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pGLO Transformation Lab
AP bio lab 8 (adapted from http://media.collegeboard.com/digitalServices/pdf/ap/bio-manual/Bio_Lab8-BiotechnologyBacterialTransformation.pdf)
Direction: for this lab, you will create a formal lab report using the “Scientific Method Lab
Report Rubric” Be sure to include all sections. Note all italicized direction for each section.
Background: summarize the background information in 1 paragraph, and answer the pre-lab
questions.
In this lab you will perform a procedure known as genetic transformation. Remember that a
gene is a piece of DNA which provides the instructions for making (codes for) a protein. This
protein gives an organism a particular trait. Genetic transformation literally means “change
caused by genes,” and involves the insertion of a gene into an organism in order to change
the organism’s trait. Genetic transformation is used in many areas of biotechnology. In
agriculture, genes coding for traits such as frost, pest, or spoilage resistance can be
genetically transformed into plants. In bioremediation, bacteria can be genetically transformed
with genes enabling them to digest oil spills. In medicine, diseases caused by defective genes
are beginning to be treated by gene therapy; that is, by genetically transforming a sick
person’s cells with healthy copies of the defective gene that causes the disease.
You will use a procedure to transform E coli bacteria (a non-harmful strain) with a gene that
codes for Green Fluorescent Protein ( GFP ). The real-life source of this gene is the
bioluminescent jellyfish Aequorea victoria. Green Fluorescent Protein causes the jellyfish to
fluoresce and glow in the dark. Following the transformation procedure, the bacteria express
their newly acquired jellyfish gene and produce the fluorescent protein, which causes them to
glow a brilliant green color under ultraviolet light.
In this activity, you will learn about the process of moving genes from one organism to
another with the aid of a plasmid. In addition to one large chromosome, bacteria naturally
contain one or more small circular pieces of DNA called plasmids. Plasmid DNA usually
contains genes for one or more traits that may be beneficial to bacterial survival. In nature,
bacteria can transfer plasmids back and forth allowing them to share these beneficial genes.
This natural mechanism allows bacteria to adapt to new environments. The recent occurrence
of bacterial resistance to antibiotics is due to the transmission of plasmids.
Plasmids can also incorporate into their DNA sequence pieces of DNA from different
organisms. Plasmids that incorporate new DNA are called recombinant plasmids.
Recombinant plasmids are used in biotechnology to carry DNA that codes for substances,
such as human insulin or growth hormone, into bacteria. Bacteria that contain the recombinant
plasmids can then be grown commercially, in bulk, to provide the needed substance. Special
enzymes, called restriction enzymes , can cut DNA fragments from almost any organism.
Many restriction enzymes work by finding palindrome sections of DNA (regions where the
order of nucleotides at one end is the reverse of the sequence at the opposite end). This way
a restriction enzyme can cut tiny sticky ends of DNA that will match and attach to sticky ends
of any other DNA that has been cut with the same enzyme. There are enzymes that cut what
are called blunt ends , or straight cuts, but theses ends do not recombine with as much
specificity. DNA ligase joins the matching sticky ends of the DNA pieces from different
sources that have been cut by the same restriction enzyme.
Once a desired DNA fragment has been isolated and cut with a specific restriction enzyme,
the sticky ends of both the desired DNA fragment, and from a plasmid that has been cut by the
same restriction enzyme, can be joined together, forming a recombinant DNA plasmid. The
plasmid must then be placed back into the bacteria that can then replicated and create many
bacteria producing the gene of interest. Special plasmids, which
have antibiotic resistance markers or fluorescent tags, are used
in this as tag for screening so that a researcher will be able to tell
that the desired DNA has been incorporated into the target
plasmid and subsequently into the host bacterium.
Bio-Rad’s unique pGLO plasmid encodes the gene for GFP
and a gene for antibiotic resistance to the antibiotic ampicillin.
pGLO also incorporates a special gene regulation system, which
can be used to control expression of the fluorescent protein in
transformed cells. The gene for GFP can be switched on in
transformed cells by adding the sugar arabinose to the cells’
nutrient medium. Selection for cells that have been transformed
with pGLO DNA is accomplished by growth on ampillicin plates.
