Transformation (genetics)
As a transformation of the non-viral transfer free DNA is referred to into competent bacterial cells, and in fungi, algae, yeasts and plant molecular biology. Transfection refers to a DNA insertion in eukaryotic animal cells. The transformation is next to the transduction and conjugation of one of the three possibilities of gene in prokaryotes.
History
This also naturally occurring phenomenon caused experimentally and described for the first time in 1928 by Frederick Griffith. 1944 succeeded Oswald Avery and his colleagues to prove that this is a transfer of deoxyribonucleic acid (DNA ).
Application
The transformation is used in genetic engineering, recombinant DNA into bacteria - to reproduce - after cloning of DNA fragments into a vector. The thus generated DNA varieties are introduced by gene transfer into other cell nuclei to produce transgenic animals or plants.
Methods
Free DNA, usually a plasmid, can be added to bacteria that can take up the DNA with a suitable treatment. This method takes advantage of the natural skills to bring the bacterial cells to the uptake of foreign DNA.
In some bacteria, such as Escherichia coli, however, there is no natural competence, so that preparatory steps for the transformation are necessary.
The simplest method of transformation is the Verwendeung chemically competent cells. Here, the bacterial cells are treated with calcium chloride or rubidium with effective. With 30 minutes incubation at 0-4 ° C, the DNA is taken up; in some E. coli strains to increase the efficiency after a short heat shock (41-43 ° C for 45-90 seconds). Whether this pores are formed in the membrane through which DNA can enter into the cells, or whether other Menchanismen cause uptake is unclear. The salt treatment may contribute to the fact that there are less repulsive forces between the negatively charged DNA and the negatively charged cell membrane. Overall, this transformation method is simple and feasible in a short time.
Another method is the so-called electroporation. Here, the bacteria are treated with an electric shock (2000-2500 V for a few milliseconds ) in order to bring the DNA through the membrane. This method is much more effective than the chemical method. However, the media containing the bacteria must be completely free of salt, as it may cause a short circuit otherwise. The resulting short circuit spark heats the transformation mixture abruptly by killing sensitive bacteria.
Mechanisms
Bacteria possess a competence to free DNA uptake. This is determined by several competence proteins, nutritional deficiencies are strongly expressed the quorum sensing by or. For the reason that gram negative and gram positive bacteria have a cell wall other structure, to distinguish between them.
Gram- positive bacteria bind free DNA in a competence protein. By endonucleases, the DNA is cleaved at the length of about 18kbp stranded and imported into the cell. The other, left strand is degraded by nucleases.
Gram- negative bacteria have to import the free DNA a secretin channel in the outer membrane as well as a DNA transporter at the inner membrane. The DNA is first imported by the secretin. Finally, the single-stranded DNA is imported by the transporter and degraded the second strand.
After recording the single-stranded DNA leads to the binding with the double-stranded DNA of the cell. This results in a triplex, wherein the RecA protein exchanging segments of DNA. This leads to insertions and deletions at the bacterial DNA. By replicating the DNA now results in two different strands, since the imported DNA was recombined with only one strand.