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Probably the greatest strength of molecular biology is the ability to manipulate genetic material, commonly called genetic engineering. Specific fragments of DNA may be isolated, cut into discrete pieces by the action of restriction endonucleases, and rejoined by the action of DNA ligase to create novel genes and other genetic constructs. This technology allows scientists to study the activity of genes to understand their function. One of the applications of this technology is the potential to treat genetic diseases, such as cancers, by gene replacement.

The genetic manipulations described above require large quantities of DNA. One of the easiest ways to get large amounts of DNA is to place the desired DNA into bacteria, grow the bacteria, then harvest the bacteria, and isolate the DNA (see Chapter 3). Bacteria can maintain DNA as plasmids: circles of DNA that usually contain a gene that allows the bacterium to grow in the presence of an antibiotic. In this experiment, students will introduce a plasmid into bacteria. This process is called transformation. Bacteria are treated so they will take the plasmid up into their cells. These are called competent cells. Transformation involves mixing competent bacteria with plasmid DNA and then selecting bacteria containing the plasmid using agar plates that contain an antibiotic.

Purpose

This laboratory protocol will allow students to demonstrate the phenomenon of transformation in a relatively simple procedure. Competent Escherichia coli (E. coli) cells which have been kept in freezer storage will be thawed on ice, treated with beta-mercaptoethanol (BME), introduced to a transforming plasmid, briefly heat shocked, and then plated out on a nutrient agar which contains the antibiotic ampicillin that selects for transformants. Using E. coli without the plasmid as a negative control (-) and E. coli with the plasmid as a treated or positive (+) sample, students will be able to directly observe the transformation of bacteria to ampicillin resistance. The (+) strain will survive on the antibiotic containing agar plate due to the laboratory procedures conducted by the student. The procedure and observation made by the student brings home the concept of genotype and phenotype being directly controlled by the genes which are made of DNA.

 

Bacterial Transformation Learner Outcomes

The following cognitive objectives should be addressed:

1. Define the vocabulary related to genetic engineering: transformation, recombinant DNA, host, plasmid, vector, and competent.
2. Describe the work of Frederick Griffith as it relates to transformation.
3. State why E. coli is used in many genetic engineering experiments.
4. Relate genetic constitution of an organism with its physical attributes: describe the relationship between genotype and phenotype.
5. Apply the technology used for DNA transformation to current work in the field of gene therapy.
6. Discuss the moral and ethical issues of recombinant DNA technology.

The following laboratory objectives should be addressed:

1. Use aseptic techniques in transferring cultures and reagents.
2. Pipette reagents.
3. Heat shock cultures with time and temperaturerestraints.
4. Use a spreader to get isolated colonies on agar plates.