In order to do transformation, the gene to be transferred is placed into a plasmid. This is done with the help of restriction enzymes, 7

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1 Fluorescent Protein Transformation Student Background Genetic transformation occurs when a cell takes up (i.e. takes inside) and expresses a new piece of genetic material DNA. Genetic transformation literally means change caused by genes and it involves the insertion of a gene(s) into an organism in order to change the organism s traits. Remember that a gene is a piece of DNA which provides the instructions for making (coding for) a particular protein. In this lab, we will transform bacteria, giving it the ability to make fluorescent proteins. These are proteins that absorb light at one wavelength (color) and re-emit the light at a different wavelength, or color. Some things will glow with a new color when you shine the "right light" on them. Most people are accustomed to seeing fluorescence produced by ultraviolet light, often called "black light." Genetic transformation is used in many areas of biotechnology. In agriculture, genes coding for traits such as frost, pest or drought resistance can be genetically transformed into plants. In bioremediation, bacteria (prokaryotes or single-celled organisms without a nucleus) can be genetically transformed with genes enabling them to digest oil spills. A medical application of transformation is in the creation of proteins, such as insulin (synthesized by Genentech) and factor VIII (blood clotting protein synthesized by Bayer). Genes can be cut out of human, animal or plant DNA and placed inside bacteria and the bacteria will produce the foreign protein coded for by the gene. For example, a healthy human gene for the hormone insulin can be put into bacteria. Under the right conditions, these bacteria can make authentic human insulin just as they would make their own proteins. This insulin can then be used to treat patients with the genetic disease, diabetes, whose insulin genes do not function properly. In this lab, you will learn about the process of moving genes form one organism to the other with the aid of a plasmid. In addition to one large circular chromosome which contains all of the genes a bacterium needs for its normal existence, bacteria naturally contain one or more tiny circular pieces of DNA called plasmids. Plasmid DNA contains genes for traits that may be beneficial to bacterial survival under certain environmental conditions. In nature, bacteria can transfer plasmids back and forth, allowing them to share these beneficial genes. This mechanism allows bacteria to adapt to new environments. The recent occurrence of bacterial resistance to antibiotics is due to transmission of plasmids. In order to do transformation, the gene to be transferred is placed into a plasmid. This is done with the help of restriction enzymes, 7

2 naturally occurring enzymes from bacteria that recognize a particular sequence of DNA bases and cut at that sequence. Bacteria use restriction enzymes to protect themselves from viruses which inject their DNA into the bacteria; the enzymes can cut the viral DNA before it can hurt the bacteria. The same restriction enzyme is used to cut the ends of the gene we want to transfer and to cut open the plasmid. Because the cuts are made at the same base sequence, the ends will match and reattach when placed together. To move the plasmid through the E. coli cell membrane you will use a transformation solution of calcium chloride (CaCl 2 ), and a procedure know as heat shock which will allow the plasmid to move through the cell wall and membrane without killing the bacteria. Although no one knows exactly how this works right now, the prevailing hypothesis is that the CaCl 2 causes pores to open in the bacterial cell wall and membrane, allowing the plasmid to enter the cell. The details of this process are one of the things that scientists still have yet to discover. In order to select only the bacteria that have received the new gene, the plasmid also has a gene for resistance to the antibiotic Ampicillin. The gene codes for the production of a protein, beta-lactamase, that allows the bacteria to digest the antibiotic before it can cause any harm. Therefore, if ampicillin is mixed into the agar on the bacterial plates, the only bacteria that can survive there will be bacteria with the gene/plasmid for ampicillin resistance. If any bacteria grow on the plate, they must have been transformed. Each colony on the plate is the offspring of one original transformed bacterial cell (a clone of the original). The bacteria reproduce very rapidly, and a colony may represent millions of cells, all of which are genetically identical, since they all came from the same original bacterium. Below is a map of the plasmid used in this lab. The gene for ampicillin resistance is labeled and FP stands for fluorescent protein. As you can see, any one of the different colored fluorescent proteins can be inserted into the same place in this plasmid. In this lab, each DNA plasmid (1-8) has the gene that codes for a different fluorescent protein. There will be 8 different colored fluorescent plasmid/proteins possible in the class. One of your tasks will be to figure out which protein s instructions were on the plasmid you received. mfruits vector ~ 3.6kb 8

3 Fluorescent Protein Student Background Fluorescent proteins (FPs) are found in light-producing cells of many coelenterates (e.g. jellyfish and corals). In most species their function is an intriguing mystery. But in general in the animal kingdom, bright coloration like this is an adaptation for survival and is used for either finding food by locating or attracting prey, for protection against predators, or for communication between individuals of a given species usually associated with attracting mates. Discosoma (dsred) Fluorescent corals To the left is a picture of the molecular structure of one fluorescent protein (Green Fluorescent Protein or GFP). You can see it has an interesting barrel shape. The part or the molecule that actually fluoresces (the chromophore) is in the middle of the barrel and is composed of a ring of only 3 amino acids. Another interesting thing about fluorescent proteins is that the organism doesn t need to use energy to make the protein light up; the protein is simply energized by light hitting the chromophore. Applications of Fluorescent Proteins FPs can be attached to many proteins without interfering with their functions. If the gene for the FP is attached to a cell s protein gene, then when the normal gene is expressed in the cell, the FP will be expressed at the same time. This makes it possible to visually detect when and where a particular protein of interest is produced and used by a cell. FPs are also being used for screening drugs, evaluating viral vectors for human gene therapy, biological pest control, and monitoring genetically altered microbes in the environment, among other applied efforts. 9

