\nAnother method of selecting recombinants from non-recombinants is their ability to produce colour in the presence of a chromogenic substrate. For this recombinant DNA is inserted within the coding sequence of an enzyme, beta-galactosidase. This results into inactivation of the enzyme, called as insertional inactivation.<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\nIf the bacteria does not have an insert, chromogenic substrate present in the medium react with betagalactosidase enzyme gives blue coloured colonies.<\/p>\n
If the plasmid have an insert, they do not produce any colour due to insertional inactivation of the gene coding for beta galactosidase, these are identified as recombinant colonies.<\/p>\n
(iv) Vectors for cloning genes in plants and animals: \nNormally Agrobacterioum tumifaciens (a pathogen of several dicot plants) transfer its \u2018T-DNA\u2019to normal plant cells and causes tumor. Similarly retroviruses in animals have the ability to transform normal cells into cancerous cells and they are used as vectors for delivering genes of interest to humans.<\/p>\n
For delivering genes of interest to plants tumor inducing (Ti) plasmid of Agrobacterium tumifaciens is modified (disarming) as non pathogenic Similarly the retroviruses are disarmed and used to deliver desirable genes into animal cells.<\/p>\n
3. Competent Host<\/span> \n(For Transformation with Recombinant DNA) \nDNA is a hydrophilic molecule, it cannot pass through cell membranes. For this, bacterial cells must have to be competent to take up DNA.<\/p>\n\n\n\nThis is done by treating them with calcium ions and incubating the cells and recombinant DNA on ice, followed by placing them at 42\u00b0C Then putting them back on ice. This helps the bacteria to take up the recombinant DNA.<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\nAnother methods:<\/p>\n
\nMicro-injection – recombinant DNA is directly injected into the nucleus of an animal cell.<\/li>\n Biolistics or gene gun -cells are bombarded with high velocity micro-particles of gold or tungsten coated with DNA. It is suitable for plants.<\/li>\n<\/ol>\nAnd the last method uses \u2018disarmed pathogen\u2019 vectors, which when allowed to infect the cell, transfer the recombinant DNA into the host.<\/p>\n
Processes Of Recombinant Dna Technology<\/span> \nRecombinant DNA technology involves several steps. They are<\/p>\n1. Isolation of the Genetic Material (DNA):<\/span> \nInitially the bacterial cells\/plant or animal tissue are treated with enzymes such as lysozyme (bacteria), cellulase (plant cells), chitinase (fungus) to open the cell to release DNA along with other macromolecules such as RNA, proteins, polysaccharides, and Other molecules can be removed by appropriate treatments and purified DNA precipitates out afterthe addition of chilled ethanol. It can be observed as collection also lipids.<\/p>\nTo get DNA in a pure form and free from other macro-molecules it is treated with enzymes. RNA can be removed by treating with ribonuclease whereas proteins can be removed by treating with protease, of fine threads in the Suspension.<\/p>\n
2. Cutting of DNA at Specific Locations:<\/span> \nIt is done by incubating purified DNA molecules with the restriction enzyme. Here Agarose gel electrophoresis is used to check the progression of a restriction enzyme digestion. DNA is a negatively charged molecule, hence it moves towards the positive electrode (anode).<\/p>\nAfter having cut at the source DNA as well as the vector DNA with a specific restriction enzyme, the cut out \u2018gene of interest\u2019 from the source DNA and the cut vector with space are mixed and ligase is added. This results in the preparation of recombinant DNA.<\/p>\n
3. Amplification of Gene of Interest using PCR:<\/span><\/p>\n\n\n\nPolymerase Chain Reaction is helpful to produce multiple copies ( eg-1 billion copies-) of the gene of interest.<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\nIt is synthesised in vitro using two sets of primers (chemically synthesised oligonucleotides that are complementary to the regions of DNA) and the enzyme thermostable DNA polymerase (isolated from a bacterium, Thermus aquaticus).<\/p>\n
This enzyme extends the primers using the nucleotides provided in the reaction mixture and the genomic DNA as template. If the process of replication of DNA is repeated many times, the segment of DNA (gene of interest) can be amplified. The amplified fragment is used to ligate with vector for further cloning.<\/p>\n
(PCR showing denaturation. annealing and extention)<\/span> \n <\/p>\n <\/p>\n
4. Insertion of Recombinant DNA into the Host Cell\/Orqanism:<\/span> \nRecombinant DNA carry gene resistant to antibiotic (e.g., ampicillin) is transferred into E. coli cells, the host cells become transformed into ampicillin-resistant cells. If spreading the transformed cells on agar plates containing ampicillin, only the transformants grow and untransformed cells die.<\/p>\nSo it is helpful to select a transformed cell in the presence of ampicillin. The ampicillin resistance gene in this case is called a selectable marker.<\/p>\n
5. Obtaining the Foreign Gene Product:<\/span> \nThe main aim of all recombinant technologies is to produce a desirable protein. Here the foreign gene is expressed under appropriate conditions. If it is necessary to produce target protein i.e recombinant protein on a small scale, rDNA transferred into the host and cloned genes of interest must be grown in the laboratory. Then the protein is extracted and purified.<\/p>\nStirred tank Bioreactor<\/span> \n \nA stirred-tank reactor is a cylindrical vessel that helps in the mixing of the reactor contents. The stirrer also facilitates oxygen availability throughout the bioreactor.<\/p>\n\n\n\nIt consist of agitator system, an oxygen delivery system and a foam control system, a temperature control system, pH control system and sampling ports, so that small volumes of the culture can be withdrawn periodically.<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n6. Downstream Processing:<\/span> \nAfter the desired product formed, it is subjected to a series of processes. These include separation and purification, which are called as downstream processing.<\/p>\nThe product is added with preservatives and undergoes clinical trials as in case of drugs. Later the strict quality control testing is done for each product.<\/p>\n
We hope the Plus Two Botany Notes Chapter 4 Biotechnology Principles and Processes help you. If you have any query regarding Plus Two Botany Notes Chapter 4 Biotechnology Principles and Processes, drop a comment below and we will get back to you at the earliest.<\/p>\n","protected":false},"excerpt":{"rendered":"
Plus Two Botany Notes Chapter 4 Biotechnology Principles and Processes is part of\u00a0Plus Two Botany Notes. Here we have given Plus Two Botany Notes Chapter 4 Biotechnology Principles and Processes. Board SCERT, Kerala Text Book NCERT Based Class Plus Two Subject Botany Notes Chapter Chapter 4 Chapter Name Biotechnology Principles and Processes Category Plus Two […]<\/p>\n","protected":false},"author":4,"featured_media":0,"comment_status":"open","ping_status":"open","sticky":false,"template":"","format":"standard","meta":{"_genesis_hide_title":false,"_genesis_hide_breadcrumbs":false,"_genesis_hide_singular_image":false,"_genesis_hide_footer_widgets":false,"_genesis_custom_body_class":"","_genesis_custom_post_class":"","_genesis_layout":"","footnotes":""},"categories":[42728],"tags":[47580],"yoast_head":"\n
Plus Two Botany Notes Chapter 4 Biotechnology Principles and Processes - A Plus Topper.com<\/title>\n \n \n \n \n \n \n \n \n \n \n \n \n \n \n\t \n\t \n\t \n