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| Getting the Plasmid In |
In nature bacteria have various enzymes that cut up the DNA of their natural enemies, such as bacteriophages (bacterial viruses). Researchers have taken advantage of these so-called restriction_enzymes to splice DNA for use in engineering bacteria. Hundreds of restriction enzymes have been isolated and each will cut a DNA strand at a specific sequence of nucleotides. Some restriction enzymes generate blunt ends, cutting across both strands of DNA. Others generate a staggered cut, producing "sticky ends." These ends anneal by hydrogen bonding to similar ends on another DNA segment cut with the same restriction enzyme.
Cloning a gene involves identifying a gene of interest in an organism, isolating DNA from that organism, and then using a restriction enzyme to snip the gene from the DNA strand. The gene?containing segment can then be spliced into a plasmid cut by the same restriction enzyme. The bacteria take up the plasmid and are allowed to replicate.
Ordinarily, bacterial cells do not readily take up plasmids. Researchers can use various tricks, however, to get cells more ready to do so. One common method holds the cells on ice in a solution of calcium chloride. The cells are then briefly heat shocked so the plasmid can cross the plasma membrane. An alternate method, electroporation, uses a short electrical pulse to open pores in the plasma membrane, allowing the plasmid to pass through.
Marker genes, such as genes for antibiotic resistance, are often engineered into plasmids. These marker genes enable researchers to know which bacteria have the plasmids. The antibiotic is added to the media used to grow the bacteria. Cells that do not contain the plasmid will fail to reproduce. In addition to marker genes, plasmids typically contain one or more genes of interest. For example, a protein not otherwise expressed by the recipient cell might be produced only when the plasmid is present. Individual colonies of bacteria, each derived from a single cell, can be evaluated for the expression of such novel gene products.
Protein production can be straightforward if the source of the novel gene was another bacterium. However, the goal of modifying bacteria might be the production of proteins encoded by eukaryotic genes from fungi, plants or animals. This presents challenges. Eukaryotic DNA contains both exons (coding sequences) and introns (intervening sequences). In eukaryotic cells this DNA is used as a template for the production of mRNA, which must then undergo mRNA splicing. Introns are removed and exons are joined to form the mRNA, which travels to the ribosome for protein production. Bacteria lack the enzymes necessary for mRNA splicing, so introducing a eukaryotic gene into bacteria requires a special procedure. First, DNA must be generated that is complementary to the already spliced mRNA. The enzyme reverse transcriptase is then used to generate a double-stranded DNA molecule called cDNA, using the mRNA as a template. Finally, this cDNA is incorporated into the cloning vector.
Expressing eukaryotic genes in bacteria presents other problems. After proteins are assembled in eukaryotic cells they are often modified. (See the Proteins and Proteomics unit.) For example, various sugars may be attached to the polypeptide so that glycoproteins are formed. Bacteria are generally unable to accomplish such post-translational modifications, and eukaryotic genes expressed in bacteria may not function properly. The inability of bacteria to perform such modifications has driven scientists to use yeast (Saccharomyces cerevisiae) and eukaryotic cell culture to produce some recombinant products.