Bruce Wallace Biotechnology Lab Program. Student Guide 5th Edition

Transcription

1 Bruce Wallace Biotechnology Lab Program Student Guide 5th Edition

2 Preface The significance of the science you are about to do is sometimes taken for granted as the protocols have been worked and reworked so that there is a high probability that you will succeed at producing the desired molecular product. The work that you are about to do is based on Nobel Prize winning science. Werner Arbor, Daniel Nathans and Hamilton Smith received the Nobel Prize for their work with restriction enzymes. Stanley Cohen, Paul Berg and Herb Boyer received the Prize for making the first recombinant DNA molecule. The recombinant DNA molecule that you are about to use extends beyond their work as it uses a gene from a eukaryote rather than prokaryotic organism. As recently as 1993, Kary Mullis received the Nobel Prize for his discovery of the Polymerase Chain Reaction, some elegant chemistry that you will be using in Laboratory 8. So the science you will be covering over the next few weeks is significant and will continue to play an important role in the development of biotechnology and medicine. Your teacher deserves a great deal of credit for making this laboratory experience possible. Although Amgen provides the equipment and the supplies needed to implement the labs, your teacher has provided many hours of preparation time, often involving weekends and evenings, to make them happen. If you ve enjoyed this laboratory experience, please remember to thank your teacher for making it a reality. This educational outreach program is largely the result of the efforts of Dr. Bruce Wallace, an Amgen scientist who strongly believed that the biotechnology industry had a responsibility to contribute to the science education of our society. Before Dr. Wallace s untimely passing, he was able to see his educational outreach program grow and evolve into the adventure of discovery upon which you are about to embark. We are able to bring this program to your school because of several key partnerships: Amgen Foundation, Foundation for Pierce College, Los Angeles/Orange County Biotechnology Center, Bio-Bridge (biobridge.ucsd.edu/a1), New England Biolabs, Fotodyne, Invitrogen, Rainin Pipettes, VWR and Bio- Rad. Should you have any questions about these laboratories, please feel to me at the address below. Martin Ikkanda Professor of Biology Los Angeles Pierce College IkkandMJ@piercecollege.edu (c) 2007, Amgen Foundation Inc. All rights reserved.

3 Table of Contents Lab manual design by Lucy Reading Cover illustration courtesy of Ken Eward

4 Table of Contents Table of Contents LAB NUMBER 1 2 2a 3 4 4a 5 5a LAB TITLE An Introduction to Microvolumetrics and Pipetting Restriction Analysis of par A and pk AN-R par A-R Restriction Digest: An Introduction to Plasmids and Restriction Enzymes Ligation of par A /pk AN-R Restriction Fragments Producing a Recombinant Plasmid, par A-R Confirmation of Restriction and Ligation Using Agarose-Gel Electrophoresis Confirmation of par A-R Restiction Digest Transforming Escherichia coli with a Recombinant Plasmid Transformation of Escherichia coli with par A-R Preparing an Overnight Culture of Escherichia coli Purification of mfp from an Overnight Culture Genomic DNA Ex traction From Buccal Epithelial Cells PAGES a.1 2a a.1 4a a.1 5a

5 Laboratory 1 An Introduction to Microvolumetrics and Pipetting The purpose of this laboratory is to provide you with a hands-on experience using some of the important tools and techniques commonly used in molecular biology and introduce you to some of the volumetric measurements that are most often used in this field of science. The laboratory will provide you with an opportunity to practice some of the skills you will need to build a recombinant DNA molecule. The instruments and supplies that you will be using over the next few weeks are identical to the ones that are used in research laboratories. While the theoretical foundations upon which biotechnology and DNA sciences have been built extend back to the early 1900s, most of the laboratory techniques utilized are relatively recent. And though the techniques you will be learning over the next few weeks have become routine in modern research laboratories, few high school and college students have an opportunity to do such sophisticated molecular biology. If and when you take a chemistry class, one of the things you will quickly notice is the differences in the quantities of reagents and chemicals that you use. In a typical chemistry lab, volumes are measured in large graduated cylinders. Solutions are often measured in 50, 100 or 200 milliliters (ml) volumes. Weights of solids are generally expressed in grams (g). In the molecular biology lab, volumes are frequently measured in microliters (µl); 1 µl is equal to ml. Weights are often expressed in terms of micrograms (µg) or nanograms (ng); 1 µg is equal to gram and 1 ng is equal to gram. You might be wondering why molecular biologists use such small volumes and amounts of materials. The reason is related to the cost of these materials and the difficulty involved with obtaining them. For example, you will be given some specially engineered plasmids (DNA) in the next laboratory. If this DNA were sold by the pound, it would cost around $360,000,000 per pound. So don t be surprised if we only give you a tiny amount of these DNA molecules. The reason why these chemicals are so expensive is related to the difficulty in preparing them in pure form. Many of these chemicals are produced within living organisms, such as bacteria, and have to be purified and separated from all of the other thousands of substances in the cell. Molecular biology, however, really requires this level of purity and precision. As you do this lab work, keep in mind that you are doing real-world molecular biology. 1.1

6 Laboratory 1 Materials REAGENT Solution 1 Solution 2 Solution 3 Distilled H 2 O (dh 2 O) 0.8% Agarose gel (pre-made) 1 x SB (or 0.5x TBE) EQUIPMENT & SUPPLIES 1.5 ml microfuge tubes P-20 micropipettor (2-20 µl) Disposable pipette tips Permanent marker Electrophoresis equipment Power supply Plastic microfuge tube rack Methods The Digital Micropipette Molecular biology protocols require the use of adjustable micropipettes. Micropipettes are used to dispense different volumes of liquids. While researchers will have several kinds of micropipettes at their lab bench, these laboratories have been designed to utilize a P-20. The P-20 is engineered to dispense liquid volumes between 2 and 20 µl. This is a high-quality, precision instrument, and it is essential that you learn to use it properly. Please read and follow these precautions: Do not set the adjustment below 2 µl or above 20 µl unless instructed to do so by your teacher. Do not use the micropipette without the proper disposable tip firmly attached to the barrel. Failure to use a pipette tip will contaminate the pipette barrel. Do not lay down a micropipette with fluid in the tip or hold it with the tip pointed upward. If the disposable tip is not firmly seated onto the barrel, fluid could leak back into the pipette. Avoid letting the plunger snap back when withdrawing or ejecting fluid; it will eventually destroy the piston. Display window Barrel Plunger button Tip ejector 1.2

7 Laboratory 1 When aspirating (drawing up) a solution, push the plunger to the first stop and lower the pipette tip below the level of the solution that you are sampling. You should be holding the tube containing the solution in your hand about eye level. It s important to actually see the solution enter the pipette tip. Slowly release the plunger and allow the liquid to move into the pipette tip. Be certain that you re not aspirating air into the tip. When dispensing (pushing out) the liquid, place the pipette tip into the tube that will receive the solution. Position the tip so that it touches the side and near the bottom of the tube. Slowly push down on the plunger to the first stop and then to the second stop. Keep your thumb on the plunger and remove the tip from the tube into which you re dispensing the liquid. This will avoid reaspirating the liquid into the pipette tip. Be certain that you see the solution leaving the tip. Pipetting Exercise Find the display window on the handle of the micropipette and note its setting. Turn the knurled knob in the handle clockwise to decrease the volume or counterclockwise to increase the volume. Turning this knob changes the distance the plunger will travel. The figures below represent some pipette settings and the volumes of liquid dispensed μl 12.4 μl 5.5 μl 2.0 μl Place a disposable tip onto the end of the pipette barrel. Using your thumb and index finger in a twisting motion, check to see that the tip is firmly seated onto the barrel. Avoid touching the pointed end, as this may contaminate the tip. Remember that you must have a tip in place when using the pipette. Place your thumb on the button that activates the plunger. Push down on this button with your thumb and notice that it has a stop position. If you exert a little more pressure with your thumb, you can push the button of the plunger to a second stop. The second stop pushes a small volume of air into the tip to eject the solution Remove the tip by ejecting it into a waste container; there is an eject button on the pipette. If you re dispensing the same reagent into separate tubes and there is no danger of cross contamination, you can use the same tip several times. To avoid contamination, it is good practice to deposit each reagent on to the sidewall near the bottom of the microfuge tube without touching any of the other reagents. This technique allows you to use the same tip to dispense a reagent into several tubes that contain a different reagent. When dispensing a new reagent, always use a fresh tip to avoid contamination. 1.3

8 Laboratory 1 Pipetting Exercise 2 1 Use a permanent marker to label three reaction tubes A, B and C. 2 The table on page 1.4 summarizes the contents of each tube, but follow the directions that begin with step 3 to set up the samples. Tube dh 2 0 Solution 1 Solution 2 Solution 3 Total volume A 2 μl 4 μl 4 μl 10 μl B 2 μl 8 μl 10 μl C 2 μl 8 μl 10 μl Set the P-20 micropipette to 2 µl and dispense dh 2 O into tubes A, B and C. Eject the tip into the plastic waste container and replace with a fresh tip. Place 4 µl of solution 1 into tube A. Eject the tip into the plastic waste container and replace with a fresh tip. Use a fresh tip and dispense 4 µl of solution 3 into tube A. Use a fresh tip and dispense 8 µl of solution 2 into tube B. Use a fresh tip and dispense 8 µl of solution 3 into tube C. Save all three tubes for the next part of the lab. Checking the Accuracy and Consistency of Pipetting Tubes A, B and C should each contain 10 µl of solution. Set your P-20 micropipette to 10 µl and place a fresh tip onto the barrel. Carefully check the volume of each of microfuge tube. There should be 10 µl in each of these tubes. Save tubes A, B and C for the next part of the lab. 1.4

