Genetic engineering has become a fast-growing field in society today. Each year, hundreds of scientists study the materials which determine our heredity, their components, and how they relate to cellular activity. Genetic research has provided doctors with powerful tools by which inherited diseases such as muscular dystrophy and cystic fibrosis can possibly be corrected. Moreover, it has shed light on the processes by which colonies of bacteria reproduce. It has provided scientists with a greater insight into the functioning of bacteria and their role in illnesses of the human body. Indeed, genetics has opened the door to the fascinating world of endo-cellular activity, a door which can take scientific study in any direction possible.

Genes, the basic units fundamental for the study of genetics, are built from molecules of DNA (deoxyribonucleic acid). Found in a cell’s nucleus, or central organelle, DNA houses the "blueprints" of the cell. In other words, the genetic instructions of DNA work to direct the developmental process of an organism. This unique acid is encoded inside the organism’s genes. An organic phosphate, DNA is constructed of four nucleotides, thymine, adenine, guanine, and cytosine. Because DNA is composed of a different sugar, deoxyribose (instead of ribose), and a distinct thymine base (instead of uracil), it is significantly a separate acid from RNA (ribonucleic acid).

Gene therapy, also referred to as genetic engineering, enables scientists to place one or more genes into cells for the purpose of providing an entirely new set of genetic instructions for those specific cells. This practice is continually used to effectively treat many diseases that currently stand without a cure. The variety of illnesses treated by gene therapy includes both inherited and non inherited illnesses, ranging from juvenile diabetes, to AIDS (Acquired Immune Deficiency Syndrome), and certain forms of cancer. Scientists treat non-inherited diseases via gene insertion, by which diseased bodily cells are programmed for entirely new functions. Gene therapy is also beneficial to the cardiovascular health of the human body. Liver cells are being treated genetically to assist the body in ridding itself of surplus cholesterol that could eventually cause heart attacks and stroke.

Certain diseases, such as cystic fibrosis, are the result of inherited genetic defects. Still other diseases are the results of the miscoding of genes. Genetic miscoding disrupts the genetic instructions of the gene itself. This process can occur when a cell’s DNA is undergoing duplication during the growth and division of the cell. In this case, the genetic mutation is specifically referred to as somatic and is commonly related to the creation of cancerous cells.

Vital to the study of genetics are enzymes, highly specialized proteins built from polymers of amino acids. Enzymes work as catalysts, which greatly hasten the reactions which take place in biological systems. Enzymes’ ability to catalyze chemical reactions depends upon the effectiveness of their original protein formation. When catalytic activity ceases or is lost by an enzyme, the particular enzyme has usually been divided into subunits, or has been disnatured. Excluding small molecules of catalytic RNA (ribonucleic acid), every enzyme is a protein.

Enzymes are the scientific keys to the many forms, or "locks" of cell survival and proliferation. These unique proteins work to provide catalization reactions in cells inside metabolic pathways. It is within these pathways that the degradation of nutrient molecules, the conservation and transformation of chemical energy, and the formation of biological macromolecules takes place.

Infact, without the process of catalysis (provided by enzymes), the reactions of cells necessary in order for the digestion of food, the sending of nerve signals, and the contraction of bodily muscles could not occur at a useful rate. Enzymes such as pepsin and trypsin control many different reactions, which include the ability of the stomach to digest meat. Urease is an enzyme which provides the acceleration for one particular reaction alone. Other enzymes yield energy for the beating of the human heart, as well as the expansion and contraction of the lungs. In certain diseases, particularly genetic disorders which have been inherited, the defectiveness or complete absence of one or more enzymes in the body is a major contributing factor to the illness.

The role of enzymes in biochemical processes inside the body is particularly significant in the continual discoveries of the medical field. A variety of enzymes help to provide energy for bodily processes such as the conversion of sugar and other food molecules, which yields entirely new substances in humans. The majority of the substances are vital for the building of bodily tissue, the replacing of blood cells, and the releasing of chemical energy required for the movement of muscles. Other enzymes have proved their effectiveness in the treatment of inflammatory areas. The specific enzyme trypsin is put to work by doctors to remove foreign matter and deceased tissue out of burns and wounds. Infact, enzymes are even important to the productivity of today’s economy! The formation of alcohol and other significant processes dealing with American industry are depend on the actions of enzymes. These enzymes, however, have usually been synthesized by certain bacteria and yeast used during the process of production.

