This exercise was designed to provide an exciting introduction to specific gene structure and function. In the experiment, students are given two plasmids (A and B) which are identified in the instructors guide. One plasmid (A) has a functional gene for the enzyme ß-galactosidase. The ß-galactosidase gene in the other plasmid (B) is inactive because it contains a segment of foreign DNA. In the first part of the exercise, students analyze restriction digests of both plasmids in order to determine which plasmid should have a functional ß-galactosidase gene.
Bacteriophage lambda is a DNA virus that attacks E. coli. Here, students dissect lambda DNA using the restriction endonucleases EcoR1 and BamH1 in order to identify specific sites, sequences, and structures along the phage genome. This single exercise enables students to explore a number of exciting topics in molecular biology, including the specificity of restriction endonucleases, DNA mapping strategies, complementary base-pairing of DNA, and the structure of a viral genome.
This two part exercise provides state-of-the-art information and practical experience with a variety of techniques that form the foundation of the biotechnology industry. In part A, students create a strain of E. coli that is resistant to the antibiotic ampicillin by introducing a plasmid that contains an ampicillin-resistance gene. The success of the transformation is monitored by growing the bacteria on an ampicillin-containing media. This experiment provides sufficient sterile materials for sixteen platings.
In l975, Edward M. Southern at the University of Edinburgh, developed a powerful technique for DNA analysis which has become known as Southern blotting. Here your students use the Southern blotting procedure to identify the major control region for transcription and replication in the lambda phage genome. Following the step-by-step procedures in their manuals, students digest lambda DNA with a restriction endonuclease, electrophorese the DNA, and then transfer the separated fragments on the gel to a nylon membrane.
Genes introduced into E.coli by plasmid-mediated transformation can confer variation in the phenotype of the bacteria. For example, E.coli containing the ampicillin-resistance gene grows in the presence of this antibiotic while the product of the ß-galactosidase gene enables the bacteria to convert the ß-galactosidase substrate X-gal to a blue product. Similarly, E.coli containing the Lux operon produce colonies that glow in the dark.
Purified DNA is most often used as a template in the PCR reaction. However, it is possible to amplify specific DNA sequences without DNA purification by starting with a single living E. coli colony. This technique is known as colony PCR and provides a powerful and reliable method for the rapid amplification and isolation of any gene in the E.coli genome or any gene on a plasmid that is carried by E.coli. In this exercise, students carry out colony PCR starting with a culture of E. coli that carries an ampicillin-resistance gene on plasmid pUC18.
The emission of light by living organisms is a fascinating process. The genetic system required for luminescence in the bacterium Photobacterium (Vibiro) fischeri is the lux operon. This operon contains a gene for luciferase (the enzyme that catalyzes the light-emitting reaction) and genes for enzymes which produce the luciferins (which are the substrates for the light-emitting reaction.). In this exercise, students create a luminescent population of bacteria by introducing into E.coli a plasmid that contains this lux operon.