9 Laboratory 1 Using Gel Electrophoresis to Separate Molecules Gel electrophoresis is a method that uses an electrical current and a gel matrix (meshwork) to separate molecules such as DNA and proteins. The molecules that are being separated are either negatively charged or are made to be negatively charged. Using an electrical current, the charged molecules are then forced through a meshwork of material that will sort out the molecules according to their sizes, although molecular shape and degree of electro-negativity will influence movement through the gel. Because the molecules are negatively charged, they will migrate through the gel toward the positive (red) electrode. The more negatively charged, the faster the molecule will migrate. In this laboratory, your teacher has made a gel composed of agarose, a polysaccharide (complex sugar). The agarose is mixed with an electrolytic solution called Sodium Borate (SB). This solution contains ions, which are electrically charged atoms. These ions help conduct the electrical current through the gel. As the molecules are drawn toward the positive electrode, the smaller molecules are able to move in and around this agarose network much more quickly than the larger molecules. Thus, over the length of the gel, the molecules become separated by size Your teacher has already prepared an agarose gel for you, but you will need to cover the agarose gel with the appropriate amount of SB buffer to run the gel properly. Two groups will share each gel. Take the box to the power supply you will use to run the gel. Check to make certain that the gel is positioned in the gel box so that the wells of the gel are located toward the negative (black) electrode. The dyes are negatively charged and they will move toward the positive (red) electrode. Fill the box with 1x SB buffer (there are several plastic containers containing this buffer in the lab) to a level that just covers the entire surface of the gel to a depth of 1 2 mm. Check to see that the gel is covered with buffer and that no dimples appear over the wells; add more buffer if needed. Set the micropipette to 10 µl and load each sample into a separate well as indicated by your teacher. Use a fresh tip for each sample. Remember that your group will be sharing this gel. One group will load their samples in three wells on the left while the other group will use the three wells on the right. You may wish to record which solution you place in each well. When loading each sample, center the pipette tip over the well and gently depress the pipette plunger to slowly expel the sample. Use your other hand to support your pipette hand to avoid shaking. Because their densities are greater than the SB buffer, the dyes will sink into the wells. SB Buffer Agarose gel Pipette tip Close the cover tightly over the electrophoresis chamber. Connect the electrical leads to the power supply. Be certain that both leads are connected to the same channel with the cathode ( ) to cathode (black to black) and anode (+) to anode (red to red). Turn on the power supply and set the voltage to v. After two or three minutes, look at the dyes to make certain they are moving toward the positive (red) electrode. You should begin to see the purple dye (called Bromophenol blue) beginning to separate from the blue dye (Xylene cyanole). In approximately 10 minutes, or when you can distinguish all three dyes, turn off the power switch and unplug the electrodes from the power supply. Do this by grasping the plug at the power supply not by yanking on the cord. Carefully remove the cover from the gel box so that you can better see the dyes in the gel. On a piece of notebook paper, record the banding or color pattern in each of the lanes containing your samples. Use this information to answer the questions in the Conclusions. Leave the gels in the gel box. Well 1.5

10 Laboratory 1 Conclusions 1a The dyes that you separated using gel electrophoresis were: Orange G (yellow), Bromophenol blue (purple) and Xylene cyanole (blue). What electrical charge did these dyes carry? 1b What evidence allowed you to arrive at this conclusion? 2a Molecular size can play a role in separation with small molecules moving through the gel matrix more rapidly than larger molecules. The formula (or molecular) weights for these dyes are Orange G (452.38), Bromophenol blue (669.98) and Xylene cyanole (538.62). From your results, did it appear that these molecules were separated clearly on the basis of size? 2b What other factors may have played a role in the separation of these dyes? 2c Which tube contained a single dye? A, B or C? 2d Name this dye. 3 When aspirating a solution, why is it important to actually see the solution enter the pipette tip? 4a After loading your gel, did any solution remain in tubes A, B or C? 4b What could account for solution remaining in these tubes? 1.6

11 Laboratory 12 Restriction Analysis of para and pkan-r Plasmids are circular pieces of DNA that are found naturally in bacterial cells. The plasmids used in molecular biology have been modified through genetic engineering to facilitate gene cloning and protein production (gene expression) in bacteria. Antibiotic resistant genes have been engineered into these plasmids and function as selectable markers that is to say, these genes allow us to select between bacteria that harbor the plasmids from those that do not. If a bacterium carries a plasmid with an antibiotic resistant gene, the bacterium will be able to grow and reproduce in the presence of that antibiotic; those bacteria without the plasmid will not be able to grow. Thus, antibiotics can be used to select bacteria that are resistant and presumably carry a plasmid with the resistant gene from those bacteria that do not carry the plasmid. Two plasmids will be used in this laboratory: par A contains a gene for ampicillin resistance, ampr, and pk AN-R contains a gene for kanamycin resistance, kanr. The purpose of this laboratory is threefold: 1) to introduce a method commonly used to analyze the genetic elements of plasmid DNA; 2) to examine the role and nature of restriction enzymes; and 3) to take the first steps in producing a recombinant DNA molecule. The plasmid para is 4058 base pairs (bp) in size. A base pair would be adenine:thymine or guanine:cytosine and is the common method used to express the size of DNA molecules. The plasmid carries the amp r gene, which encodes the protein beta lactamase, an enzyme that destroys the antibiotic ampicillin. Beta lactamase, then, enables bacteria to reproduce in the presence of ampicillin. In addition, para carries a gene for the AraC protein, a protein that helps the bacterium make proteins encoded by genes inserted into this plasmid. A gene, even a foreign one, can be expressed (produced) if it is inserted into a specific location in this plasmid. The region of para labeled p BAD, in the plasmid map, indicates para a 4058 bp mp r arac P BAD BamH I Hind III the site where RNA polymerase needs to bind to initiate transcription. The sites labeled BamH I and Hind III represents restriction sites for these two restriction enzymes. Study the plasmid map below and locate these plasmid components. The plasmid pkan-r carries the kanamycin resistant gene, kan r, which encodes a phosphotransferase, an enzyme that transfers a phosphate group to the kanamycin molecule destroying its antibiotic effects. Kanamycin is an antibiotic that kills bacteria by preventing them from making proteins. If a cell cannot synthesize proteins, it will die. The kanr gene confers resistance to kanamycin for bacteria that have taken up this gene. In addition to kanr, the plasmid carries the gene for mutated Fluorescent Protein, mfp, called red fluroescent protein or rfp. The pkan-r plasmid is approximately 5,408 bp in size. pkan-r Ka5408 bp n r rfp Hind III rfp 702 bp BamH I The fluorescent protein gene was originally isolated from Discosoma sp, a sea anemone found in the Indo- Pacific ocean. The wild-type gene has been mutated, through a process called directed evolution, to produce colors that are several times brighter than the wild-type protein. The term wild type refers to the original gene, the one that you would find in nature. The rfp gene has been engineered into the plasmid pkan-r. Note that the rfp gene for mfp has both BamH I and Hind III restriction sites on either side. A restriction site marks the specific location where an enzyme will cut the DNA plasmid. If pkan-r is digested with BamH I and Hind III, the rfp gene will be physically cut from the plasmid. During this laboratory, then, you will remove the rfp gene from pkan-r and remove the small 40bp fragment from the para plasmid using the same enzymes. During the next laboratory, you will insert the rfp gene into para producing a recombinant DNA molecule. 2.1

12 Laboratory 12 Materials REAGENT para (80 ng/ µl) pkan-r (80 ng/ µl) Restriction enzymes (BamH I + Hind III) 2.5x restriction buffer Distilled water, dh2o EQUIPMENT & SUPPLIES P-20 micropipette and tips 1.5 ml microfuge tubes Minicentrifuge 37 C water bath Permanent marker Methods Preparing the para-r restriction digest This laboratory protocol uses the restriction enzymes BamH I and Hind III to digest the plasmids para and pkan-r. This is the first step in making a recombinant DNA molecule. 1 2 Obtain the following four microfuge tubes: para, pkan-r, BamH I and Hind III (enzyme mix) and 2.5x buffer. Obtain four clean 1.5 ml microfuge tubes and use a marker to label a set of four 1.5 tubes as follows: A + = para + BamH I and Hind III A - = undigested para (para without enzyme) K+ = pkan-r+ BamH I and Hind III K- = undigested pkan-r(pkan-r without enzyme) A + = para + enzymes + buffer 3 The reaction matrix summarizes the reagents used in the restriction digest. To set up the digest, follow the specific directions beginning at step 4. Tube 2.5x Buffer dh 2 0 para A+ A- K+ K- 4μL 4μL 4μL 4μL 2μL 2μL 4μL 4μL pkan-r Enzymes Total Volume 2μL 10μL 10μL 4μL 4μL 2μL 10μL 10μL Use a fresh tip and add 4µL of 2.5x restriction buffer to all four tubes. Add 2µL of dh2o to tubes labeled A- and K -. What is the purpose of this step? Use a fresh tip and add 4µL of para to tubes labeled A+ and A-. Use a fresh tip and add 4µL of pkan-r to tubes labeled K + and K -. Add 2µL of enzyme mix, containing BamH I and Hind III, to the A+ and K + tubes. Add the enzymes directly into the solution at the bottom of the microfuge tube. Be certain to use a new tip for each tube to avoid contamination. After the addition of the enzymes, gently pump the solution in and out with the pipette to mix the reagents and cap the tubes. If there is a minicentrifuge available, set the tubes into the rotor, being certain the tubes are in a balanced configuration, and spin the tubes for four seconds. This brief spin will pool all of the reagents at the bottom of each tube. Place all four tubes into the 37 C water bath, and incubate for at least 60 minutes. Following the 60 minute incubation, the digest can be kept frozen, at 20 C, until time is available for electrophoresis. 2.2

