Gene Function and Cloning in Bacteria. (B1-3)
Occasionally, E. coli cells are found in nature that are resistant to the toxic effects of the antibiotic ampicillin. In this exercise, students create such an ampicillin-resistant population of E. coli by introducing into bacterial cells a plasmid that contains an ampicillin-resistance gene. Sufficient sterile materials are supplied for sixteen platings. The introduction of plasmid DNA into bacterial cells is called transformation and has enabled scientists to obtain large quantities of more than 1000 genes including those for human interferon, insulin, and growth hormone. This exercise requires minimal teacher preparation and provides a laboratory experience with one of the most important techniques used in gene-cloning studies.
A Sample from the Student Guide:
Penicillin is one of the most important anti-infective agents used in clinical medicine because it is inexpensive, bactericidal and its toxicity for human cells is almost nonexistent. Penicillin interferes with the synthesis of the bacterial cell wall and will thus cause osmotic lysis of susceptible microbes. Penicillin is not a single compound but a group of compounds with related structures and activities. Many of these compounds are semi-synthetic in that part of each molecule is made by a mold to which the chemist adds another chemical group. Over 500 semi-synthetic penicillins have been made during the past 30 years. Ampicillin is a broad-spectrum semi-synthetic penicillin that will kill a number of bacterial species including Escherichia coli. Occasionally, E.coli cells are found in nature that are resistant to the toxic effects of ampicillin. In today’s laboratory, you will create such an ampicillin-resistant population of E.coli.
Plasmids are small circular DNA molecules that exist apart from the chromosomes in most bacterial species. Under normal circumstances, plasmids are not essential for survival of the host bacteria. However, many plasmids contain genes that enable bacteria to survive and to prosper in certain environments. For example, some plasmids carry one or more genes that confer resistance to antibiotics. A bacterial cell containing such a plasmid can live and multiply in the presence of the drug. Indeed, antibiotic-resistant E.coli isolated in many parts of the world contain plasmids that carry the genetic information for protein products that interfere with the action of many different antibiotics. In this laboratory, you will introduce a plasmid that contains an ampicillin resistance gene into E.coli.
The plasmid that you will use in today’s laboratory is called pUC18. A map of the basic structure of pUC18 is shown in Figure 3-1. Plasmid pUC18 is a circular DNA molecule that contains only 2686 nucleotide pairs (molecular weight= 2x106). The small size of this plasmid makes it less susceptible to physical damage during handling. In addition, smaller plasmids generally replicate more efficiently in bacteria and produce larger numbers of plasmids per cell. As many as 500 copies of this plasmid may be present in a single cell. Plasmid pUC18 contains an ampicillin-resistance gene that enables to grow in the presence of the antibiotic. Bacteria lacking this plasmid, or bacteria that lose the plasmid, generally will not grow in the presence of this antibiotic. The ampicillin resistance gene of pUC18 codes for the enzyme β-lactamase (penicillinase) which inactivates ampicillin and other penicillins.