Transformed cells will appear white (wild-type phenotype) on plates not containing arabinose,
and fluorescent green under UV light when arabinose is included in the nutrient agar medium.
For this experiment, 4 plates will be used:
Plate 1: +gGLO LB/amp
Plate 2: +gGLO LB/amp/ara
Plate 3: - gGLO LB/amp
Plate 4: - gGLO LB
Prelab Questions: answer after background paragraph
1. To genetically transform an entire organism, you must insert the new gene into every cell in
the organism. Which organism is better suited for total genetic transformation, a single
celled organism that reproduced asexually or multicellular organism that reproduces
sexually.
2. Scientists often want to know if the genetically transformed organism is safe, can pass its
new traits on to its offspring, and want to see results quickly. Based on the above
considerations, which would be the best choice for a genetic transformation: a bacterium,
earthworm, fish, or mouse? Describe your reasoning.
pGLO plasmid DNA GFP Flagellum Pore Cell wall Beta-lactamase Bacterial (^ antibiotic resistance^ ) chromosomal DNA
KEY:
+pGLO : added plasma with GFP gene
- pGLO : do not add plasma with GFP LB : type of nutrient broth to grow the E. Coli bacteria Amp: antibiotic ampicillin Ara : arabinose sugar added
- Use a sterile loop to pick up 2 – 4 large colonies of bacteria from your starter plate. Select starter colonies that are "fat" (ie: 1–2 mm in diameter). It is important to take individual colonies (not a swab of bacteria from the dense portion of the plate), since the bacteria must be actively growing to achieve high transforation efficiency. Choose only bacterial colonies that are uniformly circular with smooth edges. Pick up the +pGLO tube and immerse the loop into the transformation solution at the bottom of the tube. Spin the loop between your index finger and thumb until the entire colony is dispersed in the transformation solution (with no floating chunks). Place the tube back in the tube rack in the ice. Using a new sterile loop, repeat for the - pGLO tube.
- Examine the pGLO DNA solution with the UV lamp. Note your observations. Immerse a new sterile loop into the pGLO plasmid DNA stock tube. Withdraw a loopful. There should be a film of plasmid solution across the ring. This is similar to seeing a soapy film across a ring for blowing soap bubbles. Mix the loopful into the cell suspension of the +pGLO tube. Optionally, pipet 10 μl of pGLO plasmid into the +pGLO tube & mix. Do not add plasmid DNA to the - pGLO tube. Close both the +pGLO and - pGLO tubes and return them to the rack on ice.
- Incubate the tubes on ice for 10 min. Make sure to push the tubes all the way down in the rack so the bottom of the tubes stick out and make contact with the ice.
- While the tubes are sitting on ice, label your four LB nutrient agar plates on the bottom(not the lid) as follows: Rack (^) Ice L B / a m p pGLO L B / a m p / a r a pGLO L B / a m p pGLO L B pGLO
- Heat shock. Using the foam rack as a holder, transfer both the (+) pGLO and (-) pGLO tubes into the water bath, set at 42oC, for exactly 50 sec. Make sure to push the tubes all the way down in the rack so the bottom of the tubes stick out and make contact with the warm water. Double-check the temperature of the water bath with two thermometers to ensure accuracy. When the 50 sec are done, place both tubes back on ice. For the best transformation results, the transfer from the ice (0°C) to 42°C and then back to the ice must be rapid. Incubate tubes on ice for 2 min.
- Remove the rack containing the tubes from the ice and place on the bench top.Open a tube and, using a new sterile pipet, add 250 μl of LB nutrient broth to the tube and reclose it. Repeat with a new sterile pipet for the other tube. Incubate the tubes for 10 min at room temperature.
- Gently flick the closed tubes with your finger to mix and resuspend the bacteria. Using a new sterile pipet for each tube, pipet 100 μl of the transformation and control suspensions onto the appropriate nutrient agar plates. LB broth 250 μl pGLO + pGLO -
Analysis Questions:
- Which of the traits that you originally observed for E. coli did not seem to become altered? Explain.
- Of the E. colitraits you originally noted, which seem now to be significantly different after performing the transformation procedure? Explain.