13 Laboratory 12 Conclusions Review the restriction maps of plasmids, para and pkan-r. Because BamH I and Hind III are specific restriction endonucleases, they will consistently cut DNA wherever it encounters the six-base recognition sequences indicated below. The precise location that is cut is called a restriction site. The DNA molecule consists of two strands of nucleotide building blocks. These building blocks are oriented in the opposite direction on each strand. Thus, the two stands that makeup a DNA molecule are said to be anti-parallel. For convenience, we can say that one strand is oriented in a 5 ( five prime ) to 3 ( three prime ) direction while the other strand is oriented 3 to 5. Careful examination of the restriction sequences will reveal that the sequence of nucleotides is a palindrome; that is to say, it reads the same on both strands when read in a 5 3 direction. BamH I 5,...G G A T C C...3, 3,...C C T A G G...5, Hind III 5,...A A G C T T...3, 3,...T T C G A A...5, Therefore, whenever Hind III encounters this six-base sequence, it will cut the DNA helix between the adjacent adenine bases. This leaves four unpaired bases forming a sticky end. 5,...A 3, 3,...T T C G A 5, Sticky end Sticky end 5, A G C T T...3, 3, A... 5, 1a What is the recognition sequence for BamH I? 1b In a 5 3 direction, what sequence of bases represents the sticky-ends for each? Examine the para and pkan-r plasmid maps and fill-in the following: 2a 2b 3 para digestion will yield fragments and will be base pairs in length. pkan-r digestion will yield fragments and will be bp and bp in length. Assume you were given a culture of bacteria carrying one or both of these plasmids. Design a simple experiment that you could use to determine which of these plasmids, para or pkan-r, the bacteria in the culture were carrying. 2.3

14 Laboratory 12

15 Laboratory 12a para-r Restriction Digest: An Introduction to Plasmids and Restriction Enzymes Two powerful but fundamental tools used in biotechnology are restriction enzymes and bacterial plasmids. Restriction enzymes allow molecular biologists to cut DNA molecules from different organisms and recombine the molecular pieces to produce recombinant DNA molecules. Plasmids are circular pieces of DNA that are naturally found in bacteria. Through recombinant DNA technology and restriction enzymes, recombinant DNA plasmids can be engineered to clone genes or to express proteins encoded by genes. Restriction enzymes were first observed by Werner Arbor in Arbor discovered that some bacteria appeared to use a primitive immune system that prevented viral DNA from replicating within the infected host cell. Some years later, it was revealed that this immune mechanism involved a class of proteins now known as restriction enzymes. The name is derived from the enzyme s ability to restrict the growth of viruses in the bacterial cells. Restriction enzymes accomplish this by breaking a bond in the sugar-phosphate backbone of the viral DNA- the enzymes cut the viral DNA into small fragments. The restriction enzymes that were first identified appeared to digest the DNA molecule randomly. Later, restriction enzymes were found and purified that would cut the sugar-phosphate backbone at a specific location or within a specific nucleotide sequence, commonly four to six nucleotides in length. Table 1 identifies some of these specific restriction enzymes, their source and the nucleotide sequences each recognizes. In 1978, Daniel Nathans (Johns Hopkins University), Hamilton Smith (Johns Hopkins University) and Werner Arbor received the Nobel Prize for Medicine for their work with restriction enzymes. Table 1. Restriction enzymes used in this laboratory. indicate sites where the sugar-phosphate backbone is cut or cleaved. 2a.1 Source Bacillus amyloliquefaciens Escherichia coli Haemophilus influenzae Restriction enzyme Recognition sequence BamH I 5, G G A T C C 3, 3, C C T A G G 5, EcoR I 5, G A A T T C 3, 3, C T T A A G 5, Hind III 5, A A G C T T 3, 3, T T C G A A 5, When restriction enzymes cut or digest DNA, the fragments that result called restriction fragments have several unpaired bases extending from their cut ends. These are called sticky ends. If DNA molecules from different sources are digested using the same restriction enzyme, the unpaired bases from each piece should be able to join (or anneal) together as the unpaired bases at the sticky ends will be complementary A:T and G: C. It is this unique attribute of restriction enzymes that enable genetic engineers to combine DNA fragments from different organisms to produce recombinant DNA molecules. (a) (b) Figure 1. (a) DNA molecule with BamH I and Hind III restriction sites (bold). The arrows indicate sites where enzymes will cut the sugar-phosphate backbone of the DNA molecule. (b) The lower DNA molecule indicates the location of the sticky ends (bold). Bacterial plasmids are relatively small, circular pieces of DNA that bacteria can carry in addition to their genomic DNA (single chromosome). In nature, the plasmid DNA frequently carries one to several genes that help the bacterium survive perhaps by providing resistance to an antibiotic. Bacteria can pass along plasmids during conjugation (mating). The bacteria we use in the laboratory have been mutated, so they cannot exchange plasmids during sexual reproduction. Naturally occurring plasmids have been engineered to perform specific functions: typically, gene cloning and gene expression. This laboratory examines para-r, a recombinant DNA plasmid that has been engineered to express the rfp gene to produce a mutant Red Fluorescent Protein (mfp). The plasmid contains various control elements that allow a bacterium carrying this plasmid to express this foreign gene. The gene was originally obtained from the genome of Discosoma sp., a sea anemone from the Indo-Pacific Ocean. The plasmid map below indicates some of the important control regions,

16 Laboratory 12a arac and PBAD, and the location of the rfp gene. In addition, the map indicates the location of two restriction sites: one for Hind III and one for BamH I. How might you go about cutting out the rfp gene? Also note, the plasmid carries an antibiotic resistance gene, ampr. This gene will enable a bacterium carrying this plasmid to live in an environment containing the antibiotic ampicillin. arac para-r a4720 bp P BAD mp r BamH I rfp rfp 702 bp Hind III Materials REAGENTS para (70 ng/ µl) Restriction enzymes (BamH I + Hind III) 2.5x restriction buffer Distilled water (dh2o) EQUIPMENT & SUPPLIES P-20 micropipette and tips 1.5 ml microfuge tubes Minicentrifuge 37 C water bath Permanent marker 2a.2

17 Laboratory 12a Methods Restriction Digest of para-r The purpose of this laboratory is twofold: 1) to examine the role of restriction enzymes and their importance in genetic engineering; 2) to examine a bacterial plasmid and how it is used in biotechnology. This laboratory protocol uses the restriction enzymes BamHI and Hind III to digest the recombinant plasmid, para-r. The restriction digest will isolate from para the rfp gene from the larger fragment of the plasmid that containing ampr, arac and PBAD. The protocol uses a control, undigested para-r, along with a DNA size marker or ladder that will help you identify and confirm the sizes of the restriction fragments. Preparing the para-r restriction digest Obtain the following three 1.5 ml microfuge tubes from your teacher: para-r, enzyme mix and 2.5x restriction buffer. Obtain two clean 1.5 ml microfuge tubes and use a marker to label the tubes as follows: A+ and A-. Include your group number and class period on each tube, so that you can locate them for the next lab period. The reaction matrix summarizes the reagents used in the restriction digest. To set up the digest, follow the specific directions beginning at step 4. Tube A+ A- 2.5x buffer H 2 O para-r Enzyme mix Total volume 4μL 4μL 2μL 4μL 4μL 2μL 10μL 10μL Use a fresh tip and add 4µL of 2.5x restriction buffer to both tubes. Add 2µL of dh2o to tube labeled A-. What is the purpose of this step? Use a fresh tip and add 4µL of para-r to tubes labeled A+ and A-. Bring the A+ tube to your teacher, who will dispense the enzyme mix into the tube, or if you were given this enzyme mix, carefully add 2µL of the enzyme mix directly into the solution in tube A+ containing plasmid and buffer. After the addition of the enzymes, cap the tube and gently flick the lower portion of each tube to mix the contents. If there is a minifuge available, set the tubes into the rotor, being certain the tubes are in a balanced configuration, and spin the tubes for four seconds. This brief spin will pool all of the reagents at the bottom of each tube. Place both tubes into the 37 C water bath, and incubate for at least 60 minutes. Following the 60-minute incubation, your teacher may place the tubes into the freezer until you are ready for electrophoresis (Lab 4a). Digested plasmids can be kept at -20 C indefinitely. 2a.3

18 Laboratory 12a Conclusions Review the restriction map of the para-r plasmid. BamH I and Hind III are specific restriction enzymes and will consistently cut the double-stranded DNA wherever they encounter their respective six-base recognition sequence given in the table on page 2a.1. These locations cut are called restriction sites. The DNA molecule consists of two strands of nucleotide building blocks. These building blocks are oriented in the opposite direction on each strand. Thus, the two stands that makeup a DNA molecule are said to be anti-parallel. For convenience, we can say that one strand in oriented in a 5 ( five prime ) to 3 ( three prime ) direction while the other strand is oriented 3 to 5. Careful examination of the BamH I and Hind III restriction sequences will reveal that the nucleotide sequences are palindromes; that is to say, they read the same on both strands when read in a 5 3 direction. 5,...A A G C T T...3, 3,...T T C G A A...5, Therefore, whenever Hind III encounters this six-base sequence, it will cut the DNA helix between the adjacent adenine bases. This leaves four unpaired bases forming a sticky end. 5,...A 3, 3,...T T C G A 5, Sticky end Sticky end 5, A G C T T...3, 3, A... 5, 1a What are the recognition sequences for Hind III and for BamH I? 1b In a 5 3 direction, what sequence of bases represents the sticky-ends? 2a Examine the para-r plasmid map and fill in the following: How many restriction fragments will result from the digestion of para with BamH I and Hind III? 2b What will be the approximate lengths, in base pairs, of these restriction fragments? 2c Which restriction fragment will carry the ampr gene? 2d Which restriction fragment will carry the rfp gene? 3 Assume your teacher gave you a culture of bacteria. The culture could be one containing bacteria carrying the plasmid para-r or a culture containing bacteria without the plasmid. Design a simple experiment that you could use to determine which of these cultures you were given. 2a.4

