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Download Recombinant DNA Using Bacterial Plasmids NAME: Background
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Recombinant DNA Using Bacterial Plasmids NAME:__________________________ Background Bacteria have not only their normal DNA, they have a circular DNA also. It is a wonderful ally for biologists who desire to get bacteria to produce very specific proteins. The plasmids conveniently can be cut, fused with other DNA and reabsorbed by bacteria. The bacteria easily incorporates the new DNA information into its metabolism. This “recombining” of DNA is called RECOMBINANT DNA. There are a number of RESTRICTION ENZYMES that are available to cut the plasmid DNA and the new DNA strands that need to be “pruned” closer to the specific DNA message that will be fused with the plasmid. Goals In this activity, a make-believe DNA message for the protein insulin is marked on the cell DNA. Your task will be to find an enzyme that cuts the plasmid once (and only once) and cuts the cell DNA as a close possible on both ends of the insulin code - so that the insulin code can be fused into the circle of the plasmid DNA. To do this you will need to determine which restriction enzyme to use to cut your DNA segments and which antibiotic you would use to determine if your finalized recombined DNA was absorbed by the DNA or not. *note: antibiotics are NOT the same as enzymes or insulin, they are used to kill bacteria. Materials Instructions White bacterial plasmid Non-white Cell DNA Scissors Tape Instructions 1. You will be working with a partner and will turn in ONE lab report. Make sure to READ AND FOLLOW DIRECTIONS CLOSELY. 2. Obtain scissors and a piece of tape (your teacher will come around and give you one piece that you can further cut into smaller pieces). 3. Cut the PLASMID (White) strips. Tape the strips in ANY order to make a circular plasmid. 4. As one partner works on step 4, the other partner should cut out the CELL DNA (Non-White) strips. They must be taped together in NUMERICAL ORDER. That is, strip 2 is taped to the bottom of strip 1, and 3 is taped to the bottom of strip 2, etc. Note where the DNA code for insulin (protein gene) is located. Part I Background: Your job as a biochemist would be to find a restriction enzyme that will cut open your plasmid at ONE site only (this may or may not be possible depending upon how you constructed your plasmid). The same enzyme should be able to cut your cell DNA at TWO sites, one above and one below the gene for insulin. It is very important that you find an enzyme that cuts as close to the insulin gene as possible. The tails, which are genes other unnecessary proteins need to be cut off leaving “sticky ends” that look like this 5. For this activity I will tell you that the enzyme you are to use is HindIII TA TA CG GC AT AT HindIII 6. Once you have found where the enzyme will cut the plasmid (once) trace the cut the enzyme would make and make the cut. 7. Now find the two areas that the restriction enzyme will cut the DNA. One above the desired gene and one below the desired gene. Mark directly on the Cell DNA strip where the bases will be cut apart (and leave “sticky ends”). Write the name of the enzyme next to each marking and make each cut along the line you traced. 8. After you have completed marking your plasmid and cell DNA complete each of the cuts in the staggered fashion made by the actual enzyme. This will expose the “sticky ends” where joining will be possible. (Since one enzyme was used, all “sticky ends” will be compatible.) Use tape to connect your insulin gene into the plasmid chain. You have now created a RECOMBINANT DNA!!!! Discussion Questions: 1. In this activity, you incorporated an insulin gene into the plasmid. How will the recombinant DNA be used to produce insulin? 2. Label the image to the right. Use the terms: Bacteria, Plasmid, Bacterial DNA, Human Cell, Human DNA, Desired Gene, Restriction Site, Recombinant DNA, Cloned Bacteria. 3. Describe the process that is occurring in the image to the right. Cell DNA TA GC GC GC CG CG TA AT GC GC CG AT CG AT GC GC GC CG CG CG GC GC AT GC AT TA TA CG TA TA AT AT GC TA CG AT AT GC CG AT GC GC TA TA CG GC AT AT GC GC TA AT CG AT TA AT AT CG GC TA CG TA CG TA TA CG GC TA CG AT TA GC TA GC CG CG TA TA TA TA AT AT AT TA GC TA AT AT TA AT TA TA CG CG TA CG CG TA TA AT AT GC AT AT TA TA TA CG GC AT AT CG GC GC GC GC CG CG CG TA AT GC GC AT CG CG 1 2 3 4 5 6 = protein gene Bacterial Plasmid GC CG CG CG AT GC AT GC TA TA TA CG TA TA AT AT GC GC TA CG TA AT GC AT AT AT AT TA GC TA GC TA GC TA CG CG AT GC TA AT GC GC TA AT GC GC CG CG CG CG CG TA TA TA TA TA AT GC GC GC AT CG TA = AMPICILLIN RESISTANCE = TETRACYCLIN RESISTANCE CG GC AT GC TA TA AT AT CG CG TA AT GC GC AT GC GC GC CG CG CG TA GC GC TA GC GC GC GC GC CG AT AT GC GC TA TA AT TA AT CG TA TA AT AT GC CG CG GC TA AT GC GC TA TA CG GC AT AT CG TA CG CG = KANAMYCIN RESISTANCE = PLASMID REPILCATION
