HOW-TO-'DO-IT A Visual Demonstration of Plasmid DNA Preparation from Bacteria * RALPH L. KEIL, LAURA K. PALMER Downloaded from http://online.ucpress.edu/abt/article-pdf/71/6/363/55200/20565332.pdf by guest on 02 October 2021 Unprecedented advances in unraveling mysteries of life have disease, or other areas the students or instructor want to develop. occurred as a result of the molecular biology revolution. Some Since many students know someone with diabetes (in many cases major achievements of this revolution include new methods for the they know a classmate who has diabetes or one of them may even production of therapeutic compounds, insights into mechanisms suffer from the disease), this example provides an opportunity to of gene function and regulation, and the complete sequencing of get students to actively participate in discussion, regardless of their the genomes of numerous organisms including humans. A critical academic abilities. foundation of this revolution is the ability to use the bacterium An alternative introduction is to discuss consequences of Escherichia coli (E. coli) as a factory to produce large amounts of sequencing the entire three billion base pairs of DNA of the virtually any DNA of interest by inserting it into a small, extrachro human genome (http://www.ornl.gov/sci/techresources/Human mosomal circle of DNA called a plasmid. The ease and reproducibil Genome/home.shtml). The current and potential uses of this ity of plasmid DNA isolation from bacteria permits numerous labs technology in forensic science, health care, and business decisions to conduct molecular biology experiments and greatly accelerates are all areas that may be discussed depending on what focus the progress in understanding complex biological processes. instructor wants to develop. Many students who are not normally High school biology and introductory undergraduate biology interested in biological sciences become interested through dis or molecular biology classes frequently include labs in which stu cussions regarding possible uses of personal genetic information. dents either isolate or manipulate plasmid DNA. Such labs often Students are often surprised to learn that their ability to obtain consist of a "cookbook" lab protocol that contains little, if any, health or life insurance or their employment may be affected by information as to the purpose of the various steps involved in isolat the DNA sequences they have inherited (http://www.ornl.gov/sci/ ing the plasmid DNA. A major problem with this approach is that it techresources/Human_Genome/elsi/elsi.shtml). leaves the procedure as amysterious "black box" formost students. An additional means of generating interest is to have a culture To assist in illustrating the roles of the different steps of the proto tube with an overnight growth of E. coli.Asking the students if they col, amodel bacterial cell can be constructed with readily available have heard of E. coli normally elicits recounts of news stories of materials and used in a highly interactive presentation that visually food contamination. Discussion of differences between strains of E. demonstrates the purpose of each step. This demonstration can be coli that are pathogenic and non-pathogenic can lead to the revela used as a stand-alone lesson when introducing recombinant DNA tion that E. coli are normal inhabitants of our intestines that, along or it can be used as part of an introduction to awet-lab experiment with other intestinal bacteria, are necessary for us to live as they (a simple, rapid, low-cost, and highly reliable protocol is presented help digest our food as well as provide nutrients such as vitamin K. in the Appendix). The demonstration and the discussion related to (Some brave students may be persuaded to smell the tube of bacte it provide numerous opportunities to stress concepts included in ria,which normally produces interesting facial contortions.) the National Science Education Standards. O An E. coli Model To Illustrate Plasmid O Developing Student Interest Purification Why should I care? seems to be a common question in the minds of many students in introductory courses. Depending on the time Materials available, the level of the students or their interests, a number of * about twenty 3/8" flat metal washers from a hardware approaches can be used to help capture students' attention and store engage them in class discussion. In high school freshman biology classes, we found that a means of introducing recombinant DNA * about 2 m of twine (or string) with a double-helix structure technology and the role it can play in students' lives is to use an with the ends tied together to form a "circle" example of cloning a specific gene. Because of the prevalence of * quart-size zip-lock baggie diabetes, a useful example is the cloning of the human insulin gene * 