Biotechnology -Unit 3:Recombinant Technology by Doctor-dr

by Doctor-dr

Biotechnology

Unit 3: Recombinant Technology

Introduction

• One of the few tools that transformed traditional biotechnology into “Modern Biotechnology” is recombinant DNA technology.
• In 1973, a group of scientists led by Paul Berg, Herbert Boyer, Annie Change, and Stanley Cohen created the first recombinant DNA molecule.
• When recombinant human insulin made by bacteria was first sold in the United States in 1982, the field of contemporary biotechnology was born.
• The work that led to this historic event began in the early 1970s, when researchers devised protocols for creating new types of bacterial plasmids or vectors by cutting and pasting pieces of DNA together to create a new piece of DNA (recombinant DNA) that could be inserted into a host bacterium like E. coli.
• We've also discovered that yeasts may be used to make vaccinations like hepatitis B, as well as plants with unique features like disease resistance, insect resistance, and herbicide resistance, and plants with higher nutritional attributes.
• Because of the development of recombinant DNA technology, these outstanding genetic engineering aims have been realised.

Definiton

• Recombinant DNA technology, to put it simply, is the skill of cutting and pasting genes.
• This approach refers to a set of techniques or technologies that allow us to create novel DNA combinations (recombinant DNA or rDNA) in the lab for a variety of reasons.
• The resulting rDNA molecule may then be placed into a suitable host cell, where it can multiply and create numerous copies.
• The technique of gene cloning, also known as DNA cloning, is based on this notion.
• Because of the development of recombinant DNA technology, these outstanding genetic engineering aims have been realised.

Steps in rDNA Technology

The fundamental stages involved in using the rDNA method for gene cloning are as follows:

1. Extract DNA from the organism (e.g., extraction)

2. Use restriction enzymes to cut DNA into appropriate sized pieces.

3. Insert each fragment of DNA into a cloning vector (an artificial DNA molecule capable of replicating in a host organism) by ligating or splicing it together.

4. Convert recombinant DNA (cloning vector + DNA fragment) into a host that can replicate and pass copies on to offspring.

Tools of Recombinant DNA Technology

• Since the process of making a recombinant DNA requires the precise cutting and  stitching of DNA molecules.
• it involves a number of molecular tools—the enzymes to cut and modify the DNA.

The major enzymes required for the making of an rDNA molecule are the following:

1. Type II restriction Endonucleases

2. DNA Ligase

3. Alkaline Phosphatase

1. Restriction Endonucleases

• Restriction enzymes are an essential component of the bacterial defence mechanism.
• Restriction endonucleases are enzymes that prevent bacteriophages from multiplying and multiplying their DNA in bacteria.
• Restriction endonucleases are divided into three categories. Type I, type II, and type III are the three types, each with a somewhat distinct mechanism of action on DNA.
• Recombinant DNA methods employ type II restriction endonucleases because they can detect particular DNA sequences and cleave at a point within those sequences.
• H.O. Smith, K.W. Wilcox, and T.J. Kelley isolated and characterised Hind-II, the first sequence-specific restriction endonuclease from Hemophilus influenzae bacterium, in 1968.
• These restriction endonucleases are known as molecular scissors because they are responsible for transforming DNA molecules into a particular number of pieces by cutting the molecule at specific sequences known as restriction sites.
• Since Hind-discovery, II's more than 3000 restriction enzymes have been identified from over 230 bacterium strains.

Nomenclature of Restriction Endonucleases

• The names of restriction enzymes are usually based on their origin.
• The genus is represented by the first letter of the name.
• The second and third letters refer to the prokaryotic cell species from which they were isolated.
• The fourth letter (if any) denotes the organism's strain.
• The sequence in which the enzymes were extracted from specific bacterium strains is shown by Roman numerals.

Restriction Enzymes

• The word ‘endo' or ‘exo' is added to the name of nucleases to better define them.
• The word 'endonuclease' refers to sequence-specific nucleases that break the phosphodiester link anywhere in the molecule's interior.
• Exonucleases are nucleases that work by hydrolyzing phosphodiester linkages from the molecule's ends.

