Plasmids do the “Tricking”

Bacteria can be tricked to clone an extra gene if it is combined with an extra DNA molecule called a vector. Gene cloning vectors are small groups of genes. These smaller DNA molecules can occur as circles called plasmids or they could also occur as liner molecules called viruses. The genes in a virus particle encode what is needed to make the protein coat and replicate the genes to produce more copies of the virus. The smaller groups of genes in plasmids and viruses can be genetic tools that play a critical role in cloning genes from any organism. We will focus our description of gene cloning on methods that use plasmids as cloning vectors.

Plasmids, are naturally occurring and used by bacteria to transfer genetic information among themselves. Many plasmids isolated in nature encode antibiotic resistance genes. They also have sequences called an origin of replication that are recognized by the bacteria cell and signal the bacteria’s replicating enzymes to replicate the plasmid. Because the plasmids are small (a few thousand base pairs) they are mobile. Once they enter a bacteria cell, they are replicated several to many times. The plasmids can remain as free molecules that are not part of the bacteria’s larger chromosome. Bacteria that maintain plasmids with an antibiotic resistance gene and are then exposed to an antibiotic will live while bacteria that are susceptible to the antibiotic do not survive. Bacteria that are transformed or genetically altered by the introduction of a new plasmid have an incentive to maintain this new antibiotic resistance trait. Therefore they will make copies of the plasmid.

In the 1960's the properties of these plasmids described above were discovered. In the 1970s, Stanley Cohen and other researchers at the University of California discovered that these plasmids could be altered and maintain their transformation properties. They could combine new DNA sequences with the plasmids, making recombinant plasmids in a test tube. The recombination was directed to occur in a specific way by cutting the DNA with a restriction enzyme and then ligating or attaching the cut ends of different molecules together with DNA ligase. Bacteria cells could then be transformed with these recombinant plasmids and would replicate the entire plasmids regardless of the origin of the new DNA sequences that were recombined into the plasmids. If the recombination involved the insertion of new DNA into an antibiotic resistance gene, the resistance gene would be inactivated. The insertion mutated the antibiotic resistance gene coding sequence and the bacteria could no longer use that information to express the resistance trait.

Fig. 6: Plasmid with an antibiotic resistance gene and LacZ gene is useful as a vector to introduce new DNA into bacteria for cloning. Inserting foreign DNA into the LacZ gene inactivates the gene. (Image by D. Lee)

The first gene cloners recognized that plasmids with two genes that introduced new traits into bacteria such as antibiotic resistance would be useful as cloning vectors. We will describe gene cloning with one of these plasmids.