Small, circle-shaped DNA molecules which exist separate from the chromosomes in most bacterial species are known as plasmids, particles also critical to the comprehension of genetics. More specifically, the term "plasmid" refers to any extrachromosomal genetic particle which directs cellular "traffic" back and forth through chromosomes. Strange as it may seem, plasmids are often labeled as different viruses, mitochondria, chloroplasts, and parasites.

Diseases which have been particularly classified as genetic disorders are formed as a result of the lack of one or more specific enzymes in tissues. Also, excessive activity of a certain enzyme can produce abnormal bodily conditions, some of which can be harmful to humans. Similarly, plasmids also play their role in affecting the formation of diseases due to bacteria production. A large amount of plasmids is composed of genes which allow bacteria to survive and multiply in specific environments. Certain plasmids carry one or more genes that maintain a resistance to antibiotics. Bacterial cells which contain such plasmids are able to survive and reproduce in an environment which contains a medical drug. Uncontrolled, the mechanisms of replication contained in a virus such as that of the bacteria can produce more than 100,000,000 times the amount of original DNA molecules which make up the virus - in only twenty-four hours!

A specific medical antibiotic used in the continual fight against bacteria growth is penicillin. Sir Alexander Fleming first observed the effects of the drug, which derived from a particular type of mold, Penicillium notatum. One of the most vital anti-infective medicines active in clinical medicine, penicillin proves to be a valuable drug. Not only is it bactericidal and inexpensive, but the its toxicity for human cells is practically non-existent.

Penicillin kills bacteria and causes the inhibition of their growth. It destroys life in only the bacterial organisms which are undergoing growth and reproduction. Micro-organisms like streptococci, pneumococci, and not to mention diseases such as gangrene, endocarditis, and scarlet fever are subject to the destroying effects of penicillin.

However, the drug has proved its inability to cure specific types of staphylococci, as well as other bacteria, due to the bacterial organism’s inability to produce the enzyme penicillinase. Thus, the enzymes of the bacteria themselves are able to destroy the antibiotic (penicillin) present. The colonization of bacterial species within humans, which inevitably leads to the formation of infections, results when the strains of a patient’s own bacteria establish infections in a bodily state thought to be sterile. Often, scientisits observe bacteria in laboratories by charting its growth in agar, or a gel-like material extracted Often, scientisits observe bacteria in laboratories by charting its growth in agar, or a gel-like material in certain red algae species. Bacteria such as enterococci, which have been discovered by scientists to be penicillin-resistant, can easily cause deadly infections of the urinary tract. (Often, scientisits observe

bacteria in laboratories by charting their growth in agar, or a gel-like material extracted from the walls of red algae cells).

A broad range of partly-synthetic penicillin is known as ampicillin, which will kill a variety of species of bacteria, one being Escherichia coli (E. Coli). E. Coli bacteria resistant to the toxicity of antibiotics, or drugs, including ampicillin, form a wide variety that spans the globe. In certain parts of the world, E. Coli bacteria of this type contain genetic information used to form protein products. It is these products which intervene with the actions of antibiotics as they fight illnesses and infections.

When a cell is cloned, an identical copy of itself is made. Scientists clone cells of bacterial origin in order to observe the way they form and develop into colonies. Cloning was, in the past, a process restricted to the isolation of one cell from a larger cell population, and then the allowance of the self-reproduction of the cell. DNA cloning specifically involves the separation of a gene or a gene segment from the gene’s larger chromosome. It is then attached to a molecule similar to that of the gene’s carrier DNA. The segment of modified gene can then be replicated thousands to millions of times!

