Safety Note
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In cases where special safety instructions are not given, use common safety precautions, especially when working with hot, sharp, or breakable items. Be sure to read and follow warning labels on household chemicals.
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Lesson List
Characterization of the Bioluminescence (Lux +) Gene in the Microorganism E. coli
Determining Cutting Site Locations
DNA Fingerprinting: The Great Cafeteria Caper
DNA on a Stick
Innocent or Guilty: A Lab on DNA Gel Electrophoresis
Producing a Strain of E. coli that Glows in the Dark
Restriction Analysis of Lambda DNA
Transformation of Competent Cells with a Recombinant Plasmid
Using a Controlled Experiment to Identify Two Unknown Plasmids
Lesson Descriptions |
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| Characterization of the Bioluminescence (Lux +) Gene in the Microorganism E. coli |
Students grow E. coli strain HB101, which contains the plasmid pUCD607 with the bioluminescence (Lux +) gene. The plasmid containing the Lux + gene is isolated from the E. coli, then characterized by restriction analysis. The bioluminescence biochemical process and the nature of its phenotypic expression in E. coli HB101 is explained.
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Determining Cutting Site Locations
The following activity introduces DNA restriction mapping.Students cut pieces of paper into lengths representing those produced when specific enzymes are used to cut a strand of DNA.
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DNA Fingerprinting: The Great Cafeteria Caper
Students will extract DNA from their own hair roots. A DNA fingerprinting simulation kit with standard DNA samples will also be used in this experiment.The DNA will be digested with a variety of restriction enzymes (e.g.,B am H I and Hin d III).Students will run an electrophoresis gel to examine patterns of their DNA along with standard DNA.The experiment will be based on a crime scene scenario.
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DNA on a Stick
What is the chemical basis of life? What kind of chemical is DNA? The first step in the genetic manipulations involved in biotechnology is the isolation of DNA. This project describes one method of chromosomal DNA isolation with minimum breaks. There are several basic steps in DNA extraction. The cell must first be lysed (broken open) to release the nucleus. The nucleus must also be opened to release the DNA. At this point the DNA must be protected from enzymes that will degrade it, causing shearing. Once the DNA is released, it must then be precipitated in alcohol.
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Innocent or Guilty: A Lab on DNA Gel Electrophoresis
This lesson, based on EDVOTEK Kit #109, "DNA Fingerprinting I: Identification of DNA by Restriction Fragmentation Patterns," presents a simulation of a DNA fingerprint (RFLP—Restriction Fragment Length Polymorphism). The prelab section introduces the importance of DNA fingerprinting—a form of identification that is being accepted by both scientific and legal experts. The procedure is used in forensic work, paternity suits, missing-person cases, archeology, and animal breeding. The protocol for the lab is introduced. The lab involves students preparing a gel for electrophoresis. DNA fragments, which have been predigested using two different restriction enzymes, will be run on a gel electrophoresis apparatus, and the results will be analyzed to determine which suspect committed the crime. The post-lab section concentrates on the ethical implications of DNA fingerprinting.
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Producing a Strain of E. coli that Glows in the Dark
In this exercise,students will create a luminescent population of bacteria by introducing into Escherichia coli (E.coli)a plasmid that contains the lux operon.This operon is found in the luminescent bacterium Vibrio fischeri and contains two genes that code for luciferase (the enzyme that catalyzes the light-emitting reaction)and several genes that code for enzymes that produce the luciferins (the substrates for the light-emitting reaction).The success of the transformation is readily apparent,since E.coli colonies that take up this plasmid glow in the dark.In another group,a control plasmid (pUC18)that does not contain the lux operon will be introduced into E.coli,and these cells will not glow in the dark.Both plasmids contain an ampicillin-resistant gene,and therefore both cell types will grow in the presence of the antibiotic.Each resistant colony growing on ampicillin- nutrient agar plates represents a single transformation event,and only those colonies that have taken up the plasmids will grow on the agar.The bioluminescent colonies represent bacteria that can successfully express a metabolic marker.
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Restriction Analysis of Lambda DNA
This lab is separated into two different sections due to lab time allowed for each session over a two-week period. During the first lab session,students will prepare their first digests and the control, using the three different restriction enzymes.During the second lab period,the students will get their digests from the instructor;cast the agarose gel;set up and run the electrophoresis;and photograph, analyze,and graph the results.
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Transformation of Competent Cells with a Recombinant Plasmid
This exercise demonstrates the use of competent Escherichia coli (E.coli)cells in the take-up of plasmids to cause their transformation.The strain used in this exercise is JM83;competent cells may be acquired from UC Davis,or they could be made competent in a previous exercise.These cells are made to take up pUC19,which contains two engineered genes,one for ampicillin resistance (ampicillin-resistance gene)and the other (• -galactosidase gene)to convert X-gal in nutrient agar to a blue color.
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Using a Controlled Experiment to Identify Two Unknown Plasmids
This activity can serve as an assessment following a unit on biotechnology.For a biotechnology unit including lecture and laboratory sessions on DNA structure,plasmids,restriction enzymes,gel electrophoresis,gel analysis following electrophoresis,and bacterial transformation,the student activity described below could serve as a "practicum" of sorts to assess what was learned in the unit. Alternatively,the protocol written in the instructor’s notes could be given to the student as a traditional lab exercise.