RECOMBINANT DNA TECHNOLOGY

This field of biotechnology encompases a number of methodologies that enable new combination of genetics material to be artificially construted in laboratory.

In other words

Recombinant DNA technology involves using enzymes and various laboratory techniques to manipulate and isolate DNA segments of interest. This method can be used to combine DNA from different species or to create genes with new functions. The resulting copies are often referred to as recombinant DNA.

rDNA

Recombinant DNA is a form of the artificial DNA that is made through the combination or insertion of one or more DNA strands, therefore combining DNA sequences as per your requirement.

DICOVERY

·       Discovery of DNA structure- 1953 watson and crick.

·       Isolation of DNA ligase- 1967 (for the formation of phosphodiester bond with the help of ATP).

·       Isolation of restriction endonucleases around- 1970.

·       Formation of recombinant DNA – 1972.

·       First human protein made by using rDNA technology- Somatostatin in E.coli.

·       First plasmid vector capable of replicate within host 1973.

RECOMBINANT DNA TECHNOLOGY GOALS

·       To isolate and characterize a gene.

·       To make desired and alternation in one or more isolated gene.

·       Transfer transgene into living cells.

·       Artificially synthesize new gene.

·       Understanding the heriditory disease & the cure.

·       Improving human genome.

 

 

 

 

 

 

 

 

 

 

Steps involved in rDNA experiment-

 

Identification of desired gene

 

 

 

 

Isolation of desired gene with the help of restriction enzyme

 

 

 

 

The vector cut with theb same restriction enzyme

Vector and isolated gene is ligated together with help of DNA

Amplification and transfer of desired gene into the progeny

The recombinant vector is introduce into the host cell

 

 

 

 

 

 

 

 

 

 

 

 

 

 

                         

 

                                          

 

 

 

 

 

 

 

Gene cloning

The basic steps in a gene cloning experiment are follows.

 

 

1.      A fragment of DNA containing the gene to be cloned, is inserted into a circular DNA molecule called vector, to produce a chimera or recombinant DNA molecule.

2.      The vector act as  vehicle that transport  the gene into a host cell, in which is usually a bacterium, although other types of living can be used.

3.      Within the host cell

what s gene and their definition

 

GENE

Genes are unit of inheritance. These are located on chromosome in linear form and carried information from parents to offspring by gametes. Gene control the expression of characters. Gene was first visualize by MENDEL and called it factor. The word gene introduce by Johannsen  in 1909. In a simple language gene is a segment of DNA with specific sequence of nucleotide which has coded information for specific character or for specific protein.

TYPES OF GENES

1.   House-keeping genes-

Active in every organisms and all time (example glycolysis)

2.   Luxury genes- Inactive for most of the time and expressed in certain cells or at certain time only when their products are needs.

3.   Structure genes-These  genes code for substances which are needed for the morphological or functional traits of the cell

4.   Control genes-Positive and negative control on functioning of structural genes.     

a)     Regulator genes

b)     Operator genes

c)     Promoter genes

d)     Terminator genes

 

5.   Interrupted gene and discontinuous genes-

                I.          Split genes

             II.          Introns

6.   Alternative genes- These are form different discontinuous genes get connected forming several new combinations.

7.     Overlapping genes- In overlapping sequence code for more than two different proteins. Its means same specific nucleotide sequence is shared in the transcription of two different mRNA for the synthesis of two non-homologous proteins

8.   Transposons genes- jumping genes (from one position to another position in in the genome of a cell)

9.   Smart genes- Artificially constructed genetic molecules composed of one or more genes code for functional protein or enzymes.

 

NUCLEIC ACID {DNA AND RNA}

 History of Nucleic Acid

Nucleic acid was first discovered by Friedrich Miescher in 1869. He gave its first name as nuclein. In the early 1880s Albrecht Kossel further purified the substance and discovered its highly acidic properties. He later also identified the nucleobases.

WHAT IS NUCLEIC ACID

Nucleic acids are biopolymers, macromolecules, essential to all known forms of life. They are composed of nucleotides, which are the monomers made of three components: a 5-carbon sugar, a phosphate group and a nitrogenous base. The two main classes of nucleic acids are deoxyribonucleic acid and ribonucleic acid.

5-Carbon sugar--    Pentose is a monosaccharide (simple sugar) with five carbon atoms.

Phosphate group--  Phosphate groups are attached to the 5' carbon of a nucleotide and 3'                                            carbon of adjacent nucleotide in a DNA strand.

Nitrogenous base-- 

purines- (Adenine (A) and Guanine (G))

pyrimidine- (Cytosine (C) and Thymine (T)) 

These nitrogenous bases are attached to C1' of deoxyribose through a glycosidic bond. Deoxyribose attached to a nitrogenous base is called a nucleoside.