Several important steps make up the procedure of genetic cloning. First of all, a method for cutting, or penetrating, the DNA at specific points must exist. Secondly, a technique of joining two fragments of DNA covalently must be present. Thirdly, the ability to select a small molecule of DNA capable of reproducing itself must be at hand. Moreover, a method for transporting the DNA from the test tube into a "host cell" is necessary. This cell must be capable of providing the enzymatic materials for replication of the DNA. And lastly, there must be present techniques by which specific "host cells" can be selected, or identified. Each "host cell" must contain the materials to provide the complex system of enzymatic machinery for the replication of the DNA molecule. It is also necessary that "host cells" are composed of transferred DNA.

Specific genetic transfers involving plasmids also exist. Plasmids are cable of being introduced into bacterial cells by a process known as transformation. In order to provide cells with the "compotentcy" to receive the gene being transferred, a unique process is used. Cells are placed together in an incubator while bathed in a solution of calcium chloride at only zero degrees Celsius. The cells are then placed in a position to undergo heat shock. Heat shock occurs when the cells are placed in an icy water bath. Here, cell temperatures are altered, and made either lower or higher in number (depending on the cellular temperature when the transformation process first begins). The resulting state of the cells’ temperature is sally between thirty-seven and forty-three degrees Celsius. Cells subjected to heat shock, for reasons not entirely yet known to scientist, become "competent" to receive DNA molecules. This process is effected for recognizing the cells that do become hosts of plasmid DNA, which usually constitute a very small percentage of the total cell population present.

"Competency" in cells can also be recognized by discovering whether or not the cell is resistant to the effects of an antibiotic, such as ampicillin. These "select marker" cells distinguished via this process have received a plasmid containing a gene that the cell finds necessary for growth under particular conditions. In the cloning of DNA molecule, the specific gene-containing fragment is placed inside a plasmid or virus. Thus results a recombinant DNA molecule, which is then ready for its introduction into a cell making up a colony of bacteria.

Relating once again to the display of the ability of a cell to "host" a gene, bioluminescence is a key process. Bioluminescence is the light generated by a live organism. A simple example is the common firefly, which emits light from within its abdomen. A wide variety of organisms undergo this unique process, including bacteria, fungi, sponges, protozoa, insects, fish, jellyfish, squid, crustaceans, and certain species of the lower plant population. Approximately 66%, or 2/3, of the population of deep sea fish, display bioluminescence. The bioluminescent angler fish lurks the deep seas, using an illuminated rod on the end of its head to lure unassuming prey. Symbotic bacteria found in the head are responsible for the glow it projects out into the water. The light produced by organisms in nature via bioluminescence is often used to attract mates, and also to coat the eggs of a particular species so that they are easily recognizable to the parents. Thus, eggs can be fertilized with greater ease because they can be clearly recognized.

Two main chemicals constitute bioluminescence. The first is a light-producing chemical known as luciferin. Several general luciferins are found within a variety of species. Bacterial, vargulin, dinoflagellate, and coelenterazine are examples of the different categories of luciferins. Vargulin can be found within the seed shrimp, or ostracod. The most common form of luciferase in marine organisms is coelenterazine. Coelenterazine is the well-known emitter of light in the photoprotein aequarin.

Luciferase is the second element of the process, an enzyme which provides for the catalysis of the reaction, which produces light. When linked together by a co-factor, this luciferin-luciferase complex is termed a photoprotein. The production of biological light using these molecules is created with a small amount of heat radiation. Probably the most extraordinary and beautiful occurrences in bioluminescence is that known as the "milky sea" phenomenon, which is rare indeed. It usually occurs in the Indian Ocean, when sailors report having witnessed a misty white light glowing on the water’s suface for several hours at a time. Plankton possessing the gene for bioluminescence help exhibit this eerie and unusual happening.

Two major phases, or stages, to mechanisms of bioluminescence in organisms exist. The first is the formation of a great quantum of energy. The second is the excitement of the majority of the energy in a luminescent molecule. This phase leads to the decaying of the species which has been chemically-excited. The deterioration of the species then leads to the emission of light in the form of photon molecules.

Buried inside the bacterium Photobacterium (Vibro) fischeri is the system of genetic materials needed fuel bioluminescence, dominated by the actions of the lux operon. Composed of two genes which provide genetic coding for the production of luciferase, the lux operon also provides genetic coding for enzymes which aid in luciferin production through several other genes contained within itself.

