” cells to survive in the presence of the antibiotic (e.g. ampicillin). Plating the plasmid-cell solution on antibiotic-containing media will select for these “transformants” and only allow plasmid-containing cells to grow and propagate into colonies.

Selection of transformed cells with the desired gene
Certain inserted genes within the plasmids provide visible proof of their presence. These include the antibiotic resistance genes that allow for the selection of the transformed cells within the solution. Some inserted genes also produce colored (e.g. chromogenic proteins) or fluorescent products (e.g. GFP) that label the colonies/cells with the inserted gene.

In some cases, the location of the cloning site within the plasmid is in the middle of a gene (i.e. β galactosidase, lacZ) that generates a (blue) colored product in the presence of a substrate (i.e. isopropyl β-D-1 thiogalactopyranoside, or IPTG). Cells transformed with these “empty” plasmids will turn blue in the presence of IPTG. Insertion of a gene in the cloning site disrupts the sequence of the β-galactosidase gene and prevents the generation of the colored product in the presence of the substrate. Cells transformed with the disrupted β galactosidase gene will remain “white” in the presence of IPTG. This “blue-white screening” protocol is thus able to screen for cells that were transformed with the desired gene in the cloning site.

PCR detection of plasmid DNA
Alternatively, the presence of the desired gene in the inserted plasmids may be confirmed using PCR amplification. PCR reactions specific for the desired gene may be done using DNA from cells. Amplification of the expected product would confirm the presence of the gene within the samples. PCR reactions specific for plasmid sequences will also confirm/identify the type of plasmid used for the transformation.

Genetically Modified Organisms (GMOs) With the ability to insert gene sequences, comes the possibility of providing new traits for these target organisms. This has allowed the development of GMOs. Some of these genetic modifications promise higher product yield for their targets. These include the Flavr-Savr Tomato and Bt-Corn.

The Flavr-Savr (“Flavor Savor”) tomato was the first genetically modified organism that was licensed for human consumption. The trait modified in this tomato is its ripening process. A gene for an enzyme that causes the degradation of pectin in the cell walls (i.e. polygalacturonase) normally softens the fruit as it ripens. In Flavr Savr tomatoes, an inhibitor (i.e. antisense RNA) disrupts the expression of this gene, thereby delaying the softening of the fruit and extending the time it may be kept in storage and transported to markets.

Bt-Corn was developed to incorporate the production of a toxin (i.e. Bt-endotoxin) from Bacillus thuringensis in corn plants. This toxin results in the death of pests that feed on these plants like the corn borer larvae. The toxin has been shown to be selective for Lepidoptera larvae and is non-toxic to humans, mammals, fish and birds. The selective toxicity of the toxin allows its use in foodcrops. The introduction of the toxin is believed to increase crop production due to decreased losses from pest infestation. The same technology has been applied in the Philippines for the development of Bt-Eggplant.

Despite the proposed benefits of GMOs, some people have raised their concerns regarding the consumption of these modified foods. While most of the products are tested for safety, concerns are raised for the possibility of not being able to detect hazards that are present, but are currently undetectable by today’s current technology.

Because of these issues, manufacturers are urged to provide labels that notify consumers of GMO presence in their products. While GMOs are believed to be safe when licensed by the food regulatory agencies, it is believed that the consumers must be provided with enough information to make their own choices regarding their use.

Lesson 1 - BioMolecules : Structure and Functions
Lesson 2 - BioMolecules : DNA Replication and Protein Synthesis
Lesson 4 - Biomolecules : Applications of Recombinant DNA