Recombinant "Paper" Plasmid Background: Many bacteria contain plasmids, small independent DN fragments that carry specific pieces of genetic information, such as resistance to specific antibiotics or other genetic characteristics. Plasmids can be transmitted from one bacterium to another, or from the environment into a host bacterium, a process called transformation. Plasmids can also incorporate into their DN sequence pieces of DN from different organisms. Plasmids that incorporate new DN are called recombinant plasmida Recombinant plasmids are used In biotechnology to carry DN that codes for substances, such as human insulin or growlh hormone, into bacteria. Bacteria that contain the recombinant plasmids can then be grown commercially to provide the needed substance. Special enzymes, called restriction enzymes, can cut DN fragments from almost any organism. ypically, restriction enzymes are used to cut DN molecules into individual genes. here are many different restriction enzymes, each of which recognizes one specific nucleotide sequence. Many restriction enzymes work by finding palindrome sections of DN (regions where the order of nucleotides at one end is the reverse of the sequence at the opposite end). his way a restriction enzyme can cut tiny sticky ends of DN that will match and attach to sticky ends of any other DN that has been cut with the same enzyme. DN ligase joins the matching sticky ends of the DN pieces from different sources that have been cut by the same restriction enzyme. Once a desired DN fragment has been isolated and cut with a specific restriction enzyme, the sticky ends of both the desired DN fragment, and from a plasmid that has been cut by ttie same restriction enzyme, can be joined together, forming a recombinant DN plasmid. Special plasmids, which have antibiotic resistance markers, are used in this process so that a researcher will be able to tell that the desired DN has been incorporated into the target plasmid and subsequently into the host bacterium. In this exercise, you will simulate the process of forming a recombinant plasmid using paper models. he gene of interest has been identified on the cell DN template, and the isolated plasmid template has a number of antibiotic resistance genes. You must find an appropriate restriction enzyme that can cut the gene of interest out of tlie cell DN and splice it into the piasmid using the matching sticlcy ends of DN cut by the selected enzyme. You will also discuss how you might use antibiotics to detemiine if a host bacterium has successfully incorporated the recombinant plasmid. dapted for Morton Biology Seoieniber 2007 Mr J.P. Fuller
2 Materials Required for Each roup Procedure plasmid sheet (blue paper) restriction enzyme sheet (bright pink paper) cell DN sheet (green paper) tape scissors 1. onstruct a Piasmid: he blue sheet is made up of strips representing segments of the plasmid DN. ut the plasmid strips out along the dotted lines and tape the strips together SiantHBB i-^ o(sh^. ^MfcaidiBpBifeiiijii LII'II ull iibbgiwwaj.liiiyiiiiiii;in. When the strips are connected, take the two free ends and tape them together forming a circle with the nucleotides facing out. (Be sure that the ends do not overlap covering any of the nucleotides.) he plasmid that has been chosen also has genes that code for antibiotic resistance. Bacteria that Incorporate such antibiotic resistant plasmids become resistant to those antibiotics. hese resistance "markers" are useful in identifying bacteria that successfully incorporate desired recombinant plasmids. Note the location of the antibiotic resistance sites (variousiy shaded) on the plasmid, as well as the location of the plasmid replication site. he key for these sites is at the bottom of the plasmid paper sheet. 2. ssemble the DN: ut out the strips of the cell DN from the green sheet. he strips are numbered 1-6. ape the strips together in numeric order forming one long nucleotide sequence stnp. his DN strip contains the gene, which is shaded, that will be transfen-ed to the plasmid. 3. Restriction Enzyme "ards": ut out the bright pink enzyme sheet along the thick dotted lines to form the "cards" that simulate the restriction enzymes. Each card has a segment of nucleotide base pairs that represents the code recognized by that specific restriction enzyme. 4. Locate the Restriction Sites on the Plasmid: ompare the sequences of base pairs on each of the enzyme cards with the nucleotide (base pair) sequences on the circular plasmid. Mark the places on the plasmid in pen or pencil that are identical vsoth the code of the restriction enzymes. hese are locations \*iere the enzyme can cut the plasmid. Note the cut pattern illustrated by the dotted lines on the enzyme. Refer to the Sac 1 restriction enzyme example below as a guide. Example restriction site match; Rasmid ~ f.. Enzyme Sac dapted for Morton Biology Seotember 2007,... Mr. J.P. Fuller'