19 Laboratory 3 Ligation of para/pkan-r Restriction Fragments Producing a Recombinant Plasmid, para-r In this laboratory the restriction fragments produced during Lab 2 will be ligated, or bonded together, using DNA ligase, making new recombinant plasmids. These newly formed plasmids will represent recombinant DNA molecules because the four restriction fragments have been recombined in different ways to produce new constructs. For example, assume that the four plasmid fragments were represented by the letter A, A, K and R, where A and A represent the para fragments and K and R represent the two fragments resulting from the pkan-r digest. Plasmids could be represented by any combination of two letters, such as AK or A R, and any combination of even numbered fragments, such as AKA R or ARAAKK and so forth. As you can see, there are many kinds of recombinant molecules that could result from mixing together these restriction fragments. As you will remember, the restriction enzymes we are using are BamH I and Hind III. Cutting the plasmids at the BamH I and Hind III restriction sites leave sticky ends. The sticky ends on the cut DNA can be ligated to any other fragment of DNA with a complementary sticky end. Examine the para plasmid map, below, to see the locations of the BamH I and Hind III restriction sites and the sticky ends that form on the 5 -ends of its restriction fragment. Because para has one BamH I and one Hind III restriction site, the digest will leave two fragments. The restriction fragments are depicted below. It is important to remember that the large restriction fragment carries the amp r gene, the gene that provides resistance to ampicillin. The smaller fragment does not carry any genes. pkan-r will leave two fragments, one will be 4706 bp and the other will be 702 bp. Ligation will bond any two BamH I sticky ends together and any two Hind III sticky ends together. You should be able to see that many different combinations of fragments are possible. The combination of interest to us is the 4018bp para fragment recombined (containing the amp r gene) with the 702bp pkan-r fragment (rfp gene). The combination of these two fragments will yield a recombinant plasmid we will call par A-R. The ligation of the 702bp pkan-r fragment will place the rfp gene into the plasmid at a location that will allow a bacterium to synthesize (express) the mutant Fluorescent Protein, mfp. pkan-r Ka5408 bp n r rfp Hind III rfp 702 bp BamH I 5, G A T C C 3, G 4706 bp A 3, T T C G A 5, 5 A G C T T G 3, 3 A 702 bp C C T A G 5 para a4058 bp mp r arac P BAD The restriction fragments are initially held together by the hydrogen bonding between the nucleotide bases that makeup the sticky ends. You may recall that adenine and thymine share two hydrogen bonds while cytosine and guanine share three. This helps to ensure that only complementary sticky ends will match up. BamH I arac 5, G A T C C 3, G 4018 bp A 3, T T C G A 5, 5 A G C T T G 3, 3 A 40 bp C C T A G 5 The plasmid pkan-r has one BamH I and one Hind III restriction site that flank the rfp gene. The digestion of 3.1 Hind III para-r a4720 bp mp r rfp Hind III P BAD rfp 702 bp BamH I

20 Laboratory 3 DNA Ligase + ATP 5 C T A A G G A T T C 3, T A 5 3, DNA Ligase + ATP Hydrogen bonds are weak chemical bonds, and they are inadequate to hold the sticky ends together permanently. The enzyme DNA ligase, with energy supplied by ATP, will form covalent bonds between the sugar and phosphate groups of the DNA backbone. In the diagram below, you can see the positions of these bonds on each side of the DNA molecule. When the covalent bonds are formed, the bonds complete the phosphodiester linkage between the two sugars and the phosphate group on each strand. The resulting chemical bonds are a relatively strong bond. 5 C T A A G G A T T C 3, T A 5 3, Methods 1 Materials REAGENTS Digested para (A+ from Lab 2) Digested pkan-r (K+ from Lab 2) 5x Ligation buffer with ATP T4 DNA ligase in lig tube Distilled water EQUIPMENT & SUPPLIES P-20 micropipette and tips 70 C water bath Plastic microfuge tube rack Permanent marker Obtain your A+ and K+ tubes from the rack at the front of the class. Place the two tubes in the 70 C water bath for 30 minutes. This heat exposure will denature (inactivate) any BamH I and Hind III that might be active. Why is this important? While your tubes are in the water bath, obtain the 5x buffer and a Ligase tube from the instructor. The ligase tube contains 2µL of DNA ligase. Label this tube with your initials. After the 30-minute, 70 C-incubation step, add 4µL of A+ directly into the DNA ligase at the bottom of the Ligase tube. Using a new tip, add 4µL of K+ to the solution in the Ligase tube. Using a new tip, add 3µL of 5x ligation buffer directly into the solution at the bottom of the Ligase tube. Discard the buffer tube. Add 2µL of dh2o to the Ligase tube, using a clean tip. Gently and slowly pump the plunger in and out to mix the reagents. Do this without splashing the solution onto the sides of the microfuge tube. The table below summarizes the contents of the Ligase tube. A+ K+ 5x ligation buffer dh 2 O Ligase Total volume 4μL 4μL 3μL 2μL 2μL 15μL 7 8 If you have droplets of liquid clinging to the sides of the tube, briefly centrifuge the tube to pool the reagents. Place your ligase, A+ and K+ tubes in the microfuge racks at the front of the room. Your ligase tube will be kept overnight at room temperature. 3.2

21 Laboratory 3 Conclusions 1a Why was it important to place the A+ and K+ tubes in the 70 C water bath before setting up the ligation reaction? 1b What do you think might have happened if this step was omitted? 2 Make a diagram to show how the following sticky ends would join together. (: = hydrogen bonding) See page 3.2 for base pairing example. A A G C T T.... T T C G A A 3 Although many recombinant plasmids are possible, draw three possible recombinant plasmids. Include as one of the three the combination in which we are most interested the one that combines para with the pkan-r fragment carrying the rfp gene. 4 Could two rfp fragments join together and circularize in the Ligase tube? 5 In the DNA molecule, there are two kinds of chemical bonds: covalent chemical bonds and hydrogen bonds. Briefly describe how these bonds differ in strength and where, in the DNA molecule, you would find them. 6a During ligation, which of the bonds (hydrogen or covalent) form first? Where do they form? Which bonds form next and where do they form? 6b DNA ligase is required to form which bond? 3.3

22 Laboratory 3

23 Laboratory 14 Confirmation of Restriction & Ligation Using Agarose-Gel Electrophoresis It is impor tant, at this stage of our experimental procedure, that we confirm BamH I and Hind III have digested the original pkan-r and para plasmids and the restriction fragments have been ligated together by DNA ligase. This lab will provide evidence that we have recombinant DNA molecules. Gel electrophoresis is a procedure commonly used to separate fragments of DNA according to their molecular size. Like the dyes you separated in Lab 1, DNA fragments will migrate through the agarose maze. DNA, because of the phosphate groups, is negatively charged and will move toward the positive (red) electrode. Because it is easier for small molecules to move through the agarose matrix, they will migrate faster than the larger fragments. Picture a group of cross-country runners who are racing through a dense tropical rain forest. All other factors being equal, the shorter runners will be able to navigate through the tangle of overhanging vines and dense foliage faster than the taller runners. So, smaller DNA fragments will move through the tangle of agarose molecules faster than the longer fragments. We ll take all of our plasmid samples: digested, undigested and ligated, and use electrophoresis to separate these pieces. You might have predicted that your uncut plasmids would produce only a single DNA band; there s no reason why you would think otherwise. However, it is likely that two or three bands will appear in the undigested plasmid lanes. The reason for this is that plasmids isolated from cells exist in several forms. One form of plasmid is called supercoiled. You can visualize this form by thinking of a circular piece of plastic tubing that is twisted. This twisting or supercoiling results in a very compact molecule, one that will move through the gel very quickly for its size. A second plasmid form is called a nicked-circle or opencircle. Often a plasmid will experience a break in one of the covalent bonds located in its sugar-phosphate backbone along one of the two nucleotide strands. Repeated freezing and thawing of the plasmid or other rough treatment can cause the break. When this break occurs, the tension stored in the supercoiled plasmid is released as the twisted plasmid unwinds. This circular plasmid form will not move through the agarose gel as easily as the supercoiled form. Although it is the same size, in terms of base pairs, it will be located closer to the well that the supercoiled form. The last plasmid form we are likely to see is called the multimer. When bacteria replicate plasmids, the plasmids are often replicated so fast that they end up linked together like links in a chain. If two plasmids are linked, the multimer will be twice as large as a single plasmid and will migrate very slowly through the gel. In fact, it will move slower than the nicked-circle. Your pkan-r and para samples, then, may each have three bands that appear in the gel. Starting closest to the well, you might observe a multimer, followed by a nicked-circle band and, finally, a fast traveling supercoiled band. We will use a special staining technique that permits us to see the fragments embedded within the gel, then make a photographic record of your gel to document this important step. 4.1