- If the genetically transformed cells have acquired the ability to live in the presence of the antibiotic ampicillin, then what might be inferred about the other genes on the plasmid that you used in your transformation procedure?
- From the results that you obtained, how could you prove that the changes that occurred were due to the procedure that you performed?
- Recall what you observed when you shined the UV light onto a sample of original GLO plasmid DNA. Did it glow before the transformation and what does this observation indicate about the source of the fluorescence?
- What is the “screening process” and why is it necessary?
- Describe the evidence that indicates whether your attempt at performing a genetic transformation was successful or not successful.
- Very often an organism’s traits are caused by a combination of its genes and its environment. Think about the green color you saw in the genetically transformed bacteria: a. What two factors must be present in the bacteria’s environment for you to see the green color? (Hint: one factor is in the plate and the other factor is in how you look at the bacteria). b. What do you think each of the two environmental factors you listed above are doing to cause the genetically transformed bacteria to turn green? c. What advantage would there be for an organism to be able to turn on or off particular genes in response to certain conditions? d. Conclusion: Copied from rubric: Write a paragraph or two discussing the implications of the data. Include any information not disused in the analysis questions. What do your results mean when you consider the original question or hypothesis. Discuss whether the hypothesis was supported or not supported by the data. Point out the statistical significance of your results if applicable. Relate your conclusions to the concepts learned in class. Discuss any source of error. Could improvements be made? If the results are unexpected or contradictory, you should attempt to explain and point out possible avenues for further research.
7. While the tubes are sitting on ice,
label your four agar plates on the
bottom (not the lid) as shown on the
diagram.
8. Heat shock. Using the foam rackas a
holder, transfer both the (+) pGLO and (-)
pGLO tubes into the water bath, set at
42 °C, for exactly 50 seconds. Make sure to
push the tubes all the way down inthe rack
so the bottom of the tubes stick out and
make contact with the warm water. When
the 50 seconds have passed, place both
tubes back on ice. For the best
transformation results, the change from the
ice (0°C) to 42°C and then back to the ice must be rapid. Incubate tubes on ice for 2 min.
9. Remove the rack containing the
tubes from the ice and place on the
bench top. Open a tube and, using
a new sterile pipet, add 250 μl of
LB nutrient broth to the tube and
reclose it. Repeat with a new
sterile pipet for the other tube.
Incubate the tubes for 10 min at
room temperature.
10. Gently flick the closed tubes with
your finger to mix. Using a new
sterile pipet for each tube , pipet
100 μl from each of the tubes to
the corresponding plates, as shown
on the diagram onto the
appropriate plates.
11. Use a new sterile loop for each
plate. Spread the suspensions
evenly around the surface of the
agar by quickly skating the flat
surface of a new sterile loop back
and forth across the plate surface.
12. Stack up your plates and tape them
together. Put your group name and
class period on the bottom of the
stack and place the stack upside
down in the 37°C incubator until
the next day.
QUICK GUIDE
Appendix: Additional Information that may be useful. (do not need to print)
Appendix A Historical Links to Biotechnology
Biological transformation has had an interesting history. In 1928, Frederick Griffith, a London physician working in a pathology laboratory, conducted an experiment that he would never be able to fully interpret as long as he lived. Griffith permanently changed (transformed) a safe, nonpathogenic bacterial strain of pneumococcus into a deadly pathogenic strain. He accomplished this amazing change in the bacteria by treating the safe bacteria with heat-killed deadly bacteria. In this mixture of the two bacterial strains there were no living, virulent bacteria, but the mixture killed the mice it was injected into. He repeated the experiment many times, always with the same results. He and many of his colleagues were very perplexed. What transformed safe bacteria into the deadly killers? Many years later, this would come to be known as the first recorded case of biological transformation conducted in a laboratory, and no one could explain it. Griffith did not know of DNA, but knew the transformation was inheritable. As any single point in history can be, Griffith’s experiments in transformation can be seen as the birth of analytical genetic manipulation that has led to recombinant DNA and biotechnology, and the prospects for human gene manipulation. In 1944, sixteen years after Griffith’s experiment, a research group at Rockefeller Institute, led by Oswald T. Avery, published a paper that came directly from the work of Griffith. “What is the substance responsible?” Avery would ask his coworkers. Working with the same strains of pneumonia-causing bacteria, Avery and his coworkers provided a rigorous answer to that question. They proved that the substance is DNA, and that biological transformation is produced when cells take up and express foreign DNA. Although it took many years for credit to be given to Avery, today he is universally acknowledged for this fundamental advance in biological knowledge. Building upon the work of Avery and others, Douglas Hanahan developed the technique of colony transformation used in this investigation.1, 2 Historical Context Genetic Transformation 1865 —Gregor Johann Mendel: Mendel presented his findings describing the principles by which genetic traits are passed from parent to offspring. From his work the concept of the gene as the basic unit of heredity was derived. 