page: Cloning vehicles The basic experimental techniques involved in gene cloning have now been described. A DNA molecule needs to display several features to be able to act as a vehicle for gene cloning. Most important, it must be able to replicate within the host cell, so that numerous copies of the recombinant DNA molecule can be produced and passed to the daughter cells. Two kinds of DNA molecule that satisfy these criteria can be found in bacterial cells: plasmids and bacteriophage chromosomes. We concentrate in this course only on plasmids. The greatest variety of cloning vectors exist for use with E. coli as the host organism. This is not surprising in view of its central role in basic research over the last 50 years. The tremendous wealth of information that exists concerning the microbiology, biochemistry and genetics of E. coli has meant that virtually all fundamental studies of gene structure and function have been carried out with this bacterium as the experimental organism. A cloning vehicle also needs to be relatively small, ideally less than 10 kilobases (kb) in size, as large molecules tend to break down during purification, and are also more difficult to manipulate. As already said, the most commonly used as cloning vectors are E. coli plasmids but natural plasmids range from 1.0 kb for the smallest to over 250 kb for the largest, so only a few will be useful for cloning purposes. The smaller plasmids make use of the host cell‘s own DNA replicative enzymes in order to make copies of themselves, whereas some of the larger ones carry genes that code for special enzymes that are specific for plasmid replication (Brown 1991). Figure 1. Plasmids: extrachromosomal and Figure 2. Electron micrograph independent molecules of DNA, of a circular plasmid found in bacterial cells of E. coli (Hartl and Jones 2000) (Brown 1991) Plasmids Most natural plasmids are double-stranded, covalently closed, circular DNA molecules that can be isolated from bacterial cells in a superhelical form. Plasmids behave as accessory genetic units that replicate and are inherited independently of the bacterial chromosome. Frequently they contain genes coding for enzymes that are advantageous to the bacterial host. Among the phenotypes conferred by plasmids are resistance to and production of antibiotics (see later), degradation of complex compounds, production of enterotoxins, restriction and modifying enzymes as well as enzymes for conjugal transfer of plasmids during sexual reproduction. Plasmids are circular molecules of DNA that lead an independent existence in the bacterial cell. Plasmids almost always carry one or more genes, and often these genes are responsible for a useful characteristic displayed by the host bacterium. For example, the ability to survive in normally toxic concentrations of antibiotics such as ampicillin, chloramphenicol or tetracycline is often due to the presence in the bacterium of a plasmid carrying antibiotic-resistance genes. In laboratory antibiotic resistance is used as a selectable marker to ensure that bacteria in a culture contain a particular plasmid. All plasmids possess at least one DNA sequence that can act as an origin of replication, so they are able to multiply within the cell quite independently of the main bacterial chromosome. The copy number refers to the number of molecules of an individual plasmid that are normally found in a single bacterial cell. Each plasmid has a characteristic but not well understood copy number ranging from as low as one (for the large plasmids) to as many as 50 or more. Generally speaking, a useful cloning vehicle needs to be present in the cell in multiple copies so that large quantities of the recombinant DNA molecule can be obtained (Brown 1991). Worksheet IX.12 Cloning vehicles - cloning vectors