6.5 by 9.5" manila envelope and the production of human insulin for therapeutic use in bacte ria (Ladisch & Kohlmann, 1992). This example permits student * enough marbles to fill the baggie involvement by questioning them about what they know about * 0.7 by 1m piece of carpet diabetes, the role of insulin in the disease, the health effects of this THE AMERICAN BIOLOGY TEACHER PLASMID DNA PREPARATION i Construction of the Model E. cofi Cell students can be prompted to identify the twine as representing the bacterial chromosome (the double helical structure should be The washers (representing plasmid DNA molecules), twine very helpful) and the marbles as representing components of the (representing the bacterial chromosome of E. coli), and themarbles cytoplasm, such as enzymes or ribosomes. Use the scissors to cut (representing proteins, ribosomes, and other components of the the twine into several pieces and explain that due to the extensive bacterial cytoplasm) are placed in the baggie (representing the length of the bacterial chromosome, it sheers when the plasma cell's membrane), which is then sealed. The filled baggie is then membrane is lysed. Once the twine is cut into pieces, the two inserted into the manila envelope (representing the bacterial cell strands can be pulled apart to demonstrate how DNA is denatured wall), which is then sealed. A single washer is retained to demon (separated into single strands) by the alkaline solution (due to strate what the students want to isolate and how difficult it is to the NaOH in Solution II). If appropriate, this provides a chance to locate the approximately 20 washers among the twine and marbles remind students about acids, bases, and pH as well as a chance to at the various stages of the protocol. discuss how double-stranded DNA can be denatured by alkali or Students can be provided with a copy of a protocol for plasmid heat. (One concept that can be difficult to convey is that the plas DNA isolation that has explanations for each step. The protocol mid DNA is also denatured by this treatment but since it is fairly in the Appendix is an example of an alkaline lysis protocol that small and the two single stands are intertwined, they are still held has been very successful in high school freshman biology classes together and will renature [reanneal] when the solution is neutral ranging from "applied" to "honors." The demonstration can be ized in the next step.) Point out that it is still not easy to find and conducted as a "dry lab" demonstration or as a prelude to a "wet isolate the washers from the marbles and twine pieces. Downloaded from http://online.ucpress.edu/abt/article-pdf/71/6/363/55200/20565332.pdf by guest on 02 October 2021 lab" in which the students isolate plasmid DNA and conduct gel Next, "glue" is added (the high salt and potassium in Solution electrophoresis to assess their DNA yield. The model permits dis III; Step 7 of the Appendix) that binds to the proteins (marbles) cussion regarding what the various objects in the model represent. and denatured chromosomal DNA and the detergent from Solution This discussion can be expanded to talk about the composition of II. Spinning this mixture in a tube at high speed forces all the com any of these components, depending on the learning objectives ponents that have been "glued" together to the bottom of the tube appropriate for the class. (The discussion also permits correction (Step 9; Appendix). This can be simulated by gently pushing the of students' misconceptions, e.g., in our experiences students have suggested the marbles represent mitochondria, although organelles do not exist in bacteria). Figure 1. Visual demonstration of plasmid isolation from "Dry Lab" Plasmid Purification E. coli. A) intact cell B) removal of cell wall C) plasma membrane lysis Demonstration and release of cellular contents D) removal of cellular debris E) plas A convenient way to begin the demonstration is mid concentration. See text for further details of each step. by holding the model E. coli cell (Figure 1A) in one hand and a washer ("plasmid") in the other hand and ask the class if it could easily isolate (or even see) the 20 plasmids in the bacterial cell. Ideas as towhat component of the bacterial cell is represented by the manila envelope can be solicited from the students. Their identification of it as the cell wall provides an opportunity to discuss the fact that cells of some organisms like bacteria and plants have cell walls while our cells do not. Further discussion on why that is the case is certainly recommended. Scissors can be used to cut through the manila envelope (being care ful not to cut the underlying baggie; Figure 1B) while explaining that it is first necessary to get through the cell wall (accomplished by Solutions I and II in Steps 5 and 6 of the protocol in the Appendix).