Many type II Restriction Endonucleases Recognize Palindromic Sequences

• In a 5′–3′ orientation, the palindromic sequences read the same on both strands of DNA.
• The length of these is generally four to eight nucleotides.

Type II restriction enzymes cut DNA and produce two types of fragments

• Hind-II and Sma-I are restriction enzymes that cut DNA symmetrically on both strands at the same nucleotide location, leaving blunt ends.
• Others, on the other hand, create sticky (overhanging or staggered) pieces.

2. DNA Ligase

• This enzyme is responsible for joining two double-stranded DNA fragments by forming a phosphodiester bond between two neighbouring nucleotides.
• The 3′OH group (hydroxyl group at the 3′ carbon) and the 5′ PO4 group of the pentose sugar of neighbouring nucleotides of two distinct DNA fragments or nicks produced in a double-stranded DNA molecule form the phosphodiester linkage.
• DNA ligase is employed in rDNA studies to connect two distinct DNA segments that are annealed by the sticky ends (plasmid/vector and foreign DNA).
• Ligase enzymes come in a variety of forms and come from a variety of sources. However, the T4 DNA ligase generated by T4 bacteriophage is the most commonly utilised.

3. Alkaline Phosphatase

• The problem of self-ligation and the reformation of the original plasmid or vector, which decreases the efficiency of recombinant DNA synthesis, is always present in rDNA investigations.
• Alkaline phosphatase can be used to avoid this.
• The presence of a 3′OH group and a 5′ PO4 group is required for the ligase enzyme to function.
• The DNA ligase cannot operate if any of these groups are missing.
• The phosphate group on the 5′ end of the DNA molecule can be removed by alkaline phosphatase, resulting in a 5′OH group.
• Because the insert DNA includes the 5′PO4 group, the only opportunity of ligation is between the vector and the foreign DNA, resulting in a recombinant DNA molecule.
• Bacterial alkaline phosphatase (BAP) and calf intestinal alkaline phosphatase (CAP) are two enzymes that have been isolated.

Vectors: The Vehicle for Cloning
Vectors act as a vehicle for carrying foreign DNA into a host cell for multiplication.
Usually small circular DNA molecules of bacterial origin are used as cloning  vectors.
A DNA molecule should possess the following essential characteristics to act as a  cloning vector:

• Origin of Replication
• Selectable Markers
• Multiple Cloning Sites (MCS) or Polylinker
• Small Size

Types of Cloning Vectors

There are six different types of cloning vectors commonly used in recombinant DNA experiments. They are the following:

1. Plasmid-cloning vectors
2. Bacteriophage cloning vectors
3. Cosmid-cloning vectors
4. Yeast artificial chromosomes (YACs)
5. Bacterial artificial chromosomes (BACs)
6. Animal and plant vectors (Shuttle vectors)

1. Plasmid-Cloning Vectors

• Plasmid-cloning vectors are the most frequently used, flexible, and easy-to-manipulate vectors, as they are generated from bacterial plasmids.
• Plasmid-cloning vectors like pBR322 are an example.
• This was the first plasmid vector that was widely utilised. It comes with a variety of useful features, including:
• Replication's starting point. It contains a plasmid pMB1 fragment that serves as a replication origin.
• Size. At 4,363 bp, it's rather tiny. In pBR322, there is ‘room' for an insert of at least six kbp.
• Number of copies. A reasonable amount of copies (15 copies per cell)
• Selectable marker. Ampicillin and tetracycline resistance genes are found in it.
• Sites for cloning. It has a few restriction locations that aren't seen anywhere else. Some of these are present in antimicrobial resistance genes (e.g., sites for Pst I is found in Ampr and BamHI and Hind III in Tetr)

• Similarly, cloning and expression vectors from the pUC Series are widely utilised.
• When compared to pBR322, these vectors have three key differences.
• There are a lot of copies. Blue-white screening yields 500 to 600 copies of the plasmid per cell when the origin of replication is mutated.
• A polylinker that has been synthesised. This is a fragment of synthetic DNA with multiple distinct restriction sites.