1900 —Carl Correns, Hugo De Vries, Erich Tschermak: Plant geneticists conducting inheritance studies uncovered that their work was essentially a duplicate of work performed nearly four decades earlier by an unknown Austrian Augustinian monk, Gregor Johann Mendel, who studied peas. 1928 —Frederick Griffith: Griffith transformed nonpathogenic Diplococcus pneumoniainto pathogenic bacteria using heat-killed virulent bacteria. He suggested that the transforming factor had something to do with the polysaccharide capsule synthesis. Griffith did not know of DNA, but knew the transformation was inheritable. Griffith’s experiments in transformation can be seen as the birth of analytical genetic manipulation that has led to recombinant DNA technology and the prospects for human gene manipulation. 1944—Oswald Avery, Colin MacLeod: Avery and his colleagues announced that they had isolated the transforming factor to a high purity, and it was DNA. Since this classic experiment in molecular genetics, transformation, conjugation (bacterial mating), and transduction (viral DNA transfer) have been used to transfer genes between species of bacteria, Drosophila, mice, plants and animals, mammalian cells in culture, and for human gene therapy. 1952 —Alfred Hershey, Martha Chase: Hershey and Chase used radioisotopes of sulfur and phosphorus, and bacteriophage T2 to show conclusively that DNA was the information molecule of heredity. Along with the work of Avery, MacLeod, and McCarty, the Hershey/Chase experiment sealed the understanding that DNA was the transforming material and the information molecule of heredity. 1972 —Paul Berg, Janet Mertz: Berg used the newly discovered endonuclease enzyme, EcoRI, to cut SV DNA and bacteriophage P22 DNA, and then used terminal transferase enzyme and DNA ligase to rejoin these separate pieces into one piece of DNA. Creation of the first recombinant DNA molecule was the beginning of the age of biotechnology. The new molecule was not placed inside a mammalian cell because of concerns in the scientific community regarding genetic transfers.
APPENDIX A
melanogaster, a model widely used in the laboratory, is the largest animal so far to have its genetic code deciphered. A rough draft of the human genome was completed by a team of 16 international institutions that form the Human Genome Sequencing Consortium. Researchers at Celera Genomics also announced completion of their ‘first assembly’ of the genome. 2001—On February 12, 2001, Celera Genomics and the International Human Genome Sequencing Consortium jointly announced the publishing of the nearly complete sequence of the human genome - the genetic “blueprint” for a human being. This accomplishment took the international team almost twenty years and involved the collaboration of thousands of scientists from around the world. Celera Genomics reported completing the work in approximately nine months. The two groups differed in their estimates for the number of genes in the human genome, but the range predicted by both groups, between 25,000 and 40,000 genes, is far fewer than the previous estimate of 100,000 genes. This unexpected finding suggested that an organism as complex as a human being can be made of so few genes, only twice as many as found in the worm C. elegansor the fly D. melanogaster. The unveiling of the full sequence of the human genome makes it possible for researchers all over the world to begin developing treatments for many diseases. President George Bush decided that only experiments involving the existing 64 embryonic stem cell lines would be eligible for possible federal funding. The president’s decision was disappointing to many scientists who hoped to use embryonic stem cells to develop treatments for many ailments. Advanced Cell Technology, a small company in Massachusetts, announced that it had successfully cloned human embryos for the purpose of extracting their stem cells. This method could ultimately be used to treat patients with a variety of diseases by making replacement cells, such as nerve and muscle cells, which can be transplanted back into same person without the risk of being rejected by the body. PPL Therapeutics, the company that helped to clone Dolly the sheep, announced that it had cloned five genetically modified piglets with an inactivated, or “knocked out”, gene that would make their organs much less likely to be rejected when transplanted into a human recipient. The success of PPL Therapeutics brings hope to the thousands of people who are waiting to receive donated organs such as hearts, lungs, kidneys, and livers. 2002 —Dolly the sheep, the first mammal to be cloned from an adult cell, developed arthritis at a relatively early age of five years. It is not clear whether Dolly’s condition was the result of a genetic defect caused by cloning, or whether it was a mere coincidence. The news has renewed debates on whether cloned animals are susceptible to premature aging and health problems and has also been a setback for those who argue that cloning can be used to generate a supply of organs to help patients on the transplant list. 2008 —Discovery and landmark developmental uses of GFP wins the Nobel Prize in Chemistry. Osamu Shimonura was the first to isolate GFP and found that it had fluorescent properties when exposed to UV light. Martin Chalfie used GFP as a luminous genetic tag. Roger Y. Tsien uncovered GFP's fluorescent mechanism.