Via the lux operon, bioluminescence is made useful in uncovering hidden elements, bacteria, and genetic processes otherwise unknown to exist in an environment. The process can be used to sense the presence of ATP (adenosine triphosphate) molecules. Bioluminescence is capable of doing this because ATP is necessary in its initial reaction which produces light. Bacteria bearing light genes attached to their regulons, which are resistant to ions, bioluminescence has been used t seek ions of mercury and aluminum, and also oxygen. In a past experiment directed toward new studies in the medical field, the arteries of a dog were presented in a state of exposure, along with the bacterial genes which coded for luciferase. Via bioluminescence, these genes were used to affirm whether of not genes foreign to the bodily system of a dog could be introduced into the dog’s genome. In order to determine the success of the gene transformation within the dog, luciferin was added. If this chemical emitted light, or caused bioluminescence during the experiment, than it could be confirmed that the new genes could successfully be incorporated into the dog’s system successfully.

The many aspects of gene therapy range from the treatment of diseases, to the discoveries relating to cellular activity, and to the growth and development of complex colonies of microorganisms. Yet however used, genetic engineering proves to be a vital tool in the dawn of a new future in science. By observing the actions of plasmids, operons, and enzymes, scientists can better understand the rates of and reasons for chemical reactions occurring in the human body, and in the natural world. By understanding the structure and behavior of DNA, the reproduction of organisms, including bacteria, can be further comprehended. Specific processes such as bioluminescence are used as "labels" to inform scientists

ut the success of gene insertions that could one day prevent inherited diseases and save lives. The exciting field of genetics has opened its door. It is our job, as society, to walk through with an open mind.

Lauren Midthun

Biology-H, Per. 4

11-12-98

Materials

0.5 ml E. Coli (DH5)

110 ul plasmid pUC18

110 ul plasmid lux

10 ml nutrient broth

20 petri dishes

400 ml of nutrient agar + penicillin

10 inoculating loops

10 sterile transfer pipets

5 sterile tubes

0.6 ml ampicillin (60mg/ml)

5 ml Calcium Chloride

water bath

air incubator at 37 degrees Celsius

dark rooms

sterile micropipets

Procedure A - Preparing the Agar-Ampicillin Plates

1. Loosen the cap on the bottle of agar.

2. Put the bottle in a beaker of boiling water until the agar has become a liquid.

3. Make sure that the level of water in the beaker is equal to the agar level in the bottle.

4. Allow 20 to 25 minutes in order for the agar to melt.

5. Take the agar bottle out of the bath and, at room temperature, let it cool for 10 minutes.

6. Empty the 0.6 ml of ampicllin solution into the agar bottle and replace the cap.

7. Shake the bottle to mix its contents.

8. Have three petri dishes ready, and remove their covers.

9. Pour 10 to 20 ml of the agar solution into the bottom half of each dish.

10. Replace the lids of the dishes right away.

11. Allow 1 to 2 days at room temperature for teh hardening of the agar.

Procedure B - Preparing the Cells to "Accept" the Plasmid DNA

12. While working with bacteria from this point on, gloves should be worn.

13. Put the E. Coli tube and calcium chloride vial into the ice bath.

14. Use a sterile pipette to transfer 1/2 ml calcium chloride to the E. Coli tube.

15. Use the same pipet to shift the contents of this tube back into the Calcium Chloride solution vile.

16. Mix the solution in the vial by tapping it with your index finger.

17. The cells can then be called "competent", or ready to host the DNA, after they have ben placed on ice and incubated for about 10 minutes. If necessary, the cells can be stored in the Calcium Chloride solution on ice for 20 to 24 hours.

Procedure C - The Acceptance of DNA by the "Competent" Cells

18. Label a test tube "N" for "No DNA - Control Plasmid" (these cells will have received no gene whatsover 19. Distinguish a second tube "C" for "Competent Cells" (these cells will have received the ampicillin-resistance gene). Mark yet a third test tube as "L" for "plasmid lux" (the cells in this tube will have received not only the ampicillin-resistant gene, but also the plasmid lux).

20. Put all tubes in an ice bath.

21. Add 6 drops, or approximately 120 microliters, or competent cells to each of the test tubes using a sterile micropipet.