Types of nucleic acid--

There are two types of nucleic acids: DNA and RNA

1. DNA--deoxyribonucleic acid
2. RNA--Ribonucleic acid 

1.  DNA 

1.  DNA, or deoxyribonucleic acid, is the hereditary material in humans and almost all other organisms. Nearly every cell in a person’s body has the same DNA
2. Most DNA is located in the cell nucleus (where it is called nuclear DNA), but a small amount of DNA can also be found in the mitochondria (where it is called mitochondrial DNA or mtDNA)

2.  RNA

1. RNA- A polymer of ribonucleotides, is a single stranded structure. 
2. There are three major types of RNA- m RNA,t RNA and r RNA.

STRUCTURE OF DNA--

1. The structure of DNA is illustrated by a right handed double helix, with about 10 nucleotide pairs per helical turn 
2. Each spiral strand, composed of a sugar phosphate backbone and attached bases, is connected to a complementary strand by hydrogen bonding (noncovalent) between paired bases, adenine (A) with thymine (T) and guanine (G) with cytosine (C).

https://www.nature.com/scitable/content/ne0000/ne0000/ne0000/ne0000/104573282/74---Copy_1_2.jpg

DNA DENATURATION 

• DNA denaturation is the separation of a double strand DNA molecule into two single strands DNA, which occurs when the hydrogen bonds will be breaks present between the double strand of DNA This process occurs when dsDNA expose by heat, change in pH and some chemicals etc.

 DNA REPLICATION

DNA replication is the process where DNA makes same copies of itself . Replication occurs at the rate of between 50 nucleotide per second ( in mammals ) and 500 nucleotides per second ( in bacteria). Watson and crick give this mechanism basis on their double helical structure Each strand of DNA serves as a template on which its complementary strand is synthesised . This method of DNA replication is described as semi - conservative , because each daughter DNA molecule is a hybrid conserving one parental polynucleotide strand and the other one newly synthesised strand.

Important Point of Replication Process-

1. The base pairing during DNA replication, i.e., adenine pairs with thymine and guanine with cytosine.
2. Nucleotide monomers are added one by one to the 3 ' end of the growing strand by the enzyme DNA polymerase. 
3. The sequence of nitrogenous bases in each daughter strand being formed is complementary to the base sequence in the template strand. 
4. C - 3 ' carbon of deoxyribose present on the 3 ' end of the new polynucleotide chain of DNA has OH group and is free to bind to another nucleotide . C - 5 ' carbon of deoxyribose on the 5 ' end of polynucleotide chain has a phosphate . Therefore , the new polynucleotide chain is always synthesized in 5 ' → 3 ' direction.

Requirements for DNA Replication

1. Precursor nucleotide molecules- dNTPs, dATPs, dGTPs, dCTPs, dTTPs.
2. Template DNA 
3. RNA Primer
4. Enzyme-DNA polymerase, Ligase, helicases.
5. Proteins-DNaA, SSB protein

Enzyme immobilization techniques

              Enzyme immobilization techniques 

What is enzyme immobilization?

• imprisonment of cell or enzyme in a distinct support(matrix).
• The support/matrix allows change of medium.
• The medium contain substrate or inhibitor molecule.
• First immobilization technology : amino acylases by aspergillus oryzae for the production of L-amino acid japan.
• The main advantages of enzyme immobilization:    

       1.increase functional efficiency 

       2. enhanced reproducibility 

  Advantages-

• Reuse
• Continuous use 
• minimum reaction time 
• less chance of contamination 
• more stability 
• improve in process control 
• high enzyme substrate ratio

Disadvantages-

• loss of catalytic properties in sometime 
• some enzyme become unstable 
• some enzyme are inactivated by heat generation in system 
• high cost for isolation, purification and recovery of active enzyme 

Application-

• Industrial production: Eg. Antibiotics, beverage ,amino acid etc 
• Biomedical application: treatment, diagnosis and drug delivery .
• Food industry: Production of jams, jellies, and syrup.
• Research: blotting experiment, proteases for cell lysis 
• Waste water treatment: treatment of sewage & industrial effluents

    Support/matrix-

• The enzyme hold in support/matrix 
• The should be cheap and easy available 
• Their reaction with medium and enzyme should be minimums as possible 
• A wide range of matrix are used in immobilization of enzyme/ cell 
• Matrix categories into 3 grouped 

1.  Natural polymers
2. Synthetic polymer 
3. Inorganic polymer 

Types of immobilization-

1. Adsorption 
2. Covalent bonding 
3. Entrapment 
4. Copolymerization 
5. Encapsulation 

       (1).  Adsorption-

• Oldest method of enzyme immobilization 
• Simplest method of enzyme immobilization 
• Nelson and Griffin used charcoal to adsorb invertase 
• Enzyme are adsorbed to external surface of support 
• Support/ carrier may be ;

