Bacterial hosts

In each cycle, the library of mutated genes is first inserted in a standard bacterial host such as Escherichia coli or Bacillus subtilis. Subsequently, bacterial colonies are plated out on agar plates and harvested individually by a colony picker. Each colony is placed in a separate well of a microtiter plate containing nutrient broth, so that the bacteria grow and produce the protein of interest. Because each colony originates... 

Recombinant DNA technology can also be used to design genes that encode for proteins with desired features [34]. The gene can be incorporated into a plasmid, which is then used to transform a bacterial host such as Escherichia coli. Finally, the production of the desired amino acid polymer is performed by the host with a precisely defined sequence and near absolute monodispersity [29, 35]. 

Classification of bacterial viruses In the bacterial viruses, a formal classification scheme is rarely used. Rather, each bacterial virus is designated in terms of its principal bacterial host, followed by an arbitrary alphanumeric. Thus, we speak of T4 virus of Escherichia coli or P22 virus of Salmonella typhimurium. An overview of some of the major types of bacterial viruses is given later. We should note, however, that although a bacterial virus may be designated in reference to its principal host, the actual host range of the virus may be broader. Thus, bacteriophage Mu, generally studied with Escherichia coli, also infects Citrobacter and Salmonella. 

Another viral chromosome that can be used as a vector is that of the filamentous phage Ml3. The M13 chromosome is a single-stranded DNA molecule which when inserted into the bacterial host replicates outside the bacterial chromosome in the cytoplasm. The virus is then reassembled and released from the bacterial cell without cell lysis. 

The procedure of inserting DNA into a vector and its subsequent introduction into the bacterial host is extremely inefficient and will result in the majority of bacteria in the culture either containing none of the required vector or containing a vector in which no DNA is inserted. However, vectors have been produced that, by placing the bacteria in specific media, allow only the growth of those containing the vector and the inserted DNA. 

Bacterial hosts are inappropriate choices for expression of proteins such as the blue copper proteins stellacyanin, laccase, and ceruloplasmin which are extensively glycosylated. In these cases, it may be necessary to employ tissue cultures of appropriate origin to obtain the native protein. In this regard, the amino-terminal half of human serum transferrin, which lacks carbohydrate, has been expressed in high yield in baby hamster kidney cells by Funk et al. [13], while the glycosylated carboxyl-terminus has proved to be more problematic [103]. 

The DNA or cDNA library is then introduced into a preparation of bacterial host cells. Usually, the first host selected is a laboratory strain of E. coli which has been grown and pretreated with inorganic salts to make uptake of DNA easier. The ability to take up foreign DNA is called competence, cells which have been specially prepared for the purpose are called competent cells. Other methods to transfer DNA into cells include electroporation (application of an external electric field to permeabUize the cell wall), transfection (where a recombinant bacterial virus is used to transfer the DNA to the target cell) or ballistic methods (by using DNA-coated particle projectiles). The last method has been used to introduce foreign DNA into plant cells and mammalian cells. 

Muniesa, M., Moce-Llivina, L., Katayama, H., and Jofre, J. (2003). Bacterial host strains that support replication of somatic coliphages. Ant. Van Leeuwenhoek Int. J. Gen. Mol. Microbiol. 83, 305-315. 

Y. Tang, D.A. Tirrell, Biosynthesis of a highly stable coiled coil protein containing hexafluoroleucine in an engineered bacterial host, J. Am. Chem. Soc. 123(44) (2001) 11089-11090. 

Phage T4 encodes a DNA polymerase much used in the laboratory because of its ability to polymerize using a long single-stranded template. It also depends upon accessory factors provided by the bacterial host (Section 5) 287,287a... 

Effective cloning systems are available for a variety of bacterial hosts, including Bacillus subtilis, Streptomyces spp., and Agrobacter tumefaciens. Cloning systems have also been developed for eukaryotic hosts such as the yeast Saccharomyces cerevisiae, mammalian cells in tissue culture, and plant cells. 

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