Biomolecular Engineering Solutions for Renewable Specialty Chemicals. Группа авторов
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1.2.1.4.3 Terminal Transferase
Terminal deoxy‐nucleotidyl transferase [Terminal transferase (TdT)] has the capability to transfer or add polynucleotide in 3′ terminus of the DNA molecule. It is extracted from calf thymus. TdT is a template impartial polymerase that catalyzes the addition of deoxynucleotides to the 3′ hydroxyl terminus of DNA molecules with none template strand. In rDT, blunt‐ended DNA strands are required every now and then to make it cohesive via the addition of nucleotides at 3′ of one strand (Greider and Blackburn, 1985)
1.2.1.4.4 Topoisomerases
Topoisomerases change the affirmation of covalently closed circular DNA molecule, such as plasmids with the aid of removing the supercoils present in the round DNA molecule and alternate the linking number. It performs a major role in replication and transcription when the DNA unwinds, doing away with positive and negative supercoils (Liu, 1989).
1.2.2 Vectors
A vector is a DNA molecule, which act as molecular transporter, that can replicate autonomously in an appropriate host cell and into which the gene of interest (a foreign gene) is inserted. Insertion of a foreign gene into the vector is aiming either to get numerous copies of the gene of interest or obtaining a product from this gene. Vector is basically of two types: cloning vector and expression vector. Characteristics of an ideal cloning vector are mentioned below:
1 It should have origin of replication, able to replicate autonomously.
2 It should be easily isolated and purified.
3 The vector should have suitable selection marker genes that will allow easy selection of the transformed cells from nontransformed cells.
4 For gene transfer, vector should have the ability to integrate either itself or the DNA insert it carries into the genome of the host cell.
5 The cells transformed with the vector containing the DNA insert should be easily identifiable and selectable from those transformed cells having unaltered vector.
6 Unique restriction digestion sites should be present where gene of interest can be inserted.
Expression vectors are different from cloning vectors. They are designed in such a way that they should have a promoter sequence to express the gene. Expression plasmid has all the information regarding transcription which is followed by translation to synthesize proteins from mRNA. Expression vectors should have the followings gene sequences: A strong promoter for the initiation, termination codon, adaptation of distance between the promoter and cloned gene, incorporated transcription termination sequence, and a compact translation initiation sequence.
Number of cloning vectors are also present which includes plasmid, cosmid, phage vectors, bacterial artificial chromosomes (BACs), and yeast artificial chromosomes (YACs). All the vectors mentioned have different incorporation capacity for foreign DNA. Plasmids are extra chromosomal circular double‐stranded DNA replicating elements present in bacterial cells. Plasmids size is ranging from 5.0 to 400 kb. Plasmids are inserted into bacterial calls by a transformation. Plasmids can incorporate an insert size of up to 10 kb DNA fragment. Bacteriophage infects bacteria and has a very unique mechanism for delivering its genome into bacterial cell. Hence, it can be used as a cloning vector for larger DNA segments insertion. Phage vectors can insert DNA fragments of size up to 20 kb. BACs are simple plasmid vectors that are designed to integrate large DNA fragments of size 75–300 kb (Kim et al., 1996b). BACs have antibiotic resistance marker genes and a very stable OriC that promotes the distribution of plasmid after bacterial cell division and maintaining the plasmid copy number to one or two per cell. YACs are yeast expression vectors. A very large DNA fragments sizes ranging from 100 to 3000 kb can be cloned using YACs (Riley et al., 1990). YACs have an extra benefit over BACs in expressing eukaryotic proteins which need posttranslational modifications.
1.2.3 Incorporation of Modified DNA into Host
After engineering the DNA with the help of restriction enzymes, ligase, and getting it attached to the vector, it is ready to go inside the host cells. How this vector that is a recombinant vector now is made to enter inside the host cells is explained in brief.
1.2.3.1 Introducing Recombinants into Prokaryotes
The most frequently used model organism E. coli has the potential for becoming a host for cloning purposes. A huge number of E. coli vectors are also available. Transformation is the most used and effortless method used to carry the recombinant DNA inside the host cells. There are several other methods some of which are discussed.
1.2.3.1.1 Transformation
Transformation is the most commonly used method in cloning. In this simple technique, the DNA from the surrounding is taken up by the competent cells. Some of the host cells like E. coli, yeast, and mammalian cells are not naturally competent. These cells are made competent chemically so that they can easily take external DNA. Calcium and magnesium chloride in specific concentrations in cold condition can make E. coli cells competent.
1.2.3.1.2 Transduction
Using phage particles for transferring foreign DNA into the host cells is called transduction. Therefore, it is also called as phage‐mediated gene transfer. It can be classified into generalized and specialized transduction. Generalized transduction involves phage particles attacking host cells and as the assembly of phage particles takes place host DNA gets packed inside them. When this phage particle infects another host cell, the DNA gets integrated into the host genome through recombination. While in specialized transduction, the phage genes get integrated with host genome and lysogenic cycle takes place. When some stimulus is given, the phage particles carry more than one genes from the host. These phage particles can infect a naive host cell by which the gene of interest can be inserted into the host genome.
1.2.3.1.3 Conjugation
Conjugation is the procedure of movement of hereditary material from a donor cell to a beneficiary cell when they are in close contact. It was found by Lederberg and Tatum in 1946 who indicated that two distinct strains of microscopic organisms with various development prerequisites could trade qualities. It was found by Lederberg and Tatum in 1946 who indicated that two distinct strains of microscopic organisms with various development prerequisites could trade qualities. They derived that the bacterial cells must cooperate with one another so as to move the genetic material and the procedure is currently known as sexual conjugation by direct contact. A section (not often all) of the donor’s chromosome recombines with the recipient chromosome through homologous recombination. For conjugation the key characteristic is the physical contact. The two cells should be in close proximity. Recipients containing donor DNA are called transconjugants. Genetic exchange through conjugation is unidirectional. Fertility plasmid (F plasmid) plays an important role in conjugation. The donor cells are F+ and have F plasmid, while the recipient is not having F plasmid and is F−.
1.2.3.2 Introducing Recombinants into Eukaryotic Hosts
The usage of bacterial hosts for genetic engineering laid the muse of rDT era; however, researchers have additionally had high‐quality avocation in genetically engineering eukaryotic cells, especially the ones of plant life and animals.
1.2.3.2.1 Transfection
Every other technique to transfer rDNA into host cells includes mixing the foreign DNA with charged substances like calcium phosphate, cationic liposomes, or diethylaminoethyl (DEAE)‐dextran is a polycationic derivative of the carbohydrate polymer dextran and covering on recipient