24 Laboratory 14 Materials REAGENTS Plasmid samples: K-, K+ A-, A+ Ligated plasmid ( LIG tube) 0.8% agarose gel 5x loading dye 1x SB (or 0.5x TBE) DNA size marker (25 ng/µl) EQUIPMENT & SUPPLIES P-20 micropipette and tips 1.5 ml microfuge tubes Electrophoresis apparatus Power supply Marker pen Plastic microfuge tube rack Methods Collect the five plasmid samples and the DNA marker from your teacher and place them in your plastic tube rack. You should have six tubes. Obtain five clean 1.5 ml microfuge tubes and label them as follows: A-, A+, K-, K+, and L. The microfuge tube with the marker should already be labeled. The following table summarizes plasmid sample preparation for electrophoresis. See Hints before setting up these tubes. Tube A+ A K K+ L Hints: dh 2 0 Loading dye K+ K- A- A+ LIG Total volume 4μL 4μL 4μL 4μL 3μL 2μL 2μL 2μL 2μL 2μL 4μL 4μL For example, to the tube labeled A-, add 4µL of para-, 4µL of dh 2 O and 2µL of loading dye. The loading dye should be located in your plastic microfuge tube rack next to the dh 2 O tube. 4μL 4μL 5μL 10μL 10μL 10μL 10μL 10μL If you study this table, you ll see that you can add water to all five tubes, then add the loading dye to all of the tubes without changing the tip. Then, dispense the plasmid sample into each tube, changing the tip each time to avoid contamination. Save the LIG tube that contains your ligated plasmid; there should be about 10µL remaining in this tube. Important: Return the LIG tube to the collection rack, at the front of the room, as you will need it for the next lab. Centrifuge all samples to pool the reagents at the bottom of each tube. Be certain that the tubes are placed in a balanced configuration. 4 Prepare the gel and electrophoresis box to receive these plasmid samples. Be certain the gel wells are oriented closest to the negative (black) electrode. Pour the 1x SB buffer (or 0.5x TBE) over the gel until there are no visible dimples breaking the surface of the buffer over the wells. It s important that the gel be completely under the SB buffer. However, you don t want so much buffer in the box to allow the electrical current to run through the buffer and not the gel. 5 Take your plasmid samples and marker to the gel, along with your pipette and tips. Continued on next page

25 Laboratory 14 Continued from last page.. 6 You will share this gel with another group. Unless your teacher has you load your samples in a different pattern, load your samples in the order indicated below. Follow the loading directions that begin with step seven. If you load your sample in a different order, be certain to record it in your notebook for later reference. marker K+ K- A+ A- L 7 Using a clean tip, set your P-20 micropipettor to 10 µl. Aspirate 10 µl of your DNA size marker and slowly dispense it into the well. As you do this, slowly lower the pipette tip below the surface of the buffer directly over, but not into, the well. Putting the tip into the well can damage the wall of the well or puncture the bottom of the well. These are not good things to do. SB Buffer Agarose gel Pipette tip Well Use two hands to steady the pipette. Slowly dispense the sample by pushing to the first stop of the pipette. Because of the loading dye, the sample will have a greater density than the electrophoresis buffer. This will allow the sample to sink into the well. Important: While holding the button on the first stop, slowly remove the pipette tip from the gel box. If you ve loaded your sample correctly, the well will be filled with a blue-colored solution Continue this procedure with the plasmid samples, following the order indicated on page 4.3. Change the tip for each sample. If you choose to load your samples in a different order, be certain to record the sample order in your notebook. Close the gel box lid tightly over the electrophoresis chamber. Connect the electrical leads to the power supply. Be certain that both leads are connected to the same channel (same side) with the negative (black) to negative (black) and positive (red) to positive (red). On the power supply, set the voltage to v. After two or three minutes, look at your gel and be certain that the purple dye (bromophenol blue) is moving toward to positive electrode. If it s moving in the other direction toward the negative (black) electrode check the electrical leads to see whether they are plugged into the power supply correctly. Be certain that you return your LIG tube to the front of the room. This tube should contain your recombinant plasmids and will be used for the next lab. Your teacher will explain what to do with your gels, so listen carefully. If your lab time is short, you may not have sufficient time to complete the electrophoresis. The yellow dye will need to run just to the end of the gel, about minutes. 4.3

26 Laboratory 14 Conclusions These questions are to be answered after you ve had an opportunity to analyze your gel photograph. 1 How did your actual gel results compare to your gel predictions? 2a Are there any bands, appearing in your gel photo, that are not expected? 2b What could explain the origin of these unexpected bands? 3a Do you see evidence of the three plasmid forms in the uncut lanes? 3b Is there evidence of more than one form of multimer? 4 Why are the ligated plasmids so close to the well? 5 Two of the 702 bp pkan-r fragments, rfp gene fragments, may form a circularized fragment because each end of the fragments terminates in BamH I and Hind III sticky ends. Is there evidence of a circularized 1404 bp fragment in the ligated lane? 4.4

27 Laboratory 14a Confirmation of para-r Restriction Digest The purpose of this protocol is to examine the restriction fragments that result from the double digestion of para-r by BamH I and Hind III (Lab 2a). Gel electrophoresis is a procedure commonly used to separate fragments of DNA according to molecular size of the restriction fragments or number of base pairs. Like the dyes you separated in Lab 1, DNA fragments will migrate through the agarose maze. Because of its phosphate groups, DNA is negatively charged and will migrate towards the positive (red) electrode. Because it is easier for small molecules to move through the agarose matrix, they will migrate faster than the larger fragments. Picture a group of cross-country runners that are racing through a dense tropical rain forest. All other factors being equal, shorter runners would be able to navigate through the tangle of overhanging vines and dense foliage faster than taller runners. So, smaller DNA fragments will move through the tangle of agarose molecules faster than the longer fragments. We ll take both plasmid samples- digested and undigested- and use electrophoresis to separate these restriction fragments. You might have predicted that your uncut plasmids would produce only a single DNA band, there s no reason why you would think otherwise. However, it is likely that two or three bands will appear in the undigested plasmid lane (control). Here is the reason for this: plasmids isolated from cells exist in several forms. One form of plasmid is called supercoiled. You can visualize this form by thinking of a circular piece of plastic tubing that is twisted. The twisting or supercoiling of the plasmid results in a very compact molecule, and one that will move through the gel very quickly for its size. A second plasmid form is called a nicked-circle or a relaxed-circle. Often a plasmid will experience a break in one of the covalent bonds located in its sugar-phosphate backbone along one of the two nucleotide strands. Repeated freezing and thawing of the plasmid or other rough treatment can cause the break. When this break occurs, the tension stored in the supercoiled plasmid is released as the twisted plasmid unwinds. This circular plasmid form will not move through the agarose gel as easily as the supercoiled form. Although it is the same size, in terms of base pairs, it will be located closer to the well than the supercoiled form. The last plasmid form you may observe is called the multimer. When bacteria replicate plasmids, the plasmids are often replicated so fast that they end up in linked together like links in a chain. If two plasmids are linked, the multimer will be twice as large as a single plasmid and will migrate very slowly through the gel. In fact, it will move slower than the nickedcircle. The undigested plasmid, para-r, sample may have three bands that appear in the gel. Starting closest to the well, you might observe a multimer, followed by a nicked-circle band and, finally, a fast traveling supercoiled band. We will use a special staining technique that permits us to visualize the fragments embedded within the gel, and then make a photographic record of your gel to document this important step. 4a.1

28 Laboratory 14a Materials REAGENTS Plasmid samples: A and A+ (from Lab 2a) 0.8% agarose gel 5 x loading dye 1 x SB (or 0.5x TBE) DNA size marker (25 ng/µl) EQUIPMENT & SUPPLIES P-20 micropipette and tips 1.5 ml microfuge tubes Electrophoresis apparatus Power supply Plastic microfuge tube rack Marker pen Methods Collect both plasmid samples and the DNA marker from your teacher and place them in your plastic tube rack. You should have three tubes. Add 2 µl of loading dye to the A+ and A- tubes. Take care not to contaminate the plasmid samples. The loading dye will increase the density of each sample so the DNA will sink into the gel well. The loading dye also contains visible dyes so we can track the progress of our samples during electrophoresis. The DNA size marker already contains loading dye. Without creating bubbles, gently pump the pipette several times to mix the loading dye with the plasmid samples. Remember to use a new tip for each plasmid sample to avoid contamination. Prepare the gel and electrophoresis chamber to receive these plasmid samples. Be certain the gel wells are oriented closest to the negative (black) electrode. Pour the 1x SB (or 0.5x TBE) buffer into the electrophoresis box until there are no visible dimples breaking the surface of the buffer over the wells in the gel. It s important that the gel be completely submerged below the buffer but you don t want so much buffer in the box as the electricity runs only through the buffer and not through the gel. 4 5 Take your plasmid samples and marker to the gel, along with your pipette and tips. You may share this gel with another group. Unless your teacher has you load your samples in a different pattern, load your samples in the order indicated below. Follow the loading directions that begin with step six. If you load your sample in a different order, be certain to record it in your notebook for later reference. marker A A + Continued on next page... 4a.2

29 Laboratory 14a Continued from last page.. 6 Using a clean tip and set your P-20 micropipette to 10 µl. Aspirate 10 µl of your DNA size marker and slowly dispense it into the well. SB Buffer Agarose gel Pipette tip Well As you do this, slowly lower the pipette tip below the surface of the buffer directly over, but not into, the well. Putting the tip into the well can damage the wall of the well or puncture the bottom of the well. Use two hands to steady the pipette. Slowly dispense the sample by pushing to the first stop of the pipette. Because of the loading dye, the sample will have a greater density than the SB buffer or TBE. This will allow the sample to sink into the well. Important: While holding the button on the first stop, slowly remove the pipette tip from the gel box. If you ve loaded your sample correctly, the well will be filled with a blue colored solution containing you sample Using a clean pipette tip, load 12 µl of your A- sample into the adjacent well. Change the pipette tip and load 12 µl of your A+ sample into the next well. Close the gel box lid tightly over the electrophoresis chamber. Connect the electrical leads to the power supply. Be certain that both leads are connected to the same channel (same side) with the negative (black) to negative (black) and positive (red) to positive (red). On the power supply, set the voltage to v. After two or three minutes, look at your gel and be certain that the purple dye (bromophenol blue) is moving towards to positive electrode. If it s moving in the other direction- towards the negative (black) electrode, check the electrical leads to see if they are plugged into the power supply correctly. Your teacher will explain what to do with your gels, so listen carefully. If your lab time is short, you may not have sufficient time to complete the electrophoresis. The yellow dye will need to run just to the end of the gel, about minutes. 4a.3

