Transformation (genetics)

from Wikipedia, the free encyclopedia

In molecular biology, transformation is the non-viral transfer of free DNA into competent bacterial cells as well as into fungi, algae, yeast and plants. Under transfection refers to a DNA insertion into eukaryotic animal cells. In addition to transduction and conjugation, transformation is one of three options for gene transfer in prokaryotes .

history

The naturally occurring phenomenon was first caused and described experimentally by Frederick Griffith in 1928 . In 1944, Oswald Avery and his colleagues succeeded in proving that this was a transfer of deoxyribonucleic acid (DNA).

application

The transformation occurs as a partial step in the cloning . During cloning, a segment of DNA is first incorporated into a vector . This recombinant DNA is then introduced into the bacteria by means of transformation, which then grow and in the process also multiply the vector and thus the DNA segment. The desired DNA segment can be duplicated very often in this way. Using horizontal gene transfer, it could then be introduced into foreign cell nuclei in order to generate transgenic animals or plants .

Methods

Free DNA, usually a plasmid , can be added to bacteria, which can take up the DNA with appropriate treatment. Here one makes use of the natural competence to get the bacterial cells to take up foreign DNA.

However, with some bacteria, such as Escherichia coli , there is no natural competence , so that preparatory steps for the transformation are necessary.

The simplest method of transformation is to use chemically competent cells. The bacterial cells are treated with calcium chloride or, more effectively, with rubidium chloride . The DNA is taken up by incubating for 30 minutes at 0-4 ° C; with some E. coli strains, a short heat shock afterwards (41–43 ° C for 45–90 seconds) is said to increase efficiency. It is unclear whether this creates pores in the membrane through which the DNA can get into the cells, or whether other mechanisms cause the uptake. The salt treatment may contribute to the fact that there are fewer repulsive forces between the negatively charged DNA and the negatively charged cell membrane. Overall, this transformation method is simple and can be carried out in a short time.

Another method is so-called electroporation . 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 medium with the bacteria must be completely free of salt, otherwise a short circuit can occur. The resulting short-circuit spark suddenly heats the transformation and kills the bacteria.

Mechanisms

Bacteria have the ability to take up free DNA. This is determined by various competence proteins that are increasingly expressed through quorum sensing or nutrient deficiency. Because Gram-negative and Gram-positive bacteria have a different cell wall structure, a distinction must be made between them.

Gram-positive bacteria bind free DNA to a competence protein. By endonucleases , the DNA is cleaved at the length of about 18kbp stranded and imported into the cell. The other, remaining strand is broken down by nucleases.

Gram-negative bacteria have a secretin channel on the outer membrane and a DNA transporter on the inner membrane to import the free DNA . The DNA is first imported through the secretin. Finally, the DNA is imported in one strand by the transporter and the second strand is broken down.

Once the single-stranded DNA has been absorbed, it binds to the double-stranded DNA of the cell. This creates a triple helix in which the RecA protein exchanges DNA segments. This leads to insertions and deletions in the bacterial DNA. The replication of the DNA now creates two different strands, since the imported DNA was only recombined with one strand.

Individual evidence

  1. ↑ calcium chloride method (Engl.)
  2. ↑ Pope, B. and HM Kent (1996). High efficiency 5 min transformation of Escherichia coli. Nucleic Acids Research 24 (3): 536-537.
  3. ↑ Info on electroporation
  4. ↑ Comparison of different transformation methods (PDF)
  5. ↑ M. Bakkali: Could DNA uptake be a side effect of bacterial adhesion and twitching motility? In: Archives of microbiology. Volume 195, Number 4, April 2013, pp. 279-289, doi : 10.1007 / s00203-013-0870-1 . PMID 23381940 . PMC 3597990 (free full text).
  6. ^ JF Allemand, B. Maier, DE Smith: Molecular motors for DNA translocation in prokaryotes. In: Current Opinion in Biotechnology. Volume 23, number 4, August 2012, pp. 503-509, doi : 10.1016 / j.copbio.2011.12.023 . PMID 22226958 . PMC 3381886 (free full text).
  7. ^ ST Chancey, D. Zahner, DS Stephens: Acquired inducible antimicrobial resistance in Gram-positive bacteria. In: Future microbiology. Volume 7, number 8, August 2012, pp. 959-978, doi : 10.2217 / fmb.12.63 . PMID 22913355 . PMC 3464494 (free full text).

literature

See also