22. Add 10 microliters of the control plasmid to the "C" test tube using a sterile micropipet. Do the same using the "L" test tube.

23. In order to ensure that the cells are in a state of suspension, tap gently against the tube of competent cells with your index finger.

24. Add 6 drops, or approximately 120 microliters, of competent cells to each of the wo test tubes using a sterile micropipet.

25. In order to mix these solutions, use the tip of your index finger to tap each of these tubes.

26. For 10 to 15 minutes, store both of these tubes on ice.

27. Suspended in calcium chloride, the competent cels should now begin to accept, or "take up", the plasmid DNA.

28. Retrieve a test tube and label it "N" for "No Plasmid DNA" (the cells in this tube will have received no gene whatsover).

29. Move the "N" tube to water bath which has been preheated to 37 degrees Celsius. Allow it to stay there for 5 minutes.

30. Now add 0.7 ml of nutrient broth to each tube. Allow the tubes to incubate for 30-45 minutes at 37 degrees Celsius. (Make sure the broth is dispensed only with a pipet which is sterile. By incubating within the broth, the bacteria are able to "recover" from their treatment by calcium chloride and express the gene for ampicillin resistance).

Procedure D - Distinguishing the Solutions by Their Ability to Host the Plasmid DNA

31. Retrieve three ampicillin-nutrient agar plates. Label one "C", one "N", and one "L".

32. Remove 0.25 ml of the "C" tube solutin using a sterile pipet.

33. Lift the lid from the "C" agar plate.

34. Transfer the bacteria from the "C" tube into the "C" agar plate.

35. Use an inoculating loop to evenly spread the bacteria onto the agar surface.

36. Repeat the same process with the "L" test tube and "L" agar plate.

37. Repeat the same process with the "N" test tube and "N" agar plate.

38. Replace the lids of the three plates, and leave them at room temperature until their solutions have been absorbed (this process should take approximately 10 to 15 minutes).

39. Turn the agar plates upside down.

40. Allow the plates to incubate, at room temperature, in the dark.

41. Upon completion of the procedure, wash your hands with an anti-bacterial soap.

Dispose of gloves in a container designated specifically for bio-hazardous wastes.

42. When the entire exeriment has been completed, autoclave the bactiera or expose them to a concentrated bleach (such as Clorox) for one hour.

The following additional experiments can only be completed if bioluminescence successfully occurs in the main experiment. Therefore, at this point, early in experimentation, it cannot be determined whether these experiments will be accompanied by written procedures and actually conducted.

1. Abaza, Ronney. "Bioluminescence." http://www.biology.lsa.umich.edu/~www/bio 311/projects/ronney/biochem.shtml (15 October 1998)

2. Author Unknown. "Chemistry of Bioluminescence." http://lifesci.ucsb.edu/~biolum/chem/

(15 October 1998)

3. Blaese, Michael R. "Gene Therapy." Microscoft (R) Encarta (R) 97 Encyclopedia. 1996, Volume Not Applicable, Pages Not Applicable.

4. Evers, Chris. "Birds Do It, Bees Do It." Lederberg and Tatum Discover Bacterial and Phage Recombination. http://outcast.gene.com/ae/AB/BC/Birds_Bees.html

5. Kleinsmith, Lewis J. and Kish, Valerie M. Principles of Cell and Molecular Biology: Second Edition. New York: Harper Collins College Publishers, 1995.

6. Kornberg, Arthur and Baker, Tania A. DNA Replication: Second Edition. New York: W.H. Freeman and Company, 1992.

7. Lehninger, Albert L. and others. Principles of Biochemistry: Second Edition. New York: Worth Publishers, 1993.

8. Maniatis, T. and others. Molecular Cloning: A Laboratory Manual. New York: Cold Spring Harbor Laboratory, 1982.

9. Northrop, John H. "Structure and Function of an Enzyme." Microsoft (R) Encarta (R) Encyclopedia. 1996, Volume Not Applicable, Pages Not Applicable.

10. Stryer, Lubert. Biochemistry: Fourth Edition. New York: W.H. Freeman and Company, 1995.