30 Laboratory 14a Conclusions Questions 1 and 2 should be answered before or during electrophoresis. 1 Besides using electrophoresis to separate DNA fragments, it can be used to estimate their actual size. For example, we might be looking for a gene and we suspect it is of a certain size. Electrophoresis can be used to locate fragments in that size range. In order to do this, we would need to run a gel with a mixture of DNA fragments of known sizes. This mixture, called a marker or ladder, serves as a control or a standard to which we can compare the positions of other DNA bands in the same gel. In the diagram, below, the marker lane contains 10 DNA bands of known sizes. The fragment sizes are given below. Using this information and the plasmid map of para- R, predict the positions of DNA bands produced the A- and A+. You might want to review the different plasmid forms described on page 4a.1 and the para-r plasmid map described on page 2a.3. marker A- A+ Marker fragments kilobase pair (thick band) Questions answered following electrophoresis documentation. 2 Compare your gel photo with your prediction. Do you see any unexpected DNA bands? 3 Relative to the DNA ladder, between what two bands is the rfp gene located? Is this where you would have predicted it to be located? 4 In the A- lane, do you see evidence of different plasmid forms? Which plasmid conformation shape migrates the fastest? Which is the slowest? 5 Does the A+ lane indicate complete digestion? Explain your answer. 6 Which DNA fragment contains the ampr gene? What is the size of this DNA restriction fragment? 4a.4

31 Laboratory 15 Transforming Escherichia coli with a Recombinant Plasmid Thus far, you ve produced ligated, recombinant plasmids. Hopefully, some of these DNA recombinants will have the 702bp pkan-r fragment, the rfp gene, ligated into the large para restriction fragment. This plasmid is referred to as para-r. Now, we want to get these recombinant plasmids into bacterial cells so that we can get the cells to express rfp gene and make the mutant fluorescent protein. The process of taking up foreign pieces of DNA, like a plasmid, into a bacterial cell is called transformation. Transformation is a process that occurs in nature, although it is probably somewhat rare. A British medical officer, Frederick Griffith first studied the process, in Bacteria usually pass on extra chromosomal genetic material, like plasmids, during conjugation (bacterial sex) rather than relying on luck. But taking up plasmids can provide bacteria with certain genes that confer selective advantage, for example, antibiotic resistance. Under experimental conditions, however, it is possible to prepare cells so that about one cell in a thousand will take in a plasmid from the surrounding environment. There are several factors that determine transformation efficiency. Two of these are related directly to the plasmid used for transformation. The larger the plasmid, the less likely it will be taken up by the bacterium. Remember, in order for the bacterium to take in foreign DNA, the plasmid must pass through bacteria s plasma membrane and cell wall. Therefore, small plasmids are more likely to pass through the bacterium s plasma membranes (E. coli has two) and its cell wall than large plasmids. Plasma membranes Plasmid Genomic DNA Cell wall Plasmids Plasmids can assume different shapes. The supercoiled form is the easiest to get into the cell while the nickedcircle or the multimer, two or more plasmids linked together, are more difficult. The ligation tube, containing the recombinant plasmids you prepared, does not contain any supercoiled plasmids. Supercoiling of a plasmid requires an enzyme that is found in the bacterial cell; it was not included in your ligation tube. The recombinant plasmids you prepared are primarily nicked-circled, but there is a wide variation in sizes. In nature, transformation is a relatively rare event. To increase our chances of getting our recombinant plasmids into bacterial cells, we will use competent cells. When cells are competent, it means that they are ready to receive plasmids. For the most part, you don t find competent cells in nature; instead, cells have to be made competent in the laboratory. One common way this is done is by soaking the cells in calcium chloride. Remember that DNA is negatively charged. Do you remember why? The plasma membranes surrounding the bacterial cell also contain phosphate groups and are negatively charged. The problem of trying to get negatively charged DNA past a negatively charged membrane is that like electrical charges tend to repel each other. When cells are made competent, they are suspended in a solution of calcium chloride because calcium ions (positively charged atoms of calcium, Ca++) help to neutralize the negative electrical charges of the plasma membrane and the plasmid. With these repulsive charges neutralized by the calcium ions, the plasmid DNA has an easier time passing by the plasma membrane of the bacterial cell. The cells have already been made competent for you and your teacher will give you an aliquot. You will, however, need to do the next step. Now that we have the negative charges on the DNA and the plasma membranes neutralized, we need to create a bit of a pressure difference between the inside and the outside of the bacterial cell. This is done by first getting the bacteria really cold and then quickly putting them into warm water. This is called heat shock, and it creates a situation in which the pressure outside the cell is a tiny bit higher than inside the cell. This pressure gradient will help to move the plasmid DNA from the outside to the inside of the bacterial cell. Following this brutal treatment, we ll need to feed our bacteria and let them recover for a few minutes before we spread them onto agar plates. 5.1

32 Laboratory 15 Once the cells have recovered, you ll take samples of these cells and spread them on a series of sterile agar plates. One of these plates will contain only bacterial food; the plate contains no antibiotic. This plate is marked LB. A second plate contains LB and ampicillin; this plate is marked amp. The third plate contains LB, ampicillin and a simple sugar called arabinose; this plate is marked ara. Ampicillin is an antibiotic that prevents bacteria from fully forming its cell wall. Cells that are not ampicillin resistant cannot grow in its presence; the new cells simply rupture or lyse. If a cell receives an ampicillin-resistant gene, ampr, it will produce a protein that will chemically destroy ampicillin and, therefore, will be able to grow with ampicillin in its environment. Arabinose, a simple sugar, is needed by the bacterium to express the rfp gene. If a bacterium takes up para- R, arabinose helps the enzyme RNA polymerase, needed to transcribe the rfp gene, to align itself correctly on the plasmid. This relationship will be discussed in the next lab. Although the E. coli strain that you are using in these labs is relatively benign, it is important that you use proper techniques when handling them. Materials REAGENTS AND CULTURES LIG tube (recombinant plasmids) 100 µl of competent cells (LMG) 350 µl of LB broth (sterile) Crushed ice (in a styrofoam cup) Agar plates, sterile 1 LB, 1 LB/amp, 1 LB/amp/ara EQUIPMENT & SUPPLIES P-20 micropipette and tips P-200 micropipette and tips 42 C water bath 1 pack cell spreaders (shared) Plastic microfuge tube rack 1.5 ml microfuge tubes Marking pen 5.2

33 Laboratory 15 Methods In order to make this lab run smoothly, it s important that you know which tasks have been assigned to each group member before the beginning of the lab. One member should prepare the ice and get the competent cells, another can retrieve your ligated plasmids and another can get the agar plates, LB broth and clean microfuge tubes. 1 2 Pick up two clean microfuge tubes. Label one P+ and the other P-. P+ P- Pick up a Styrofoam cup with crushed ice and place one tube containing 100 µl of competent cells into the ice. It s important that the cells remain at 0 C. Also, place your P+ and P- tubes into the ice. 9 Label the bottoms (plate containing the agar) of all three plates with your group number and class period. Write small and on the edge of the plate. Then divide the LB and amp plates down the middle using two lines. Label one half of each plate P+ and the other half with a P-. See the diagram below. Do not divide the ara plate. P- P+ P- P+ P Pick up your ligated plasmids from the microfuge tube rack labeled LIG tubes. Your LIG tube should be labeled with your group number and class period. Set the P-200 pipettor to 50 µl (set to ) and place a clean tip onto its barrel. Very carefully resuspend the cells by gently pumping the cells in and out two times. Hold the tube by the upper rim to avoid warming the cells with your fingers. Aliquot 50 µl of the resuspended cells into the prechilled P+ and P- microfuge tubes. Immediately return the aliquoted cells to the wet ice. Hold the tubes by the upper rim to avoid warming the cells with your fingers. Using the P-20 pipette, add 10 µl of your ligated plasmid to the tube labeled P+. Gently mix the plasmid with the cell suspension by pumping the cell suspension two times. Immediately return the P+ tube into the ice. Do not add plasmid to the P tube. The cells in this tube will serve as the plasmid control LB plate LB/amp plate LB/amp/ara Following the 15-minute incubation in ice, carry the ice cup containing the cells to the 42 C water bath. Take the tubes from the ice and hold them in the water bath for 45 seconds. After the 45-second heat shock, place them back into the wet ice immediately for at least one minute. After one minute, use the P-200 pipette to add 150 µl (set to ) of LB broth to the P- tube. Cap the tube and gently flick the lower por tion of the tube two or three times to mix. Use a new tip and transfer 150 µl of LB broth to the P+ tube. Close the cap and gently flick the tube to mix. Obtain one package of sterile cell spreaders from your teacher. Two groups will share this package. You are now ready to spread your bacterial cells onto the sterile agar plates. 7 8 Keep the cells in ice for 15 minutes. While the cells are incubating in ice, obtain the following: One each of these agar plates: LB, LB/amp (LB + ampicillin) and LB/amp/ara (LB + amp + arabinose) a Using the P-200 pipette (set to ), gently pump the pipette two or three times to resuspend the cells then aspirate 50 µl of cells from the P- tube. Open the lid from the LB plate like a clamshell. Dispense these cells on the half of the plate marked P-. Close the lid. 5.3