1. Mineral support (aluminum oxide, clay)
2. Organic support (starch)
3. Modified sephareose and ion exchange  resins               

     

Methods of adsorption ;

1.static process-

immobilization to be perform by the using solution which containing enzyme to contact with the carrier (without stirring)

2.dynamic batch process-

carrier is placed in a enzyme solution and mixed by using agitation 

3.Reactor loading process-

when enzyme solution is transfer to reactor the carrier will already present in the reactor 

4.Electrode position process-

 carrier is placed proximal to an electrode in enzyme bath and the current is put on , the enzyme migrate to the carrier and deposition on the surface 

Advantages-

• Easy to carry out 
• no reagent are required 
• minimum activation step is involve 
• comparatively cheap method 
• less disruptive to protein than chemical method 

Disadvantages-

• desoption of enzyme from the carrier 
• efficiency is less 

2.Covalent bonding;

• In this process involve the formation of covalent bond between the enzyme and support/ matrix
• widely used method for enzyme immobilization 
• chemical group in enzyme that form covalent bonds with support are;

1. Amino acid 
2. Hydroxyl group 
3. carboxyl group 
4. Thiol group and methyl group 
5. Guanidyl group and imidazole group 
6. phenol rings 

Matrix used for covalent bonding-

• Carbohydrates -cellulose, DEDE cellulose , agarose 
• synthetic agent - polyacrylamide 
• protein carrier 
• amino group bearing carrier -amino benzyl cellulose 
• inorganic carrier- porous glass , silica 
• cyanogen bromide (CNBr) - agarose and CNBr sepharose 
• hydroxyl and amino group form covalent bond more easily 

Methods of covalent bonding 

1. Diazoation ;bonding between amino groups of support and thyrosil or histidyl groups of enzyme 

2. Peptide bond;bonding between carboxyl /amino groups of support and enzyme 

3. Poly functional reagent ;use of bi-functional or multifunctional reagent (glutaradehyde) which form bonding between the amino group of the support and amino group of the enzyme

3. Entrapment-

• Enzyme are physically entrapped inside the support 
• Bonds are involve in entrapment such as covalent and non-covalent bond
• matrix used will be water soluble polymer like-

1. polyacrylamide gels 
2. cellulose triacetate 
3. agar 
4. gelatin
5. alginate 

• Form and nature of matrix varies 
• pore size of matrix is adjusted to prevent loss of enzyme 
• agar have larger pore sizes 
• easy to practice at small scale 

Method of entrapment-

1.Inclusion in the gels- enzyme are trapped in gels 

2.Inclusion of fibers- enzyme support in fiber format

3.Inclusion in microcapsules - enzyme are trapped in microcapsules formed by the using monomer mixture such as polyamine, calcium alginate .

Advantages-

• This is fast method and cheap compare to another method of immobilization 
• Mild condition are required 
• No conformation change in this method 

Disadvantages-

• chance of microbial contamination 
• leakage of enzyme 

4. Cross linking or co-polymerization ;

• In this method involve covalent bonding between various groups of enzyme via poly-functional reagent 
• there are not involve matrix or support
• Commonly used poly-functional reagents are -Glutaradehyde , Diazonium salt 

 5. Encapsulation 

• enzyme are enclosed in semi-permeable membrane capsule 
• capsule is made up of nitro cellulose or nylon 
• effectiveness depends on the stability of enzymes 

Advantages-

• Simple and cheap method , large quantity of enzyme can immobilized by the using this method 

Disadvantages-

• Pore size is limitation 
• only small size of substrate is able to pass through the membrane 

Enzymes

                                              Introduction-

• Enzymes are work as a biological catalysts that increase the rate of reaction(biochemical reaction). 
• Mainly enzymes are in 3D structure globular proteins(tertiary and quaternary structure)
• Enzyme are macromolecular biological catalysts, enzyme accelerate or catalyze chemical reaction.
• During the reaction substrate bind with the enzyme at active site of enzyme , converts into different molecule called products.
• microbial enzyme are the biological catalysts for the biolchemical reaction leading to microbial growth and respiration ,as well as formation of fermentation products. 

Structure of enzyme-

  The active site of an enzyme is a region that binds substrates ,co-factors and prosthetic groups. Active site is less than 5% of the total surface area of enzyme.active site have tertiary structure of proteins.

active site further divided into two types 

Binding site- it choose the substrate and binds to active sites 

Catalytic site- it performs the catalytic action of enzyme 

Characteristics;

• Enzyme increase the rate of reaction 
• The presence of enzyme does not effects the nature and properties of end products
• Small amount of enzyme accelerate the chemical reaction 
• Enzyme are sensitive to change in pH, temp, and substrate concentration\.