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
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.
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 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.
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
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. 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.
- An extensive description of DNA Recombination
DNA recombination is referred to as the making of a functional composite DNA strand that is obtained from two or three fragments from more than one organism. The composite DNA is often called recombinant DNA. This is made possible through genetic engineering. An aspect of genetic engineering known as molecular cloning makes recombinant DNA production possible.
In molecular cloning procedure, the following are to be considered:
- Choice of host organism and cloning vector;
- Preparation of vector DNA;
- Preparation of DNA to be cloned;
- Creation of recombinant DNA;
- Introduction of recombinant DNA into the host organism;
- Selection of organisms containing recombinant DNA;
- Screening for clones with desired DNA inserts and biological properties.
The host organisms are usually bacteria, Escherichia coli is the most preferred bacteria host in molecular cloning due to its shorter replication time. The cloning vectors are mostly plasmids (pBR322, pU8, etc).
Molecular cloning process starts with DNA extraction which involve selecting of broth medium of interested microorganism or cell (in case of eukaryotes) and treating them with lysozyme to degrade the cells followed by the addition of protease to degrade the protein components of the intracellular components of the cells, addition of RNAse to degrade the RNA components and purification by centrifugation. The figure below represents the schematic process of DNA extraction from cells
DNA Digest / DNA Cutting
This is done by treating both the extracted DNA and cloning vector with the same restriction enzyme. There are a series of restriction enzymes, but the most used is EcoRI. The restriction enzyme cut both the cloning vector (plasmid) and the extracted DNA at a particular sequence giving rise to sticky ends which are easily joined together by DNA ligase towards the end of the experiment.
Polymerase Chain Reaction
The digested DNA fragment is polymerized using the appropriate primer, under appropriate temperature condition.
The mixture of the polymerized DNA fragment, the cloning vector, and DNA ligase are mixed. Fragments having the same sticky ends as the cloning vector are annealed by DNA ligase a
Escherichia coli preferably is used as the host for carrying the cloning vector. A mixture of the bacterial culture and cloning vector are mixed and then and then subjected to heat shock in the presence of divalent calcium chloride, or by using electroporation, cell squeezing or gene gun technique.
Screening and selection of transformed bacteria
Most plasmids do have antibiotic resistance gene marker. Bacteria are plated on culture media containing the genes coded for in the cloning vectors, survivors are said to have transformed and are selected and sub-cultured. The final screening stage is the culturing of the survivor bacteria on a culture media on which the gene-spliced into the cloning vector can be expressed in the form of recombinant protein. For example, if a bacteria carrying a cloning vector with a gene for insulin is plated on a medium containing Isopropyl β- d-1-thiogalactopyranoside (IPTG) which allows the expression of insulin.
The product of the successful transformation of bacteria is known as a recombinant protein. Molecular cloning technology has been utilized in the commercial production of pharmaceutical products such as recombinant chymosin, recombinant clotting factor vii, recombinant hepatitis vaccine, recombinant human growth hormone, and recombinant insulin among others. Genetic Modified Foods (GMO) such as golden rice that is rich in beta- carotene and vitamin A, as well as disease resistant plants, has also been discovered through the knowledge of DNA recombination.
If you like this post, please share and patronize us!PATRONIZE US