34 Laboratory 15 b Resuspend the cells by gently pumping the pipette then aspirate a second 50 µl aliquot for the LB/amp plate. Remember, you want to deposit the P cells on the half of the plate you labeled P-. Cover the plate. 15 g Repeat steps 14 a-e to inoculate the LB and LB/amp plates with the P+ culture. Be cer tain to use the + pipette and a new spreader to avoid contamination. Now you re ready to inoculate the LB/amp/ara plate. 50 μl P- 50 μl P- P+ P- P+ LB plate LB/amp plate a b Using the P-200 pipette (set to ), transfer 100 µl of the P+ culture onto the surface of the LB/amp/ara plate. Deposit the 100µL of cells on several areas across the agar surface rather than a single spot. Lift the lid, clamshell style, and spread the cells evenly over the surface of the plate. P+ 100 μl of cells c d e f Open the package of sterile cell spreaders at the end closest to the spreader handles. You will share this package with another group. Remove only one spreader, keeping the others sterile. Hold the spreader by the handle and do not allow the bent end to touch any surface, as this will contaminate the spreader. Close the package to avoid contaminating any of the other spreaders. Open the lid to the LB plate, like a clamshell, and gently Handle using a light, gliding motion spread the cells across the surface of the agar, keeping the cells on the P side of the plate. Try to spread them evenly and along the sides of the plate as well. Spreading surface Carefully spread the P- cells on the LB/amp plate using the same spreader and technique. Place the used spreader into the biohazard bag c d Gently rotate the plate beneath the P+ spreader so that the cells can be spread over the entire sur face of this plate. Try to get the cells spread along the wall of the plate as well. Cover the plate when finished. Allow all three plates to sit right-side up for five minutes. LB/amp/ara plate Using colored tape, tape all three plates together and place them in the incubator, gel-side up. Be cer tain that you have clearly labeled your plates with your group number and class period. You can mark the tape to help you f ind them for the nex t lab. Discard cell-contaminated waste: spreaders, cell tubes, pipette tips, by placing them into the cellcontaminated waste bag provided by your teacher. P+ 5.4

35 Laboratory 15 Conclusions Answer questions 1-3 before seeing the results of your transformation. 1 Predict the growth, if any, on the following plates. Remember the cells from the P+ culture were given recombinant plasmids while those from the P were not. Use a + if you expect growth and a - if you expect no growth. P- P+ P- P+ P+ LB plate LB/amp plate LB/amp/ara 2a What do all of the cells growing on the LB/amp and LB/amp/ara plates have in common? 2b What single restriction fragment must they all contain to grow on plates with ampicillin? 3a Would you expect that all of the cells growing on the LB/amp/ara plate were transformed with the same plasmid? Explain. 3b How might you determine which of the cells on the LB/amp/ara plate contain para-r, the recombinant plasmid that you ve made by ligating the rfp gene with the large para restriction fragment? 5.5

36 Laboratory 15 Answer these questions after viewing the results of your transformation. 4a Use the following table to compare how your actual transformation results differed from your predicted results. See page 5.5 for predicted results. Plate Predicted results Actual results LB LB/Amp LB/Amp/Ara 4b If your actual results differed from your expected, propose some reasons that might explain these differences. 5a How many red colonies were present on your LB/amp/ara plate. 5b Why did the red colonies only appear on this plate and not the LB/amp plate? 5c Would you expect that some of the bacteria on the LB/amp plate were transformed with para-r? Briefly describe how you might test your answer. 5.6

37 Laboratory 15a Transformation of Escherichia coli with para-r The process of taking up foreign pieces of DNA, like a plasmid, into a bacterial cell is called transformation. Transformation is a process that occurs in nature, although it is probably somewhat rare. A British medical officer, Frederick Griffith first studied the process, in Bacteria usually pass on extra chromosomal genetic material, like plasmids, during conjugation (bacterial sex) rather than relying on luck. But taking up plasmids can provide bacteria with certain genes that confer selective advantage, for example, antibiotic resistance. Under experimental conditions, however, it is possible to prepare cells so that about one cell in a thousand will take in a plasmid from the surrounding environment. There are several factors that determine transformation efficiency. Two of these are related directly to the plasmid used for transformation. The larger the plasmid, the less likely it will be taken up by the bacterium. Remember, in order for the bacterium to take in foreign DNA, the plasmid must pass through bacteria s plasma membrane and cell wall. Therefore, small plasmids are more likely to pass through the bacterium s plasma membranes (E. coli has two) and its cell wall than large plasmids. Plasmids can assume different shapes. The supercoiled form is the easiest to get into the cell while the nickedcircle or the multimer, two or more plasmids linked together, are more difficult. In nature, transformation is a relatively rare event. To increase our chances of getting our recombinant plasmids into bacterial cells, we will use competent cells. When cells are competent, it means that they are ready to receive plasmids. For the most part, you don t find competent cells in nature; instead, cells have to be made competent in the laboratory. One common way this is done is by soaking the cells in calcium chloride. 5a.1 Genomic DNA Plasma membranes Plasmid Cell wall Plasmids Remember that DNA is negatively charged. Do you remember why? The plasma membranes surrounding the bacterial cell also contain phosphate groups and are negatively charged. The problem of trying to get negatively charged DNA past a negatively charged membrane is that like electrical charges tend to repel each other. When cells are made competent, they are suspended in a solution of calcium chloride because calcium ions (positively charged atoms of calcium, Ca++) help to neutralize the negative electrical charges of the plasma membrane and the plasmid. With these repulsive charges neutralized by the calcium ions, the plasmid DNA has an easier time passing by the plasma membrane of the bacterial cell. Now that we have the negative charges on the DNA and the plasma membranes neutralized, we need to create a bit of a pressure difference between the inside and the outside of the bacterial cell. This is done by first getting the bacteria really cold and then quickly putting them into warm water. This is called heat shock, and it creates a situation in which the pressure outside the cell is a tiny bit higher than inside the cell. This pressure gradient will help to move the plasmid DNA from the outside to the inside of the bacterial cell. Once the cells have recovered, you ll take samples of these cells and spread them on a series of sterile agar plates. One of these plates will contain only bacterial food. The plate contains no antibiotic. This plate is marked LB. A second plate contains LB and ampicillin; this plate is marked amp. The third plate contains LB, ampicillin and a simple sugar called arabinose; this plate is marked ara. Ampicillin is an antibiotic that prevents bacteria from fully forming its cell wall. Cells that are not ampicillin resistant cannot grow in its presence, the new cells simply rupture or lyse. If a cell receives an ampicillin-resistant gene, ampr, it will produce a protein that will chemically destroy ampicillin and, therefore, will be able to grow with ampicillin in its environment. Arabinose, a simple sugar, is needed by the bacterium to express the rfp gene. If a bacterium takes up para- R, arabinose helps the enzyme RNA polymerase, needed to transcribe the rfp gene, to align itself correctly on the plasmid. This relationship will be discussed in the next lab. Although the E. coli strain that you are using in these labs is relatively benign, it s important that you use proper techniques when handling them.

38 Laboratory 5a 1 Materials REAGENTS AND CULTURES E. coli (LMG) plate Crushed ice 10 µl para-r (10 ng/µl) 1 ml 50 mm CaCl 2 1 LB plate 1 LB/amp plate 1 LB/amp/ara plate EQUIPMENT & SUPPLIES 1.5 ml microfuge tubes Sterile inoculating loop Disposable cell spreaders (2) Microfuge tube rack Permanent marker P-20 micropipette and tips P-200 micropipette and tips Beaker with disinfectant 42 C water bath Methods Preparing competent cells for transformation Bacterial transformation requires sterile techniques. It is essential that directions be followed precisely. Use the marker to label one of the 1.5 ml sterile microfuge tubes P+ and the other tube mark P-. Plasmid DNA will be added only to the P+ tube. The P- tube will represent a negative plasmid control. Pick up a tube containing CaCl2. Use your P-200 pipette and a clean tip to transfer 250 µl of CaCl2 to the P+ and P- tubes. Hint: Set the P-200 pipette to 125 µl and transfer two aliquots to each of the labeled tubes. The pipette should read in the display window. Your instructor will provide the class with a Petri plate containing colonies of E. coli cells. Use a sterile inoculating loop to gently scrape two or three large bacteria colonies from the Petri plate and transfer them to the P+ tube. Knock the loop against the side of the tube to dislodge the colonies from the loop. Cap the tube and vigorously drag the tube across the surface of your microfuge tube rack to suspend the cells in the CaCl2. Continue to do this until you can no longer see any visible clumps of bacteria. Place this tube into your crushed ice. P- P+ P- P+ P Repeat this procedure using the same inoculating loop, but transfer the colony and suspend the cells in the P- tube. Place this tube in the crushed ice. Place the inoculating loop in disinfectant or cell waste bag for proper disposal. Transfer 10 µl of plasmid (para-r) directly into the cell suspension in the P+ tube. Briefly finger vortex the mixture by gently flicking the bottom of the microfuge tube with your index finger. Avoid splashing the mixture on the sidewall of the transformation tube. Return the P+ tube to the crushed ice. Incubate both tubes in crushed ice for 15 minutes. Be certain the tubes are in contact with the ice. It is important that the cells get very cold. Obtain one each of the following plates: LB, LB/amp and LB/amp/ara. Using a marker and straight edge, draw a line down the center of the LB and LB/amp plates, but not the LB/amp/ara plate. Make this division on the bottom gel side of the two plates. Label a P- and a P+ on each half of LB and LB/amp plate bottoms and a + on the LB/amp/ara plate bottom. LB plate LB/amp plate LB/amp/ara 5a.2