Note: Not all of the restriction enzymes may have matches with the piasmid. You may set aside any enzyme cards that do not match; they cannot be used in this exercise. 5. Locate the Restriction Sites on the DN: Using only the enzymes that had matches on the plasmid, locate and mark restriction sites for each of the enzymes on the cell DN. he enzyme must have a match in two places on the cell DN: one above the gene and the second below the gene to be useful. Discard any enzyme that cannot cut the cell DN both above and below the gene. Select one enzyme that can cut the plasmid in one place and the cew DN in two places. Ideally, you would choose the enzyme that can cut closest to the gene on both sides, so that less "extraneous" cell DN will be transferred to the plasmid. he same enzyme must be used to make the cut in the plasmid and the two cuts on the DN molecule that removes the gene to be inserted into the plasmid. 6. Make your recombinant piasmid: ut along the fine dotted line of the enzyme card you have selected, so that the enzyme can slip into the cell DN strip and into the plasmid strip. Fit the restriction enzyme into the plasmid and cut the plasmid along the restriction site marked by the dotted lines on the enzyme. his cut forms the "sticky ends" characteristic of restriction enzymes, and facilitates the splicing of the DN into the plasmid. Repeat this process with the cell DN at the restriction site above the gene, and at the restriction site below the gene. Once you have made your cuts, insert the gene into the plasmid by matching the "sticky ends" of the gene with the "sticky ends" of the plasmid and fastening the ends together with tape. Your recombinant plasmid should be circular with a portion of the cell DN included. 7. Locate the antibiotic resistant sites on the recombinant plasmid, along mth the replication site. If you spliced the DN gene into the middle of the plasmid replication site, the plasmid will not be able to replicate, and cannot be of use. In a similar fashion, any antibiotic resistant sites that were destroyed in forming the recombinant piasmid will not be of use. Drawing of a Plasmid Mao: Draw a ring symbolizing your plasmid that has been recombined with another gene. Show where the origin of replication is located. o do this, write the letter "O" (origin) in the 12 o'clock position of the ring you just drew to represent the ongin of replication. Now, using your circular paper plasmid, vrark clockwise from "O" and write on your drawn nng the following information as it appears in your paper plasmid: (a) Show vi^ere the inserted gene is located on your loop. (b) Show which antibiotic resistant genes (R - genes) you preserved and their sequential order on your ring. (c) Name and show where your restriction enzymes (endonucleases) cut. QuestJons (nswer on e separate piece of paper in compie^^entences). 1. Howtr^^y enzymes did you end up using^wiich ones? 2. Which antibiotics would you use tojestrar the successful insertion of your piasmid? 3. What is the diffei:^ce betweerj^a^cteriai chromosome and a plasmid? 4. What Is a gene irtrhotecylarlerms? 5. What benefits do piasml^offer to bacteria? (ive a specific example.) 6. In what way dp'tfiese endohudeases cut the DN? What is the significance of this way? 7. What rol^s^uld restriction eni:yi^s play in nature? 8. Wh^t'fJoiypeptide sequence does tfte^ne inserted code for? (Review protein synthesis artowite the full amino acid sequenceror^lje inserted gene.) dapted for Morton Biology September 2007 Mr. J.P. Fuller
9. In Whatj,i;ays are bacterial transfomriation usefultojjumatfis? Provide as many specific examplesa5~yoiican find. ^^^-^-'^'""^ 10. ssume you haveaadte4^four recombinant plasmid culture to a culture of bacteria in a biotechnology lab. You neea^ertcnowjivlich of the bacteria incorporated the plasmid if they are to be of uset9.yalfmanufacturii ~the.^es^ gene product. How would you use antibiotics to IdentifSn^ich bacteria have incorporafsthhe recombinant plasmid so they can be copmercially grown to produce the substance being code^jocby the gene? Whiph-^ibiotics could you use to test this? What would you do with the bacteria that you -t^nfimied contained the recombinant plasmid? o pass in: your recombinant plasmid the restriction enzyme that was used to cut the plasmid and cell DN plasmid map answers to questions Sources: Onginal activity appeared as "Recombinant Paper Plasmids," by. Jenl<ins, in he eacher, pr. 1987, pp. 44-48. Science Rewrite of the paper plasmid model assignment were provided by the Winter2000 Biology 101 D and E students at Beilevue ommunity ollege, and refined by students in subsequent terms. Questions provided by Erol D. ltug, he Stony Brook School, Stony Brook, New York during an P Summer Institute at St. lement's School, oronto, anada, July 2007. dapted for Morton Biology September 2007 Mr. J.P. Fuller _,
Lab: Recombinant Paper Plasmid Data/ nalysis Record your test results for each enzyme in table 1.2 Name: Date: HR: abic 1.2 RESRIION ENZYMES Restriction Enzyme Used Not used Reason why or why not chosen vail Hin dm Bam HI Bgl II Hpa II Eco RI Sad Xmal LISSE PLSMID MP: Draw a ring similar to p.l. Mark the top of your plasmid "O" for origin. hen in a clockwise fashion, include the following: where the inserted gene is located, where the restriction enzymes cut and which antibiotic resistant genes you preserved.
Questions 1. What is a plasmid? 2. Define transformation. 3. What is a recombinant DN or plasmid? 4. Explain the function of restriction enzymes used during the process of recombining DN. 5. How are antibiotics used to test whether or not a plasmid has successfully been recombined? 6. In your example, which antibiotics could be used to test for successful recombination? 7. What is the role of DN ligase? 8. Discovery. Use the internet to find 3 ways recombinant DN has been used in modern medicine to benefit mankind.
5 Use BLUE paper for this page.! I III = ampidlljn rtsjsunce P5 = ksnainynn resistance dapted for Morton Biologv Seotember 2007 Mr. J.F. Fulle-^'
Use REEN paper for this page F ' c ell DN n '!, { J i - ' tss protein I me. m r ^a ^ ] ' ' /V 1 I I I 1! ^ 3 3 ^ tr~t: i i i i I c 1 dapted for Morton Biology Seotemoer 2007 Mr J.P. Fuller..
Use PINK paper for this page. Enzymes dapted for Morton Biology Seotember 2007 Mr J.P. Fuller '