Recombinant "Paper" Plasmid

Background:

Many bacteria contain plasmids, small independent DNA 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 DNA sequence pieces of DNA from different organisms. Plasmids that incorporate new DNA are called recombinant plasmida Recombinant plasmids are used In biotechnology to carry DNA 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 DNA fragments from almost any organism. Typically, restriction enzymes are used to cut DNA molecules into individual genes. There are many different restriction enzymes, each of which recognizes one specific nucleotide sequence. Many restriction enzymes work by finding palindrome sections of DNA (regions where the order of nucleotides at one end is the reverse of the sequence at the opposite end). This way a restriction enzyme can cut tiny sticky ends of DNA that will match and attach to sticky ends of any other DNA that has been cut with the same enzyme. DNA ligase joins the matching sticky ends of the DNA pieces from different sources that have been cut by the same restriction enzyme.

Once a desired DNA fragment has been isolated and cut with a specific restriction enzyme, the sticky ends of both the desired DNA fragment, and from a plasmid that has been cut by ttie same restriction enzyme, can be joined together, forming a recombinant DNA 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 DNA 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. The gene of interest has been identified on the cell DNA 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 DNA and splice it into the piasmid using the matching sticlcy ends of DNA 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.

Adapted for Morton Biology Seoieniber 2007 Mr J.P. Fuller

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Materials Required for Each Group

plasmid sheet (blue paper) restriction enzyme sheet (bright pink paper) cell DNA sheet (green paper) tape scissors

Procedure

1. Construct a Piasmid: The blue sheet is made up of strips representing segments of the plasmid DNA. Cut the plasmid strips out along the dotted lines and tape the strips together S i a n G t H B B 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.) The 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. These 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. The key for these sites is at the bottom of the plasmid paper sheet.

2. Assemble the DNA: Cut out the strips of the cell DNA from the green sheet. The strips are numbered 1 - 6 . Tape the strips together in numeric order forming one long nucleotide sequence stnp. This DNA strip contains the gene, which is shaded, that will be transfen-ed to the plasmid.

3. Restriction Enzyme "Cards": Cut 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: Compare 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. These are locations \A*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 G T C T C G A G T

Enzyme C A G

G

T C •A T

C T~ f

G ..A G

A G C

C

Sac

Adapted 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 DNA: Using only the enzymes that had matches on the plasmid, locate and mark restriction sites for each of the enzymes on the cell DNA. The enzyme must have a match in two places on the cell DNA: one above the gene and the second below the gene to be useful. Discard any enzyme that cannot cut the cell DNA both above and below the gene. Select one enzyme that can cut the plasmid in one place and the ceW DNA in two places. Ideally, you would choose the enzyme that can cut closest to the gene on both sides, so that less "extraneous" cell DNA will be transferred to the plasmid. The same enzyme must be used to make the cut in the plasmid and the two cuts on the DNA molecule that removes the gene to be inserted into the plasmid.

6. Make your recombinant piasmid: Cut along the fine dotted line of the enzyme card you have selected, so that the enzyme can slip into the cell DNA 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. This cut forms the "sticky ends" characteristic of restriction enzymes, and facilitates the splicing of the DNA into the plasmid. Repeat this process with the cell DNA 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 DNA included.

7. Locate the antibiotic resistant sites on the recombinant plasmid, along mth the replication site. If you spliced the DNA 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. To 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 (Answer on e separate piece of paper in compie^^entences). 1. Howt r ^^y enzymes did you end up us ing^w i i ch ones? 2. Which antibiotics would you use toJestTrar the successful insertion of your piasmid? 3. What is the di f fei :^ce betweerj^a^cteriai chromosome and a plasmid? 4. What Is a gene irtrhotecylarlerms? 5. What benefits do p iasml^o f fe r to bacteria? (Give a specific example.) 6. In what way dp'tfiese endohudeases cut the DNA? What is the significance of this way? 7. What r o l ^ s ^ u l d restriction eni:yiT^s play in nature? 8. Wh^t'fJoiypeptide sequence does t f t e ^ n e inserted code for? (Review protein synthesis

artowTite the full amino acid sequenceror^lje inserted gene.)

Adapted 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. Assume you haveaadte4^four recombinant plasmid culture to a culture of bacteria in a biotechnology lab. You neea^ErtcnowjivlTich of the bacteria incorporated the plasmid if they are to be of uset9.yalfmanufacturiiT§~the.^es^ gene product. How would you use antibiotics to IdentifSnA^ich bacteria have incorporafStHhe recombinant plasmid so they can be copmercially grown to produce the substance being code^Jocby the gene? Whiph-^ ib iot ics could you use to test this? What would you do with the bacteria that you -t^nfimied contained the recombinant plasmid?

To pass in: your recombinant plasmid the restriction enzyme that was used to cut the plasmid and cell DNA

• plasmid map answers to questions

Sources:

Onginal activity appeared as "Recombinant Paper Plasmids," by C. Jenl<ins, in The Science Teacher, Apr. 1987, pp. 44-48.

Rewrite of the paper plasmid model assignment were provided by the Winter2000 Biology 101 D and E students at Beilevue Community College, and refined by students in subsequent terms.

Questions provided by Erol D. Altug, The Stony Brook School, Stony Brook, New York during an AP Summer Institute at St. Clement's School, Toronto, Canada, July 2007.

Adapted for Morton Biology September 2007 Mr. J.P. Fuller _ ,

Lab: Recombinant Paper Plasmid Name: Date: HR:

Data/ Analysis Record your test results for each enzyme in table 1.2

Tabic 1.2 RESTRICTION ENZYMES

Restriction Enzyme

Used Not used

Reason why or why not chosen

A v a i l

Hin dm Bam H I Bgl I I Hpa I I Eco RI S a d Xma l

LISASE

PLASMID MAP: Draw a ring similar to p.l. Mark the top of your plasmid "O" for origin. Then 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 DNA or plasmid?

4. Explain the function of restriction enzymes used during the process of recombining DNA.

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 DNA ligase?

8. Discovery. Use the internet to find 3 ways recombinant DNA has been used in modern medicine to benefit mankind.

5

Use BLUE paper for this page.

A T ! T A C G I C G

III = ampidlljn rtsjsunce P5 = ksnainynn resistance

Adapted for Morton Biologv Seotember 2007 Mr. J.F. Fulle-^'

Use GREEN paper for this page

Cell DNA •n ^

F ' G

G G C C T A G G C A C A G G G C C c G

A G C ' A T T A C ! G C , C G

C T A

C{ A G T GJ T Ai C T | T C C G-T ' G T

T A A G T

.C

T A A G A A T T C A G m

G C A T A T G C G C

^ a r A •]•' A'

/V 1 I I A I T A 1

T A !

^ 3 3 ^

t r~ t :

C G C G T A C G C G T A T A A T A T G C A T A T T A

A C G G G G

i G C i T Ai T Ai C G I G C A T

T G C c c c

C G C G C G T A A T G C G C A T C G C G 1

tss • protein I me

Adapted for Morton Biology Seotemoer 2007 Mr J.P. Fuller . .

Use PINK paper for this page.

Enzymes

Adapted for Morton Biology Seotember 2007 Mr J.P. Fuller '