39 Laboratory 15a Transforming E. coli with para-r 1 Following the 15-minute chilling in ice, heat shock the cells in both tubes using the following procedures: Take the ice container containing the transformation tubes P+ and P- from step 7 to the 42 C water bath. It s important that the bacteria receive a distinctly abrupt change in temperature. Hold both tubes into the water bath for exactly 45 seconds. Return both tubes to the crushed ice immediately for at least one minute. 2 Following the one-minute cooldown, return both tubes to the test tube rack and maintain at room temperature. Spreading transformed cells on agar plates Position the three plates in the following order: LB, LB/amp, LB/ amp/ara. Set the P-200 pipette to 50µL. The pipette will read Holding the P- (control) cells between your thumb and index finger. Gently flick the bottom of the tube to resuspend the cells. Deposit 50 µl of these cells onto the - half of the LB and LB/amp plates. Do not deposit these cells on the LB/amp/ara plate. Spreading surface Open the package of sterile Handle cell spreaders at the end closest to the spreader handles. You will share this package with another group. Remove only one spreader, keeping the others sterile. Hold the spreader by the handle and do not allow the bent end to touch any surface as this will contaminate the spreader. Close the package to avoid contaminating any of the other spreaders Using the same spreader, repeat this spreading procedure for the LB/amp plate. Discard the cell spreader in disinfectant or the cell waste bag following its use on the LB/amp plate. After resuspending the cells in the P+ tube, use a new tip and deposit 50 µl of cells to the + side of the LB and LB/amp plates; deposit 150 µl of cells to the LB/amp/ ara plate. Using a new cell spreader, repeat steps 4 and 5, spreading the cells on the + side only of the LB and LB/ amp plates and over the entire surface of the LB/amp/ ara plate. Discard this cell spreader in disinfectant or the cell waste bag. Use masking tape to keep your plates together. Place your name on the tape so that you can locate your plates later. Place the plates upside down into a 37 C incubator. Incubate the plates for hours at 37 C. 4 Open the lid to the LB plate, like a clamshell, and gently using a light, gliding motion spread the cells across the surface of the agar keeping the cells on the side only of the plate. Try to spread them evenly and along the sides of the plate as well. 5a.3

40 Laboratory 15a Conclusions Answer questions 1-3 before seeing the results of your transformation. 1 Predict the growth, if any, on the following plates. Remember the cells from the P+ culture were given the plasmid while those from the P were not. Use a + if you expect growth and a if you expect no growth. P- P+ P- P+ P+ 2a LB plate LB/amp plate LB/amp/ara What do all the cells growing on the LB/amp and LB/amp/ara plates have in common? 2b What fragment of the para-r plasmid allows these cells to grow on these plates? 2c What is the size, in base pairs, of this fragment? 3 On which plate(s) would you expect the cells to express the rfp gene? Answer these questions after viewing the results of your transformation. 4a 4b Use the following table to compare how your actual transformation results differed from your predicted results. Plate Predicted results Actual results LB LB/Amp LB/Amp/Ara If your actual results differed from your expected, propose some reasons that might explain these differences. 5a How many red colonies were present on your LB/amp/ara plate? 5b Why did the red colonies only appear on this plate and not the LB/amp plate? 5a.4

41 Laboratory 16 Preparing an Overnight Culture of Escherichia coli The purpose of this lab is to star t a bacterial culture that will produce a sufficient quantity of mutant fluorescent protein to enable you to isolate and purify the protein. From your LB/amp/ara plate, your teacher will select one of the red colonies transformed with the para-r plasmid and use it to inoculate an overnight culture. The gene for mfp was originally isolated from a sea anemone. The mfp is used extensively in research as the protein can be fused to other proteins and then followed through the cell using fluorescent microscopy. The original fluorescent protein gene was mutated to produce a molecule that fluoresces many times brighter. The plasmid para-r was then engineered for gene expression. The diagram below depicts the region of para-r containing the major control elements required to express the rfp gene. It s important for you to note that only a small portion of the para-r plasmid is represented in this diagram and the DNA is depicted as a straight line rather than a circle. The diagram identifies three important regions: 1) arac gene; 2) the promoter region (PBAD); and 3) the location of the rfp gene relative to the other control elements in the plasmid. arac gene Transcription mrna Translation Promoter AraC protein rfp plasmid, the promoter site and a region near the arac gene. This causes the DNA molecule to bend around, forming a loop. When the DNA is in this configuration, mrna transcription cannot occur as it prevents RNA polymerase from binding to the promoter site. Without mrna, the bacterium cannot produce mfp. Promoter arac gene rfp gene AraC protein prevents rfp transcription by causing a loop to form in the region of the rfp gene. When arabinose is present in the bacterium s environment, arabinose binds with the AraC protein, forming a complex. This prevents the DNA loop from forming. The binding of arabinose also causes a change in the protein s conformation (shape) resulting in the formation of a small pocket that will help a third molecule, RNA polymerase, to join the complex. This complex of three molecules binds to the promoter site, and RNA polymerase is aligned on the DNA molecule in a way that it can transcribe the rfp gene. This transcription produces mrna, which is translated into mfp. The AraC protein, then, serves a duel function: It can inhibit mfp synthesis by looping the DNA and preventing RNA polymerase from binding to the promoter region, and it can turn on the rfp gene transcription and, therefore, mfp production, if it binds to arabinose. AraC protein arabinose RNA polymerase The arac gene codes for a regulatory protein known as the AraC protein. The AraC protein is involved with turning the rfp gene off and on. The above diagram summarizes the relationship between the AraC gene, transcription, messenger RNA, translation and the arac protein. arac gene arabinose AraC protein complex rfp Promoter Transcription The promoter site is that portion of DNA where regulation of rfp expression occurs. When there is no arabinose in the bacterium s environment, the AraC protein will physically bind to two regions of Arabinose AraC protein complex prevents DNA looping and helps to align RNA polymerase on the promoter site. mrna mfp 6.1

42 Laboratory 16 Inclusion body mfp molecules Genomic DNA Plasma membranes para-r Cell wall Cytoplasm Other proteins When the bacterium expresses the rfp gene and produces mutant fluorescent protein, the cell takes these mfp molecules and concentrates them into inclusion bodies. Inclusion bodies are concentrated granules of mfp molecules and are not bound by a membrane. Methods Because you will be working with bacteria, it will be impor tant that you work quickly to avoid contamination. 1 2 Your instructor will use a clean toothpick to transfer a few cells from an mfp-expressing colony (red) into a flask containing LB/amp/ara broth. The flask will be placed on a shaker and shaken overnight to encourage both cell division and rfp expression. Conclusions 1 Although your instructor worked quickly to transfer a sample of bacteria expressing rfp, there is a good chance that some non-rfp-expressing bacteria were transferred as well. What would prevent the growth of these bacteria in the LB/amp/ara broth? Materials REAGENT LB/amp/ara plate (from Lab 5 or 5a) Sterile flask with LB/amp/ara broth EQUIPMENT & SUPPLIES Shaker Clean toothpick 2 The purpose of this overnight culture is to clone the bacteria expressing the rfp gene and to have them produce sufficient mfp to purify the protein from the other proteins in the cell. As the cells are cultured, would you expect to find the mfp within the bacterial cells or in the nutrient broth surrounding the cells? 6.2

43 Laboratory 17 Purification of mfp from an Overnight Culture When scientists at a therapeutics company, like Amgen, have successfully identified a promising therapeutic protein, two objectives would be to locate and isolate the gene that encodes the protein. Once isolated, the gene is inserted into a plasmid so that the gene can be cloned, as additional copies of the gene will be needed for ongoing studies. The rfp gene was cloned in a plasmid called pkan-r. pkan-r is a cloning vector, a plasmid that has been engineered to replicate in high numbers within the bacterial cell. Later, cloned genes are inserted into plasmids that have been engineered specifically for protein expression in bacteria or other suitable organism. Such plasmids are known as expression vectors. para-r is an expression vector and carries the cloned rfp gene in a specific plasmid location, which allows the bacterial cell to produce mutant fluorescent protein. Transformed cells are allowed to express the protein in an overnight culture and then lysed (broken open) to release the newly synthesized protein from the cell. The protein is isolated from the other cytoplasmic proteins, purified and tested for activity. You have already completed much of the work that parallels this drug discovery scenario. The bacterial cells that have been growing in the LB/amp/ ara broth have been expressing mfp and are now ready to be lysed (day one of Lab 7) and the mfp purified (day two of Lab 7) using column chromatography. Mutant fluorescent protein is a molecule that is about 238 amino acids in size. The native (as it exists in Discosoma) protein is shaped like a cylinder with the fluorescent region, called the fluorophore, located in the center of the cylinder. In order to purify a molecule from other proteins present in the cell, one needs to look at how groups of molecules differ from one another and how these differences can be used to effect separation. Inclusion body mfp molecules Plasma membranes Genomic DNA para-r Cell wall Cytoplasm Other proteins One molecular attribute commonly used in purification is protein hydrophobicity. The term hydrophobicity is related to the behavior of a molecule in water. If a molecule is hydrophobic, it fears water while hydrophilic molecules love water. For example, oils, waxes and fats are hydrophobic; they do not dissolve in water. Table sugar and table salt are hydrophilic, and they dissolve quickly in water. It is not uncommon for large molecules, such as proteins, to have regions that are hydrophobic and other regions that are hydrophilic. If these proteins are placed in water, the hydrophobic regions tend to bend away from water while their hydrophilic regions try to bend toward the water. To a large extent, it s the bending of the protein s amino acid chain that is responsible for its overall conformation or molecular shape, with hydrophobic regions hiding in the interior of the molecule and water-loving regions on the outside. Buffer It s important for you to know that a bacterial cell contains many different kinds of proteins. The diagram below is greatly simplified as it indicates only a few kinds. The problem, however, is how do you separate a single protein, like mfp, from all of the others? A typical bacterium may contain more than a 1000 different kinds of protein. The use of the recombinant expression Column mfp Hydrophobic resin Stop cock Other proteins 7.1