Appendix B Glossary of Terms
Agar A gelatinous substance derived from seaweed. Provides a solid matrix to support bacterial growth. Contains nutrient mixture of carbohydrates, amino acids, nucleotides, salts, and vitamins.
APPENDIX B
Antibiotic Selection Use of an antibiotic to select bacteria containing the DNA of interest. The pGLO plasmid DNA contains the gene for beta-lactamase that provides resistance to the antibiotic ampicillin. Once bacteria are transformed with the pGLO plasmid, they begin producing and secreting beta- lactamaseprotein. Secreted beta-lactamase breaks down ampicillin, rendering the antibiotic harmless to the bacterial host. Only bacteria containing the pGLO plasmid can grow and form colonies in nutrient medium containing ampicillin, while untransformed cells that have not taken up the pGLO plasmid cannot grow on the ampicillin selection plates. Arabinose A carbohydrate isolated from plants that is normally used as source of food by bacteria. In this experiment, arabinose initiates transcription of the GFP gene resulting in fluorescent green cells under UV light. Beta- Lactamase Beta-lactamase is a protein that provides resistance to the antibiotic ampicillin. The beta- lactamase protein is producedand secreted by bacteria that have been transformed with a plasmid containing the gene for beta-lactamase. The secreted beta-lactamase inactivates the ampicillin present in the LB nutrient agar, which allows for bacterial growth and expression of newly acquired genes also contained on the plasmid, such as GFP. Biotechnology Applying biology in the real world by the specific manipulation of living organisms, especially at the genetic level, to produce potentially beneficial products. Cloning Cloning is the process of generating virtually endless copies or clones of an organism or segment of DNA. Cloning produces a population that has an identical genetic makeup. Colony A clump of genetically identical bacterial cells growing on an agar plate. Because all the cells in a single colony are genetically identical, they are called clones. Culture Media The liquid and solid media referred to as LB (named after Luria and Bertani) broth and agar are made from an extract of yeast and an enzymatic digest of meat byproducts which provide a mixture of carbohydrates, amino acids, nucleotides, salts, and vitamins, all of which are nutrients for bacterial growth. Agar, which is from seaweed, polymerizes when heated and cooled to form a solid gel (similar to Jell-O gelatin), and functions to provide a solid support on which to culture the bacteria. Genetic Engineering The manipulation of an organism’s genetic material (DNA) by introducing or eliminating specific genes. Gene Regulation Gene expression in all organisms is carefully regulated to allow for differing conditions and to prevent wasteful overproduction of unneeded proteins. The genes involved in the transport and breakdown of food are good examples of highly regulated genes. For example, the simple sugar, arabinose, can be used as a source of energy and carbon by bacteria. The bacterial enzymes that are needed to break down or digest arabinose for food are only expressed in the absence of arabinose but are expressed when arabinose is present in the environment. In other words when arabinose is around, the genes for these digestive enzymes are turned on. When arabinose runs out these genes are turned back off. See Appendix D for a more detailed explanation of the role that arabinose plays in the regulation and expression of the Green Fluorescent Protein gene. Green Fluorescent Protein Green Fluorescent Protein (GFP) was originally isolated from the bioluminescent jellyfish, Aequorea victoria. The gene for GFP has recently been cloned. The unique three-dimensional conformation of GFP causes it to
Appendix C Basic Molecular Biology Concepts and Terminology
