Introduction Plasmid Simulation Kit Catalog No. F1635 Publication No. 10447 enetic engineering is a 21st century reality and is wrought with many controversial issues. What is really possible? ow is this engineering accomplished? Making recombinant DN molecules is at the heart of the genetic engineering controversy. Concepts Restriction enzymes lunt ends vs. sticky ends ransformation Cloning ackground Deoxyribonucleic cid Less than fifty years ago the nature of the genetic code still eluded scientists. In the fifty years since the structure of DN was first unraveled, it has become the most significant biological topic of the century. Understanding the structure of DN helps to explain many life processes and leads to greater knowledge of why we are who we are. In addition, the uniqueness of every individual organism s DN can be used as a tool to discover relationships between organisms. simplified diagram of a short section of DN is shown in Figure 1. he diagrammed segment contains seven base pairs. real chromosome may contain a single DN molecule with as many as 10 8 (100 million) base pairs or even more! Since the base pairs represent the genetic code, the chromosomes can store a lot of messages! he two sugar-phosphate backbones in a double standard DN molecule have opposite entations. his results because the individual sugar-phosphate backbones have unequal ends. One end is called the 5 (pronounced 5-prime) end, while the other is called the 3 end. hese ends are chemically different. In a double-stranded molecule, one backbone is arranged 5 to 3 from top to bottom, and the other is arranged 3 to 5 from top to bottom. ases, recognize and bind to specific base sequences in a DN molecule and cut the DN at or near the recognition sequence in a consistent way. he restriction enzymes commonly used in laborates generally recognize specific Restriction Enzymes Restriction enzymes, also called restriction endonucle- C C DN sequences of 4 or 6 base pairs. hese recognition Figure 1. Short DN sequence. sites are palindromic in that the 5 -to-3 base sequence on each of the two strands is the same. Most of the enzymes make a cut in the backbone of DN at a specific position within the recognition site, resulting in a break in the DN. hese recognition cleavage sites are called restriction sites. C C IO-FX makes science teaching easier. IN10447 070114
Figure 2 shows some examples of restriction enzymes and their recognition sequences. he arrows indicate the cut sites and the names indicate the organism from which the enzymes were purified (for example, EcoRI from Escherichia coli). EcoRI: 5 C 3 indiii: 5 C 3 3 C 5 3 C 5 ami: 5 CC 3 lui: 5 C 3 3 CC 5 3 C 5 SmaI: 5 CCC 3 bai: 5 CC 3 3 CCC 5 3 CC 5 Figure 2. Restriction Enzyme Examples Notice that the top and bottom strands read the same from 5 to 3 ; this characteristic defines a DN palindrome. lso notice that some of the enzymes introduce two staggered cuts in the DN, while others cut each strand at the same place. Enzymes like SmaI that cut both strands at the same place are said to produce blunt ends. Enzymes like EcoRI that leave DN fragments with single-stranded protrusions are said to produce sticky ends. DN Ligase 5 C 3 3 C 5 If a DN molecule has been digested by different restriction enzymes into fragments, the ends of the DN fragments will clearly not all be the same. If, per chance, the ends of the fragments from different DN molecules do match perfectly, then the question is: Can they be reunited into a new recombinant DN molecule? Cells contain an enzyme that can expedite this reuniting process. he enzyme is called DN ligase. If two pieces of DN line up perfectly, DN ligase can help form the bonds between the sugars and phosphates to seal up the chemical backbone of the molecule and create a new DN molecule. Recombinant DN Some of the most important techniques used in biotechnology laborates today involves making recombinant DN molecules. Recombinant DN molecules are DN structures that have been reassembled from DN fragments taken from more than one ginal source. Restriction enzymes and DN ligase are important tools in executing the recombinant work. Look at the two molecules below (R and S) to visualize how a ami restriction enzyme might help to produce a new, recombinant R/S DN molecule. Note that the R s and S s represent base pairs in R and S DN molecules. RRRRCCRRRR ami RRRR CCRRRR + RRRRCCRRRR RRRRCC RRRR (R) (1) (2) SSSSCCSSSS ami SSSS CCSSSS + SSSSCCSSSS SSSSCC SSSS (S) (3) (4) RRRR CCSSSS DN + RRRRCCSSSS RRRRCC SSSS Ligase RRRRCCSSSS (1) (4) (R/S Recombinant DN) When biologists make recombinant DN molecules, they usually purify the starting DN molecules and work with them in test tubes. he starting DN molecules are first digested using restriction enzymes. he resulting pieces and DN ligase are then mixed together. he ligase forms bonds between fragments with complementary ends. 2 IN10447 2008 Flinn Scientific, Inc. ll Rights Reserved. Reproduction permission is granted only to science teachers who have purchased Plasmid Simulation Kit, Catalog No. F1635, from Flinn Scientific,