2. Expression Vectors

• The basic goal of the rDNA experiment and cloning in most situations is to sufficiently multiply or amplify the inserted DNA fragment.
• However, in certain cases, the goal of the procedure is to create huge amounts of recombinant protein encoded by the inserted gene.
• This may be done by including the appropriate regulatory elements in the vector along with the gene.
• Signals for transcription initiation and termination, such as appropriate promoters and terminator sequences, as well as signals for translation initiation, such as a start codon and a ribosome binding site, are included in such vectors upstream of the multiple cloning site (MCS).
• Expression vectors are the names given to these vectors.
• Expression vectors include the pUC series of vectors.

3. Bacteriophage Vectors

• These are the genetically modified bacteriophages that infect E. coli.
• This vector has a 48,514-bp double-stranded linear DNA genome (48.5 kb).
• About 12 unpaired and complementary bases at the extremities of this DNA molecule are sticky or cohesive, and are referred to as cos sites (cohesive end sites).
• During the lytic cycle, these cohesive sites are critical for packaging the DNA into the viral particle.

• The phage vector is constructed in such a manner that a restriction enzyme cuts the central region of the chromosome (linear) and replaces it with foreign DNA digested with the same restriction enzyme.
• To create viral particles, this recombinant DNA is packed in phage heads.
• Infecting E. coli with a 37 to 52 kb insert allows the phage to proliferate.
• Phages need 37 to 52 kb cloned DNA to replicate and create huge amounts of it.

4. Cosmid-cloning Vectors

• Certain plasmid characteristics and the cos site of the phage were used to create cosmid-cloning vectors.
• A plasmid that contains a cos site from the lambda phage genome is known as a cosmid.
• The vector replicates as a plasmid (due to the presence of a ColE1 replication origin) and may carry foreign DNA inserts up to 45 kb in size.
• The most basic cosmid vector contains a ColE1 replication origin, selectable markers like as the antibiotic-resistance gene and the -galactosidase gene, and appropriate polylinker sites and lambda cos sites.

5. Yeast Artificial Chromosomes (YACs)

• Yeast artificial chromosomes (YACs) are vectors that allow foreign DNA segments to be cloned into yeast.
• These are used to clone very big DNA segments ranging from 500 to 1,000 kb in size.
• These vectors have yeast telomere sequences on both ends, yeast centromere sequences, and yeast ARS (autonomously replicating sequences) for replication.
• On each arm, there are also restriction sites for DNA insertion as well as selection indicators like amino acid dependency and antibiotic resistance.

6. Bacterial Artificial Chromosomes (BACs)

• Bacterial artificial chromosomes (BACs) are cloning vectors based on E.coli F factor or fertility factor extrachromosomal plasmids.
• These vectors make it possible to create artificial chromosomes that can be cloned in E.coli.
• This vector may be used to clone DNA segments up to 350 kb and can be handled in the same way as conventional bacterial plasmid vectors.
• BACs include ori sequences generated from E.coli plasmid F factor, multiple cloning sites (MCS) with distinct restriction sites, and appropriate selectable markers, much like any other vector.
• In genome-sequencing initiatives like the Human Genome Project, both YACs and BACs proved extremely valuable.

7. Animal and Plant Vectors (Shuttle Vectors)

• Vectors for transforming plant and animal cells have been created.
• For usage in eukaryotic systems, a variety of vector systems have been created.
• Shuttle vectors are the most frequent of these, as they replicate in both prokaryotic and eukaryotic hosts.
• DNA modifications and characterizations are often carried out in prokaryotic systems before being reintroduced to eukaryotic species for functional investigation.
• The following are some of the typical characteristics of shuttle vectors:
• They can replicate in both prokaryotic and eukaryotic cells and in two or more kinds of hosts.
• They can replicate independently or they can integrate into the host DNA and reproduce when the host cell multiplies.
• Genes are routinely transported from one creature to another using these vectors (i.e., transforming animal and plant cells).