A study of the living world reveals that all living organisms organize themselves in some unique fashion. A detailed blueprint of this organization is passed on to offspring. Cells are the smallest functional units capable of independent reproduction. Many bacteria, for instance, can survive as single cells. The chemical molecules within each cell are organized to perform in concert. Cells can be grown in culture and harvested Cell culture is the process by which cells can be gathered from their natural locations and grown inside laboratory containers under controlled conditions. Appropriate food and environment must be provided for the cells to grow. Bacteria and yeast are very easy to grow in culture. Cells taken from plants, insects and animals can also be grown, but are more difficult to care for. After growth is complete, cells in culture can be harvested and studied. Cloning When a population of cells is prepared by growth from a single cell, all the cells in the population will be genetically identical. Such a population is called clonal. The process of creating a clonal population is called cloning. The purpose of streaking bacteria on agar is to generate single colonies, each arising from a single cell. Looking inside cells The molecules inside a cell each perform a given function. For instance, DNA molecules store information (like the hard drive in a computer). Proteins are the workhorses of the cell. To study these molecules we prepare a clonal population from a cell type of interest, break open the cells and sort the contents. For instance, it is fairly easy to separate all the proteins from all the DNA molecules. Purifying a single species of protein out of the mixture of proteins found inside a cell type is also possible. Each type of protein has unique physical and chemical properties. These properties allow the separation of protein species based on size, charge, or hydrophobicity, for instance. Special molecules, specialized functions We will take a close look at three very special kinds of molecules found inside cells: DNA, RNA and proteins. Each of these molecules performs a different function. DNA molecules are like file cabinets in which information is stored. RNA helps to retrieve and execute the instructions which are stored in DNA. Proteins are designed to perform chemical chores inside (and often outside) the cell. DNA—The universal template for biological information The master script for each organism is encoded within its deoxyribonucleic acid (DNA). The information within the DNA molecule/s of each cell is sufficient to initiate every function that cell will perform. DNA molecules are very long chains composed of repeating subunits. Each subunit (nucleotide) contains one of four possible bases protruding from its side: adenine ( A ) cytosine ( C ) thymine ( T ) guanine ( G ) Since nucleotides are joined head-to-tail, a long strand of DNA essentially consists of a chemical backbone with bases protruding along its side. The information carried by this molecule is encoded in the sequence of the bases A , G , C , and T along its length. Some further points to note about DNA structure
APPENDIX C
- Because the subunits of DNA chains are joined head-to-tail, the sequence is directional.By convention, we write DNA sequence from the free 5' (pronounced "5 prime") end of the backbone and work our way toward the other end, the "3 prime" end, or 3'. i.e. 5'... AACTG ...3'
- The protruding bases along the chain are free to form spontaneous hydrogen bondswith available bases on other DNA strands according to the following rules: (i) A pairs with T (ii) C pairs with G Because of these rules, A and T are said to be complementary bases; G and C are also complementary. (iii) For two DNA strands to pair up, they must be complementary andrun in opposite directions. i.e. (5'... AGGTC ...3') can pair with (5'... GACCT ...3'). These two strands have complementary sequences. The double-stranded pair is written as follows: 5'... AGGTC ...3' 3'... TCCAG ...5' The above molecule contains five base pairs. Indeed, in nature, DNA almost always occurs in double- stranded form with the two strands containing complementary sequences.
- DNA molecules are typically thousands, sometimes millions of base pairs long.Sometimes the two ends of a DNA molecule are joined to form circular DNA.