ransformation iologists try to introduce new DN into cells by trying to get the cells to absorb the DN that they have placed in their surrounding environment. If a cell absorbs the foreign DN and incorporates it into its genome, it is said to be transformed. Cell types vary considerably in their ability to absorb DN from their environment. Some bacterial cells are relatively adept at transformation and have become the favte vehicle for geneticists. It is possible to introduce plasmids (circular DN molecules with an gin of replication) into bacterial cells through the process of transformation. acteria that can be transformed (can take up new DN) are called competent. Some bacteria are naturally competent and others can be made competent by chemical and physical treatments. Once the bacteria absorbs the plasmid DN, they copy it along with their normal DN when they reproduce. he net result is that the geneticist has a continuing source of the new DN (by recovering it from the transformed cells). ecause many identical copies of the new DN are generated in this process it is often called cloning. If only this process were as easy as it sounds. Most cells will not maintain and copy just any DN molecule. he DN molecule must meet specific conditions of the host cell. In the case of E. coli, a very commonly used host, the DN must be a circular molecule like that in the bacteria and it must have an gin of replication. n gin of replication is a special sequence of bases where the copying (replication) begins. typical experiment used for proof of transformation involves adding a plasmid with a gene for antibiotic resistance to serve as a marker in a newly transformed bacteria. bacteria that cannot normally grow on a media containing an antibiotic is transformed into a bacteria that can grow on a media with the antibiotic. he actual growth on the media is considered to be proof that the bacteria has been transformed. Using this marker is very effective since the transformed bacteria are the only ones that can grow on the media containing the antibiotic bacteria that have not been transformed cannot grow! he other reason this technique is so significant is that transformation is usually very inefficient. In a typical experiment, less than one cell in 1,000 will be transformed, but once transformed, it can multiply and produce many more bacteria with the desired gene. Materials wist ties, 8 10 Pop beads, green, 10 Pop beads, blue, 58 Pop beads, orange, 12 Pop beads, pink, 8 Pop beads, red, 8 Pop beads, yellow, 8 Safety Precautions Plasmid Simulation Worksheet his simulation activity is considered nonhazardous. Follow all normal laboratory safety rules and procedures. Procedure Modeling Plasmids Your team will model one ampicillin-resistant plasmid (pmp) and one kanamycin-resistant plasmid (pkn). Each plasmid will have two identically-colored DN strands. e sure that one strand is put together 5 to 3 while the other is 3 to 5. he entation will be modeled with the hole (3 ) and the protrusion (5 ) end on the pop beads. efore the plasmids are formed into circles, the two strands should be lined up perfectly and tied together with twist ties as shown in Figure 3. Wrap the beads with twist ties about every three to four beads but do not wrap them between the two red or the two yellow beads. hese two colors are reserved for the restriction (separation) sites. 3 5 wist ies Figure 3. Plasmid Model Double Stranded Circular Molecules 5 3 3 IN10447 2008 Flinn Scientific, Inc. ll Rights Reserved. Reproduction permission is granted only to science teachers who have purchased Plasmid Simulation Kit, Catalog No. F1635, from Flinn Scientific,
Examine the sequences shown in Figure 4 for the order of the pop beads in the plasmid models. Each pop bead represents approximately 200 bases of DN, except for the restriction sites. Each restriction site is represented by a pair of uniquely-colored beads (yellow for indiii and red for ami). Once the parent plasmids are constructed (completed double-stranded circular plasmid) go to the Digesting Plasmid section. Orange beads designate the gin of of each plasmid. reen beads represent the KN-resistant gene and pink beads MP-resistant gene. R R O pkn O O Y Y P P P P R R pmp Y Y O O O Digesting Plasmids Figure 4. Model Plasmid Sequence 1. Digest each plasmid with indiii. Simulate this by popping apart the two yellow bead pairs representing the indiii recognition sites. Each plasmid should give a linear chain of beads with yellow beads on either end. 2. Next digest each plasmid with ami by separating the chain in between the two red beads in each chain. his should result in a total of four linear DN fragments. Each fragment should have a pair of red beads at one end and a pair of yellow beads on the other end. here should be a 5 (protrusion) and a 3 (hole) end at each end of the fragments. 