A. Mammalian Vectors

• Shuttle vectors for mammalian tissue culture have been produced.
• These eukaryotic replication sources are usually derived from well-studied mammalian viruses like simian virus 40 (SV-40) with sv-40.
• In 1979, the SV-40 vector was utilised in the first mammalian cell culture cloning experiment.
• Since then, a number of virus-based mammalian vectors have been created to transfer genes in mammals for a variety of applications, including gene therapy.
• Retrovirus-based vectors are the most widely utilised vectors for cloning genes in mammalian cells at the moment.
• In addition to the replication origin, these shuttle vectors contain antibiotic resistance genes that operate in eukaryotic cells (e.g., neomycin (G418) resistance, hygromycin resistance, and so on).

B. Plant Vectors

• For the genetic transformation of plant systems, a number of shuttle vectors have been created.
• The Ti plasmid of Agrobacterium tumifaciens, a bacterium that promotes tumour development in plants, has shown to be the most successful shuttle vector for the plant system.
• This has been used to generate A. tumifaciens-based vectors for plant genetic engineering with great success.

Host Cells

The ultimate aim of the development of recombinant DNA molecules or vectors is  the multiplication of rDNA by cloning in a suitable host cell

There are many types of host cells available for the purpose of cloning

The host cell can be:

• Bacteria
• Yeast
• Plant or
• Animal cells

MAKING RECOMBINANT DNA

1. The extraction and purification of vectors and DNA fragments carrying the gene to be cloned is the first stage in the creation of a recombinant DNA.
2. First, make the vector DNA linear with or without sticky ends by digesting it with an appropriate restriction endonuclease.
3. Then, using the same RE that was used to cut the vector, digest the genome or the donor DNA strand to extract the gene-carrying DNA fragment.
4. In the presence of DNA ligase, the linearized (digested) vector and the target DNA cut with the same restriction enzyme are incubated together.
5. During incubation, the sticky ends of the two DNA strands come closer by the base complementation and become  hydrogen bonded to each other.
6. At this point, a phosphodiester linkage is established between them, mediated by the DNA ligase enzyme.
7. This results in the formation of recombinant DNA molecule of vector and the target DNA.
8. The drawback of this technique is that the sticky ends of the linearized vector may re-anneal, resulting in the creation of the vector itself without any foreign DNA fragment.
9. Any of the following techniques can help to significantly decrease this problem:
10. Using alkaline phosphatase to remove the 5′ phosphate group restriction digestion of the vector and separation of the foreign DNA fragment using two restriction enzymes instead of one (double digestion).
11. As a result, the two sticky endpoints of the vector will not be identical, preventing some re-annealing.

Transgenics

Introduction of recombinant DNA into host cells

After the recombinant DNA molecule has been constructed, it must be delivered into an appropriate host cell for the DNA to proliferate.

The following are some of the most frequent ways to get the rDNA molecule into the host cell:

1. Transformation
2. Transfection
3. Electroporation
4. Microinjection
5. The biolistic method
6. Lipofection

1. Transformation

• The most frequent technique for introducing recombinant DNA into a host cell is transformation.
• Cells take in foreign DNA from their surroundings throughout the transformation process.
• However, many organisms, such as E. coli, yeast, and human cells, are unable to spontaneously take up DNA from their surroundings.
• Certain chemical treatments can improve a cell's ability to absorb foreign DNA or make the cell transformation-ready.
• When E. coli cells are incubated in a cold calcium chloride solution, for example, they become more competent.

2. Transfection

• Another technique for transforming cultivated cells is transfection.
• In this technique, DNA (recombinant vector) is combined with charged substances like calcium phosphate, cationic liposomes, or DEAE dextran and then over-layered on host cells.
• External DNA is eventually taken up by these host cells as a result of this process.