- Double-stranded DNA, in its native form, occurs as a coiled spring, or helix. Because itis two- stranded, it is often referred to as a double helix. The architecture of DNA allows for a very simple strategy during reproduction: The two strands of each DNA molecule unwind and "unzip"; then, each strand allows a new complementary copy of itself to be made by an enzyme called DNA polymerase. This results in two daughter molecules, each double-stranded, and each identical to the parent molecule. Proteins and RNA are the workhorses of the cell The biochemistry of life requires hundreds of very specific and efficient chemical interactions, all happening simultaneously. The major players in these interactions are short-lived protein and RNA molecules which can work together or independently to serve a variety of functions. Like DNA, RNA and proteins are also long chains of repeating units. RNA RNA (ribonucleic acid), like DNA, consists of four types of building blocks strung together in a chain. It differs from DNA in the following respects: The four bases in RNA are A , G , C , and U (uracil); the pairing rules are the same as for DNA except that A pairs with U. Although RNA can pair with complementary RNA or DNA, in cells RNA is usually single-stranded. The sugar in the RNA backbone is ribose, not deoxyribose. RNA molecules are generally short, compared to DNA molecules; this is because each RNA is itself a copy of a short segment from a DNA molecule. The process of copying segments of DNA into RNA is called transcription, and is performed by a protein called RNA polymerase. Proteins Proteins (more precisely, polypeptides) are also long, chain-like molecules but are more structurally diverse than either DNA or RNA. This is because the subunits of proteins, called amino acids, come in twenty different types. The exact sequence of amino acids along a polypeptide chain determines how that chain will fold into its three-dimensional structure. The precise three-dimensional features of this structure, in turn, determine its function. APPENDIX C
All genes in a given cell are not copied into RNA (i.e.“expressed”) at the same time or at the same rate. Thus, when speaking of gene function, one refers to its expression level. This rate can be controlled by the cell, according to predetermined rules which are themselves written into the DNA. An example: The cells in our bodies (all 100 trillion of them) each contain identical DNA molecules. Yet liver cells, for example, express only those genes required for liver function, whereas skin cells express a quite different subset of genes. DNA can be cut into pieces with restriction enzymes Restriction enzymes are proteins made by bacteria as a defense against foreign, invading DNA (for example, viral DNA). Each restriction enzyme recognizes a unique sequence of typically 4–6 base pairs, and will cut any DNA whenever that sequence occurs. For example, the restriction enzyme BamH I recognizes the sequence (5'.. GGATCC ..3') and cuts the DNA strand between the two G nucleotides in that sequence. Restriction enzymes will cut DNA from any source, provided the recognition sequence is present. It does not matter if the DNA is of bacterial, plant or human origin. Pieces of DNA can be joined by DNA ligase DNA ligase is an enzyme that glues pieces of DNA together, provided the ends are compatible. Thus, a piece of human or frog or tomato DNA cut with BamH I can be easily joined to a piece of bacterial DNA also cut with BamH I. This allows the creation of recombinant DNAs, or hybrids, created by joining pieces of DNA from two different sources. Genes can be cut out of human DNA or plant DNA, and placed inside bacteria. For example, the human gene for the hormone insulin can be put into bacteria. Under the right conditions, these bacteria can make authentic human insulin. Plasmids are small circular pieces of DNA Plasmids are small circular DNAs found inside some bacterial cells. They replicate their own DNA by borrowing the cells’ polymerases. Thus they can persist indefinitely inside cells without doing very much work of their own. Because of their small size, plasmid DNAs are easy to extract and purify from bacterial cells. When cut with a restriction enzyme, they can be joined to foreign DNAs—from any source—which have been cut with the same enzyme. The resulting hybrid DNAs can be reintroduced into bacterial cells by a procedure called transformation. Now the hybrid plasmids can perpetuate themselves in the bacteria just as before except that the foreign DNA which was joined to it is also being perpetuated. The foreign DNA gets a free ride, so to speak. Every hybrid plasmid now contains a perfect copy of the piece of foreign DNA originally joined to it. We say that foreign piece of DNA has been cloned; the plasmid which carried the foreign DNA is called a cloning vehicle or vector. In addition to their usefulness for cloning foreign genes, plasmids sometimes carry genes of their own. Bacteria die when exposed to antibiotics. However, antibiotic-resistance genes allow bacteria to grow in the presence of an antibiotic such as ampicillin. Such genes are often found on plasmids. When foreign DNA is inserted into such plasmids, and the hybrids introduced into bacterial cells by transformation, it is easy to select those bacteria that have received the plasmid—because they have acquired the ability to grow in the presence of the antibiotic, whereas all other bacterial cells are killed. DNA libraries When DNA is extracted from a given cell type, it can be cut into pieces and the pieces can be cloned en masse into a population of plasmids. This process produces a population of hybrid (recombinant) DNAs. After introducing these hybrids back into cells, each transformed cell will have received and propagated one unique hybrid. Every hybrid will contain the same vector DNA but a different insert DNA. AP PENDIX C
If there are 1,000 different DNA molecules in the original mixture, 1,000 different hybrids will be formed; 1,000 different transformant cells will be recovered, each carrying one of the original 1,000 pieces of genetic information. Such a collection is called a DNA library. If the original extract came from human cells, the library is a human library. Individual DNAs of interest can be fished out of such a library by screening the library with an appropriate probe.