4 One to Two days before the lab: 1. Label eppendorf (microfuge) tubes CaCl 2 and aliquot 1 ml for each lab group. 2. Copy instructions for students 3. Streak starter plates at least one for each class Touch sterile loop to E. coli culture in stab Use sterile loop to streak starter plates, then incubate at 37 o C for 24 hours or 2 days on desktop 4

5 Student Transformation Lab Protocol Period Individuals in my lab group and their assigned tasks: NAME TASK COMPLETED (me) Checklist of materials at each lab station: 1 tube of CaCl 2 on ice 2 empty microtubes 1 waterproof pen 4 disposable transfer pipette (or you can use p20 s with yellow tips) sterile innoculating loops OR sterile tips 2 cotton swabs tape for sealing plates after innoculation 1 LB plate 2 LB/AMP plates cup with ice and water One tube of plasmid labeled either PM1 or PM2 on ice. Class or lab station waste containers with 10% bleach solution. float rack (optional) Checklist for Procedure: 1. Check to be sure you have all the needed materials at your lab station. 2. If your plates have not already been labeled, label one LB/amp plate +, one LB/amp (negative) and LB plate (negative). Be sure to only write near the edge of the plate. 3. Label two microtubes, one with a (+) and one with a ( -) along with your initials and class period. Using a plastic pipette, place 500µl of CaCl 2 (transformation solution) in each tube, close it and place the tube on ice for at least 2 minutes. 4. Using a sterile loop/tip, pick up at least five colonies (5 dots, or part of a streak ) of bacteria from the class starter plate by gently scraping it off from the top of the LB agar on which it is growing. Transfer the colonies to each cold tube of CaCl 2 by swizzling or twisting it around. Mix by inverting the tube to be sure the bacteria are dissolved. The solution should look cloudy with no chunks. If the solution isn t cloudy, you can add more colonies. Place the tubes back on ice. 10

6 5. Using the transfer pipette, add all of the plasmid solution (in small, clear microtube labeled either PM1 or PM2) into the positive (+) tube with CaCl 2 that you had placed on ice. BE SURE THAT THE PLASMID IS ONLY TRANSFERRED TO THE + TUBE. Tap gently to mix. Using a clean transfer pipette, add all of the buffer control to the negative (-) tube. 6. Incubate both tubes on ice for 10 minutes. Make sure the tubes are immersed in the ice water. Tap your tubes gently to mix once or twice during this incubation. 7. Heat shock: Take your ice water bath with its tubes to the 42 o C hot water bath. Transfer the tubes to the hot water and time for exactly 45 seconds. Make sure that the tubes are in contact with the hot water. Immediately return the tube to the ice. 8. Incubate on ice for 2 minutes. 9. Tap your tubes gently to mix. Open the negative (-) tube, and with a clean pipette, transfer 300µL or about half of the cell mixture to the negative (-) LB/amp petri dish you prepared previously. Add the rest of this tube to the LB plate. From the positive (+) tube, and with the same pipette, transfer 300µl or about half of the mixture to ONLY the LB/amp positive (+) plate. 10. Spread the cells on each plate with a clean, sterile loop or cotton swab. Let this sit for a few minutes to absorb the liquid. 11. Stack your plates and tape together. Place in the incubator or follow your teacher s directions. 11

7 Student Pre Lab Questions: Name Period 1. What is the purpose of using ampicillin on one of the plates? 2. What is the purpose of a control in a lab? What was (were) the control(s) in this lab? 3. What genes are present in the plasmid and what is the function of each protein product? 4. In your own words, outline the steps in this procedure and explain the purpose of each step. You may use sketches or cartoons to supplement your answer. (Do this on another piece of paper) 12

8 Results and Discussion Name Period 1. How many fluorescent colonies grew on your plates? What color(s) were they? 2. Use this chart to decide which unknown fluorescent protein your DNA sample codes for. Record your conclusion below the chart. BFP (Y66H) EGFP (S65T) YFP (T203Y) mtangerine mcherry mgrape1 excitation emission 3. Did you have (or see in the classroom) colonies that appeared different in visible v. UV light? Why do you think this might occur? 4. Suppose that a group did not get any transformed colonies. What possible errors could have caused this? 13

9 Student Questions 1. Identify and discuss an ethical issue that was raised in this lab. 2. What is the purpose of developing these fluorescent proteins in biological research? 3. What would you do with fluorescent proteins if it was up to you? 14

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