Escherichia coli

Microorganism Escherichia coli can be conveniently used for production of transgenic plants, preparation of transformation vectors, in DNA recombination technology, and for genetic manipulations.

E.coli is a rod-shaped bacterium with a circular chromosome of 3 million bp. Most bacteria stems used in the DNA recombination work are E.coli derivative stems K-12.

Bacterial plasmids are often used in cloning methods. These circular extrachromosomal DNA molecules are capable of replicating themselves. They are found in naturally occurring E. coli stems. On the basis of these plasmids artificial plasmids have been created, which are now widely used in the DNA recombination technology. All of these plasmids must have the following three elements: replication elements, selective marker, and cloning area.

Protein synthesis in E.coli

In order to synthesize extraneous proteins in heterologic system, first, the gene, whose product is intended for expressing, is cloned into the expression vector. The resulting structure is introduced into cells, in which the target protein will be expressed. Expression vector should have a promotor sequence as well as autonomous replication sequences to enable his replication in the host cell. It should also have sequences creating resistance to antibiotics, to enable identification of the cells containing this vector. Here the E.coli expression system is one of the most popular ones.

Protein expression vector should have the following main elements: sequences conding the marker, which ensures support of the vector in a cell; regulating promotor, which after induction would ensure synthesis of a large amounts of mRNA from the cloned-in gene; sequences necessary for translation, such as rybosomes attachment areas; and translation initiation codone ATG and multiple cloning area to facilitate insertion of the gene into the vector. The constructed vector is used to transform the respective E.coli stem. Vectors of the pET are used the most often. In this case the target gene is inserted under the promotor of T7 RNA polymerase.

Extraneous genes are cloned into the pET vector using stems, which do not have a source of T7 RNA polymerase. In the absence of T7 RNA polymerase, expression of the target gene is minimal and RNA polymerase of a host cell can not initiate transcription from a T7 promotor, and multiple cloning zone is transcription-wise inactive. In some cases, the same stems, containing the T7 RNA polymerase gene, which are used in protein expression, may be directly used in cloning. However, this is not recommended, because if the gene turned out to be toxic to E.coli cells (for example, due to the base level of transcription), growth of cells may be slow and plasmids may be unstable.

For protein expression recombinant plasmid is introdced into a stem of E.coli, which contains the T7 RNA polymerase gene. Such stems are bacteriophagus  lisogenes BL21 (DE3). Upon introduction into the growth medium of IPTG, synthesis of T7 RNA polymerase is induced and transcription of the necessary gene is initiated.