Biomolecular Engineering Solutions for Renewable Specialty Chemicals. Группа авторов

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      1.2.1.3 Ligases

      Ligases are considered to be molecular glue in rDT, used to join two DNA segments. It also has role in various aspects of molecular biology such as replication, recombination, and cloning. In the presence of ligase enzyme when two DNA fragments are mixed under a certain condition, base pairing between two fragments occur which results in sealing of two different DNA fragments to make a chimera (Pascal et al., 2004). It occurs due to covalent bonds formation between 2′‐PO₄ group and 3′‐OH group of adjustment strands.

       1.2.1.3.1 Mechanism of Action

The DNA ligation reaction has two main steps. First, the DNA ends collide with each other by chance and reside together for enough time for the ligase to join them. For sticky overhangs, there is an additional reason; lower temperature stabilizes the hydrogen bonding between the complementary nucleotides, which really helps to join easily. DNA ligase catalyzes the joining of the 3′‐OH to the 5′‐phosphate via a two‐step process. First, the AMP nucleotide, which is linked to a lysine residue in the enzyme’s active site, is shifted to the 5′‐phosphate. Then the AMP‐phosphate bond is attacked by the 3′‐OH, forming the covalent bond and discharging AMP. To allow the enzyme to conduct further reactions, the AMP in the enzyme’s active site must be restocked by ATP. The DNA ligase enzyme has optimal activity at 25 °C so the ligation reaction is carried out at a temperature range of 16–25 °C. Normally, 1 hour at 16 °C is best for ligation but since bringing the DNA ends together is the least efficient part of the reaction favoring this by lowering the temperature to 4 °C can give even more efficiency. Two types of ligases are there that are used in genetic engineering. E. coli ligases which is generally used to join two sticky or cohesive ends. It is mainly used to catalyze a phosphodiester bond between duplex DNA‐containing cohesive ends. It will not efficiently ligate blunt‐ended fragments as much as it efficient for sticky end ligation. NAD⁺ is required as a cofactor for the ligation reaction. T4 DNA ligase that is generally used for the ligation of blunt‐ended DNA molecules. This enzyme can join blunt‐ended termini as well as ends with cohesive overhangs (either 3′ or 5′ complementary overhangs). This enzyme will also function for the repair of single‐stranded nicks in duplex DNA, RNA, or DNA/RNA duplexes. ATP is required as a cofactor for the ligation reaction. All the reactions are generally carried out at 16 °C. Sometimes PEG (polyethylene glycol) can be added that helps in fusion and increases the frequency of collision between two DNA molecules.

      1.2.1.4 DNA‐modifying Enzymes

       1.2.1.4.1 Alkaline Phosphatase

      Alkaline phosphatase prevents self‐ligation of DNA molecule (vectors and gene of interest) in genetic engineering experiment by dephosphorylating phosphate group on 3′ end of the DNA molecule. It is extracted from E. coli or calf intestine. The enzymes catalyze the hydrolysis of monoesters in phosphoric acid which can moreover catalyze a trans‐phosphorylation reaction with large concentrations of phosphate acceptors. It can be used to prevent self‐ligation of vectors in the cloning experiments because alkaline phosphate‐treated DNA fragments lack the 5′‐phosphophate in the terminal, required for the actions of DNA ligases (Tamás et al., 2002).

       1.2.1.4.2 T4 Poly Nucleotide Kinase

Polynucleotide kinase enzyme is exact reverse of alkaline phosphatase. It adds phosphate group to on the 5′ end of the DNA molecule and is extracted from phage‐infected E. coli. The reaction with polynucleotides can be made reversible, which consents the exchange of the γ‐phosphate of ATP with the 5′‐terminal phosphate of a polynucleotide, thus entangled the need of dephosphorylation the substrate DNA molecule with alkaline phosphatase (Cameron and Uhlenbeck, 1977).

       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 СКАЧАТЬ