3. Now pool the DN fragments with one or more other work teams as directed by your instructor. Place the fragments on a table top (this will simulate a test tube full of digested fragments). Recombination Patterns 1. Now simulate the activity of DN ligase. Remember DN ligase will only reformulate complementary base pairs like those ginally split by indiii and ami. Further, it is important to note that once two indiii ends have been rejoined that the new DN molecule will have two free ami ends. It is more likely that these free ami ends on the same molecule will join each other to form a new circular plasmid than it is for an additional fragment to join in a linear fashion. In other words, it is unlikely that any product DN will be formed from more than the four starting pieces. In fact, most new DN molecules will result from random selections of two fragments. 2. Focus on all the different circular products that can be made by joining any two of the pooled fragments together. int: here are 10 products possible. Use the information on top of the Plasmid Simulation Worksheet and draw all ten possible products and fill in the answers required on the worksheet. 4 IN10447 2008 Flinn Scientific, Inc. ll Rights Reserved. Reproduction permission is granted only to science teachers who have purchased Plasmid Simulation Kit, Catalog No. F1635, from Flinn Scientific,
Name: Plasmid Simulation Worksheet Circular plasmids can be diagrammed in a shorthand way. he parent plasmids can be drawn as follows: Where: = indiii site = ami site = gin of replication = ampicillin-resistant gene pkn pmp = kanamycin-resistant gene Use the same abbreviations shown above to diagram the recombinant products possible from the pooled fragments. Use this information to answer the questions for each new plasmid. pproximate Can it reproduce? Resistant to Plasmid Diagram Size (bp) (have an gin) Kanamycin? mpicillin? 5 IN10447 2008 Flinn Scientific, Inc. ll Rights Reserved. Reproduction permission is granted only to science teachers who have purchased Plasmid Simulation Kit, Catalog No. F1635, from Flinn Scientific,
Plasmid Simulation Worksheet, con t. Name: pproximate Can it reproduce? Resistant to Plasmid Diagram Size (bp) (have an gin) Kanamycin? mpicillin? 6 IN10447 2008 Flinn Scientific, Inc. ll Rights Reserved. Reproduction permission is granted only to science teachers who have purchased Plasmid Simulation Kit, Catalog No. F1635, from Flinn Scientific,
Materials Included in Kit eacher s Notes Plasmid Simulation Kit wist ties, 320 Pop beads, green, 150 Pop beads, blue, 870 Pop beads, orange, 180 Pop beads, pink, 120 Pop beads, red, 120 Pop beads, yellow, 120 dditional Materials Needed (for each lab group) Plasmid Simulation Worksheet (copy) Pencil Pe-Lab Preparation Make copies of the Plasmid Simulation worksheet for every student. Safety Precautions his simulation activity is considered nonhazardous. Follow all normal laboratory safety rules and procedures. Disposal ll items in this simulation can be reused many times. Connecting to the National Standards ips his laboratory activity relates to the following National Science Education Standards (1996): Unifying Concepts and Processes: rades K 12 Systems, order, and organization Evidence, models, and explanation Content Standards: rades 9 12 Content Standard C: Life Science, molecular basis of heredity, matter, energy, and organization in living systems Enough materials are provided in this kit for 30 students working in pairs, or for 15 groups of students. ll materials are reusable. fter the initial parent plasmids have been constructed, student teams should be reformed to include at least two groups utilizing all of the parent plasmids. Only if the pool of fragments is large enough can all 10 plasmids be constructed at once. his laboratory activity can reasonably be completed in one 50-minute class period. he sizes of the plasmid models (in base pairs) are approximate and only useful for this simulation. When estimating the size of the plasmids, each pop bead represents 200 base pairs excluding the restriction site beads. 7 2008 Flinn Scientific, Inc. ll Rights Reserved. IN10447
eacher s Notes continued nswers to Questions pproximate Can it reproduce? Resistant to Plasmid Diagram Size (bp) Does it have an gin? Kanamycin? mpicillin? 6400 two gin of s contain gene gene 8000 two gin of s contain gene gene 4600 gin of contain gene gene 5800 gin of gene gene 2400 contain gin of lthough it contains kanr gene, it contains no gin and will not reproduce No Contains no gene nor gin of 1200 contain gin of contain gene contain gene 8 2008 Flinn Scientific, Inc. ll Rights Reserved. IN10447
eacher s Notes continued nswers to Questions, con t. pproximate Can it reproduce? Resistant to Plasmid Diagram Size (bp) Does it have an gin? Kanamycin? mpicillin? 4800 gin of contain gene contain gene 3600 contain gin of lthough it contains kanr gene, it contains no gin and will not reproduce No Contains no gene nor gin of 3000 gin of contain gene contain gene 4200 gin of gene contain gene he Plasmid Simulation Super Value Kit is available from Flinn Scientific, Inc. Catalog No. Description F1635 Plasmid Simulation Super Value Kit Consult your Flinn Scientific Catalog/Reference Manual for current prices. 9 2008 Flinn Scientific, Inc. ll Rights Reserved. IN10447