1. Transformation
Change caused by genes and involves the insertion of one or more genes into an organism in order to change the organism’s traits.

2. How does genetic transformation occur?
When a cell takes up (inside) and expresses a new piece of genetic material- DNA.
Provides the organism with a new trait that is identifiable after transformation.

3. How is genetic transformation used?
Agriculture: genes coding for traits such as frost, pest, or drought resistance can be transformed into plants.
Bioremediation: bacteria can be genetically transformed with genes enabling them to digest oil spills
Medicine: diseases caused by defective genes are beginning to be treated by gene therapy, by genetically transforming a sick person’s cells with healthy copies of the defective genes that cause their disease.

4. What is the relationship between genes and proteins
What is the relationship between genes and proteins? – Protein Synthesis
DNA made of genes
Transcribed to mRNA - occurs in the nucleus
Translated to proteins – occurs at the ribosome
Proteins express your genes

5. Sterile Techniques
Always open wrappers of sterile pipets and loops so that you DO NOT touch the working end.
Do not allow the working end to touch any non-sterile object.
Do not reuse pipets or transfer loops. Once materials come in contact with E.coli put them directly into the plastic garbage bag at your station. DO NOT set them on the work table.
Wash your hands thoroughly before and after working with the cultures.
Wipe your work area with 10% bleach solution.
When putting loops or transfer pipets into tubes or vials while transferring material do not touch the side of the tube or vial.
When adding or collecting bacteria from agar plates, hold the lid over the plate to prevent contaminants from falling onto the surface of the plate.
Open the lid the minimum amount of time needed for the manipulation.

6. Handling E. coli
Naturally occurring E. coli strains of E.coli bacteria inhabit the gut of various animals, including humans.
The lab version of E. coli does not contain disease causing genes.
This version (strain) is harmless under normal conditions.
But, if introduced into eyes or a cut, laboratory strains may conceivably cause an infection
Always use a new inoculating loop
Keep your nose and mouth away from the tip of the pipet.
Wipe down your table with 10% bleach solution
Wash your hands before leaving the lab
Dispose of all materials in the plastic bag at your work station.

7. Background Information : Antibiotics and Mechanisms of Antibiotic Resistance
Ampicillin: synthetic drug of the penicillin family of antibiotics that interferes with the formation of bacterial cell walls, preventing bacteria from growing and replicating. These antibiotics contain a chemical group called a beta-lactam ring.
Ampicillin resistant bacteria destroy the antibiotic by cleaving the ring using an enzyme called beta lactamase. Beta lactamase is coded for by the ampicillin-resistant gene.

8. Background continued:
Kanamycin: a member of the aminoglycoside antibiotic family that hinders the functioning of the prokaryote ribosome and therefore interferes with protein translation.
Kanamycin resistant genes code for enzymes that inactivate kanamycin by adding a chemical group to the antibiotic.

9. Background continued:
Satellite Colonies: small colonies that group up around ampicillin resistant colonies on your ampicillin plates.
Some ampicillin sensitive bacteria may remain living but unable to replicate in the presence of the antibiotic.
As the beta-lactamase secreted by the resistant colony diffuses into the surrounding agar, it destroys the ampicillin there and allows the non-resistant bacteria to form satellite colonies.

10. Transformation and Its Role in Discovering the Function of DNA
The discovery of transformation was crucial in understanding that DNA is the material of inheritance.
Transformation : the uptake of exogenous, naked DNA by a cell. This newly adopted DNA can become a heritable part of the cell’s genetic material.
Natural Transformation: these bacteria have mechanisms for transporting the DNA into the bacterial cell.
Artificial Transformation: subjecting bacteria to specific artificial conditions can cause bacteria to consistently take up DNA. These bacteria are referred to as competent.
Once DNA is taken up into the host bacterial cell the genes coded for by the DNA need to be expressed (transcribed and translated), and the DNA needs to persist.

11. Plasmids
Small, usually circular pieces of extra-chromosomal DNA that exists in nature in bacteria. Bacteria transfer them in a process called conjugation (physical contact between the two cells).
Each plasmid has an origin of replication (a sequence of bases at which replication begins) which allows the DNA to replicate in the host bacteria.
Each daughter cell will receive copies of the plasma upon cell division. So, DNA inserted into a plasmid introduced into a bacterial cell can persist in the cell and in the cell’s offspring.
Plasmids are ideal for introducing foreign DNA into bacterial cells.

12. Selective Markers
Bacteria in nature and in culture are in intense competition for survival.
Plasmids don’t always contain genes beneficial for survival and they take extra energy to maintain.
There is a tendency for bacteria to lose their plasmids unless there is an advantage to retaining them.
To force a bacterium to keep a plasmid is to maintain the bacterium in the presence of an antibiotic that will kill it unless it develops resistance to the antibiotic.
The gene for antibiotic resistance is called a selective marker.
Only the bacteria that have taken up the desired plasmid will survive.
The plasmids you will work with are either ampicillin or kanamycin as a selective marker.

13. Antibiotics, Antibiotic Resistance, and Evolution
Many naturally occurring plasmids contain antibiotic resistance genes, which is a serious problem.
The effectiveness of antibiotics has decreased over the years because bacteria have developed resistance to them. This is an example of evolution at work.
Bacterial populations include a few individuals with genes resistant to antibiotics. There is selective pressure in favor of those resistant individual bacteria. Those bacteria survive, divide, pass on the resistant trait to their offspring.
Those offspring can come to dominate and eventually constitute most, if not all, of the population.
Another problem is doctors prescribing antibiotics to patients who don’t really need them or for viral infections (viruses are not killed by antibiotics.)