Electroporation bacteria cells

Electroporate eukaryotic cells with DNA grown in dam+ bacteria... 

Culture conditions and electroporation buffer Cells are cultured under normal culture conditions. A medium of low ionic strength should be used as buffer solution for electroporation. PBS or HBS is preferred for mammalian cells and pure distilled water or 10% glycerol for bacteria. Successful use of chemical stimulators added to the electroporation buffer has been reported (Satyabhama and Estonia, 1988). Additives increasing cell surface binding of the molecules to be electroporated may be taken into consideration. For the electroporation, cells are kept either on ice or at room temperature. To avoid contamination, sterile working conditions are important for mammalian cells. 

The uptake of the plasmid into the bacterial cell is called transformation and in the laboratory can be induced in two ways. In one method the bacteria and DNA are placed in ice-cold CaCl2 (50 mmol l-1). This induces the DNA to stick to the outside of the bacteria. The temperature is then increased to 42 °C and the DNA enters the cell. The second method is by electroporation,... 

Available methods for carrying DNA into an animal cell vary in efficiency and convenience. Some success has been achieved with spontaneous uptake of DNA or electroporation, techniques roughly comparable to the common methods used to transform bacteria. They are inefficient in animal cells, however, transforming only 1 in 100 to 10,000 cells. Microiqjection—the injection of DNA directly into a nucleus, using a very fine needle—has a high success rate for skilled practitioners, but the total number of cells that can be treated is small, because each must be injected individually. 

Cell lysis under a high electric field is referred to as electroporation [6], Under these conditions, the cell membrane experiences dramatic changes in permeability to macromolecules. The main applications of the electroporation include the electrotransformation of cells and the electroporative gene transfer by the uptake of foreign DNA or RNA (in plants, animals, bacteria, and yeast). The electric field generates permeable microspores at the cell membrane, so that the nucleic acid can be introduced by electroosmosis or diffusion. 

Passing a direct current (1V applied, in milliamperes) through a solution containing bacteria causes the instant demise of between within the current lines (Stoner, 1979). Why do they die under such mild conditions Is it because of electroporation, in which the solution ions enter the bacteria s cell and separate the contents ... 

The continuous interest and growth of the various new industrial processes related to the life sciences will also require significant contributions from membrane engineering. It is hoped that future research would provide deeper insight on the precise mechanisms involved, which would show new direction for research in membrane science and applications. It is, however, important to recognize that applicability of electroporation has been demonstrated in a variety of bacteria, yeast, and mammalian cells and some applications are ready for exploitation while many new technologies seem potentially possible. 

For the identical experimental conditions, electroporation efficiency depends on the type of cells the composition of the membrane, shape, and size of cells strongly influences the electroporation efficiency [40-42]. In electroporation of bacteria, the growth phase of cell has significant influence on transformation efficiency, which is higher for cells harvested and electroporated from mid-log phase. However, cells from stationary phase can also be transected with reasonably good efficiency. Mammalian cell can be electroporated at relatively lower fields but pulse length controls the entry of external molecules into cells. 

Electroporation, however is the most generalized way of introducing DNA not only in bacteria but also in eukaryotic cells. This technique is based on the induction of free DNA uptake by the bacterium after subjecting it to a high electric field. Electroporation allows the uptake of most sizes of plasmids. 

As is known to molecular biologists, DNA electrotransfer to bacteria is a standard method, and a small pulse generator is everyday equipment in most laboratories. However, whereas the molecular biologist would not hesitate to let billions of bacteria succumb to side effects of high-field electroporation, this view is of course not shared by those working with mammalian cells-and doctors working in the clinic would have safety as the major priority [2]. 

In some circumstances the introduction of a cloning vector into a host cell is a trivial process. For example, phage vectors are designed so they introduce recombinant DNA in an infective process called transfection, and some bacteria take up plasmids unaided. However, most host cells must be induced to take up foreign DNA. Several methods are used. In some prokaryotic and eukaryotic cells, the addition of Ca2+ to the medium promotes uptake. In others, a process called electroporation, in which cells are treated with an electric current, is used. One of the most effective methods for transforming animal and plant cells is the direct microinjection of genetic material. Transgenic animals, for example, are created by the microinjection of recombinant DNA into fertilized ova. 

Dunican, L.K. and Shivnan, E. (1989) High frequency transformation of whole cells of amino acid producing Coryneform bacteria using high voltage electroporation. Nat. Biotechnoi, 7, 1067-1070. 

On the following day, randomly pick eight colonies per transformation, amplify them and purify DNA using a standard commercial kit (e.g., QIAprep Miniprep Kit). Sequence the DNA and select the clone with the right sequence for next steps. Electroporate TGI bacteria with this DNA (as explained in previous paragraph). TGI cells containing the mutated sequence of the protein of interest can now be used in ELISA assays. 

Transform 100 pL of supercompetent XL-1 Blue E. colt with 1-5 pL of the ligation reaction as directed by the supplier (Stratagene) It is essential to obtain high-transfection efficiency, especially when looking for rare clones I found it very reliable and convenient to purchase supercompetent cells for this purpose. If this is not possible, I recommend using electrocompetent cells and electroporation to transform bacteria (68). 

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