3. Electroporation

• An electric current is utilised to produce temporary tiny holes in the cell membrane of the host cell during the electroporation procedure.
• Foreign DNA enters the cell through these brief holes.
• This technique works well with yeast, mammalian cells, and plant protoplasts in particular.

4. Microinjection

• Microinjection is a specialised technique for directly injecting DNA fragments or genes into the nucleus of plant and animal cells.
• The method entails injecting the DNA directly into the nucleus of the host cell using a glass microinjection tube or syringe.
• The microinjection equipment comprises of a high-magnification microscope with fine needles, syringes, and other accessories to carry out the automated delivery of DNA into the chosen cells.

5. Biolistic Method

• With the aid of a gene or particle cannon, the biolistic technique was created for delivering foreign DNA into plant cells.
• During the biolistic technique, high-velocity bombardment of tiny gold or tungsten particles coated with the desired DNA is used.
• The particle is bombarded with the assistance of a mechanical instrument known as a gene cannon.

6. Lipofection

• It's utilised to change all kinds of cells.
• Liposomes, which are tiny lipid vesicles, are used to transport DNA. The liposome fuses with a portion of the host cell membrane, allowing the contents - fresh DNA - to enter the cells.
• A liposome is a spherical lipid bilayer encapsulating targeted DNA.
• Endocytosis occurs when PEG is present.
• The DNA is free to recombine and integrate into the host genome after endocytosis.

Identification of Recombinants

• Only a small fraction of the overall cell population takes the recombinant DNA throughout the genetic transformation process.
• As a result, having an effective screening strategy for selecting altered cells is critical.
• Various screening approaches are based on the expression or lack thereof of some of the characteristics contained in the vector or in combination with the cloned gene.
• Antibiotic resistance is one of these characteristics.
• It is extremely straightforward to select recombinant transformants directly on a medium treated with the relevant antibiotic if the antibiotic-resistance gene is present together with the cloned gene.

• In most of the cases there are two stages of selection
• Selection of transformed cells (i.e., the cells that have taken a plasmid)
• Identify the transformed cells that have the recombinant plasmid

Stage 1

• The positive selection approach or the negative selection method can be used to identify transformed cells that contain a plasmid.
• Antibiotic resistance genes included in cloning vectors are the most prevalent indicators utilised for positive selection.
• By immediately plating the transformation products on antibiotic plates, all untransformed bacteria die, leaving only those with the plasmid encoding the antibiotic-resistance marker gene to thrive and form colonies.
• We will be able to obtain transformed cells with plasmids using this selection approach.However, distinguishing transformed cells or colonies with recombinant plasmids from transformed cells or colonies with parent plasmids without any insert DNA fragment is impossible (non-recombinant vector)

Stage 2

• For detecting the presence of recombinant vector among transformed cells, a negative-selection technique can be used.
• The multiple cloning site in the vector is designed in such a manner that the insertion of a foreign DNA strand interrupts the expression of a selectable flag gene—a process known as insertional inactivation—in order to identify those plasmids containing a foreign DNA fragment.
• For negative selection, two types of selectable markers are utilised.
• One is the insertional inactivation of antibiotic resistance
• the second is the insertional inactivation of an enzymatic activity

A. Insertional inactivation of antibiotic resistance

B. Insertional inactivation of enzymatic activity

• This system is based on the insertional inactivation of lac Z gene
• The lac Z gene is a component of the lac operon and it produces an enzyme β-galactosidase
• This enzyme can cleave a colorless, synthetic substrate, X-gal into a blue-colored product
• If the β-galactosidase gene (lac Z gene) is disrupted and inactivated by inserting the foreign  DNA fragment into it, X-gal will not be cleaved and the development of the blue color will  be prevented
• Thus, the selection medium is provided with the X-gal and the host cells that carry normal  vectors (without insert DNA) will produce blue-colored colonies, and those cells which carry  the recombinant plasmids will develop white-colored colonies
• Here, the positive selection (expression of antibiotic resistance) and the negative selection  (inactivation of an enzyme activity—β-galactosidase) can be combined together and carried  out in a single experiment