Appendix D Gene Regulation
Our bodies contain thousands of different proteins which perform many different jobs. Digestive enzymes are proteins; some of the hormone signals that run through our bodies and the antibodies protecting us from disease are proteins. The information for assembling a protein is carried in our DNA. The section of DNA which contains the code for making a protein is called a gene. There are over 30,000–100,000 genes in the human genome. Each gene codes for a unique protein: one gene, one protein. The gene that codes for a digestive enzyme in your mouth is different from one that codes for an antibody or the pigment that colors your eyes. Organisms regulate expression of their genes and ultimately the amounts and kinds of proteins present within their cells for a myriad of reasons, including developmental changes, cellular specialization, and adaptation to the environment. Gene regulation not only allows for adaptation to differing conditions, but also prevents wasteful overproduction of unneeded proteins which would put the organism at a competitive disadvantage. The genes involved in the transport and breakdown (catabolism) of food are good examples of highly regulated genes. For example, the sugar arabinose is both a source of energy and a source of carbon. E. coli bacteria produce three enzymes (proteins) needed to digest arabinose as a food source. The genes which code for these enzymes are not expressed when arabinose is absent, but they are expressed when arabinose is present in their environment. How is this so? Regulation of the expression of proteins often occurs at the level of transcription from DNA into RNA. This regulation takes place at a very specific location on the DNA template, called a promoter, where RNA polymerase sits down on the DNA and begins transcription of the gene. In bacteria, groups of related genes are often clustered together and transcribed into RNA from one promoter. These clusters of genes controlled by a single promoter are called operons. The three genes (araB, araA andaraD) that code for three digestive enzymes involved in the breakdown of arabinose are clustered together in what is known as the arabinose operon.^3 These three proteins are dependent on initiation of transcription from a single promoter, PBAD. Transcription of these three genes requires the simultaneous presence of the DNA template (promoter and operon), RNA polymerase, a DNA binding protein called araC and arabinose. araCbinds to the DNA at the binding site for the RNA polymerase (the beginning of the arabinose operon). When arabinose is present in the environment, bacteria take it up. Once inside, the arabinose interacts directly with araCwhich is bound to the DNA. The interaction causes araCto change its shape which in turn promotes (actually helps) the binding of RNA polymerase and the three genes araB, Aand D, are transcribed. Three enzymes are produced, they break down arabinose, and eventually the arabinose runs out. In the absence of arabinose the araCreturns to its original shape and transcription is shut off. The DNA code of the pGLO plasmid has been engineered to incorporate aspects of the arabinose operon. Both the promoter (PBAD) and the araCgene are present. However, the genes which code for arabinose catabolism, araB, A and D, have been replaced by the single gene which codes for GFP. Therefore, in the presence of arabinose, araCprotein promotes the binding of RNA polymerase and GFP is produced. Cells fluoresce brilliant green as they produce more and more GFP. In the absence of arabinose, araCno longer facilitates the binding of RNA polymerase and the GFP gene is not transcribed. When GFP is not made, bacteria colonies will appear to have a wild-type (natural) phenotype—of white colonies with no fluorescence. This is an excellent example of the central molecular framework of biology in action: DNAèRNAèPROTEINèTRAIT.
APPENDIX D
