methods of making competent cells

Methods of making competent cells in prokaryotes

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methods of making competent cells

The ability of bacteria to take up exogenous DNA usually from closely related species which are exposed into their environment and incorporate it into their genome is known as competence, the process known as transformation. Some bacteria naturally can incorporate exogeneous DNA into their genome; these bacteria are called naturally competent bacteria. Examples include Streptococcus pneumonia, Bacillus subtillis, Helicobacter pylori Pseudomonas and Acinetobacter species.

The uptake of exogenous DNA from the environment often helps transformants to utilize certain substrate in the environment or become antibiotics resistance. However, it should be noted that transformation in naturally competent bacteria is usually one in a million or more. Thus, there is a need for induced transformation through artificial means.

Induced transformation is a method of making bacteria competence (i.e to uptake desired DNA strand). When a bacterium can successfully incorporate the up took DNA into its genome. The resultant DNA strand of the genome is referred to as recombinant DNA. The process can as well be referred to as DNA recombination. Interestingly, recombinant DNA can be made in-vitro in the laboratory nowadays and made to be uptaken by bacteria through a process called transformation.

The knowledge of DNA recombination and horizontal gene transfer in bacteria generally has helped the mass production of useful pharmaceutical products such as recombinant insulin, recombinant growth hormone, as well in the study of mechanisms of antibiotics resistance and in the study of antibiotic resistance during epidemiological studies.

Different methods of making competent cells

Methods of making competent cells in prokaryotes

DNA extraction, purification, and amplification using the PCR machine are essential methods step before inducing bacteria to uptake a DNA strand or inducing competence. There are a series of methods of inducing transformation in bacteria cells. However, it should be noted that on the completion of the screening process. The medium containing the bacteria cells are screened for transformants.

Electroporation

In this method, the solution containing amplified bacteria DNA is mixed with a medium of bacteria suspension or culture, placed inside an electroporator and subjected to about 1.0 to 1.5 voltage for five seconds. The essence of electric shock is to create a pore in the cell membrane of bacteria and enhance permeability and inflow of DNA fragments in the medium

Cell Squeezing

Cell squeezing help in the mobilization of DNA fragments into bacteria of interest in that when a bacteria cell is passed through a microfluidic device that is made up of channels that allows that is initially free but becomes narrow in the progression. The cell membrane becomes flexible and disrupted in such a way that it won’t die but allow the passage of DNA particles into the cells. As the cell goes back to their normal shape. The openings are closed up and the uptaken genes are expressed in transformed bacteria.

Heat Shock

Divalent calcium chloride (CaCl2), amplified DNA fragments and bacteria solution of interest are mixed. The solution is then subjected to heat shock by exposing the mixture to cold condition by placing it in ice for a few seconds and then exposing to a slightly warm condition. The presence of CaCl2 in the medium help to waken the cell membrane of the bacteria while the exposure cold condition followed by sudden exposure to slightly warm condition create pores and enable the inflow of DNA particles into the bacteria sample

Gene gun

This is a method which involves the shooting of small particles of gold-coated small particles of DNA or RNA directly into a bacteria, yeast, plants and animal cells by using device known gene gun. Once the gold-coated DNA enters the recipient cell, it is incorporated into the genome. This biolistic method was initially designed for transfecting plants with resistance gene to Agrobacterium tumefaciens.

Methods of making competent cells in eukaryotes

The process of inducing eukaryotic cells to uptake exogenous DNA materials is known as transfection. Protoplast fusion, sonication, and micro-injection are the main methods used in transfection processes.

Cite this article as: Fagbohun, S.O., "Methods of making competent cells in prokaryotes," in ATG Ventures, 03/01/2020, https://atgventure.com/methods-of-making-competent-cells-in-prokaryotes/.

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