- 1 Role of Recombinant DNA Technology to Improve Life – Introduction to Genetic Engineering and Its Applications
- 1.1 History of Recombinant DNA Technology
- 1.2 Nature of Genetic Engineering
- 1.3 Tools of Recombinant DNA Technology
- 1.4 1. Restriction Endonuclease
- 1.5 Discovery of DNA Technology
- 1.6 Naming of Restriction Endonucleases
- 1.7 Types of Restriction Endonucleases
- 1.8 Cutting Sites of Restriction Endonucleases
- 1.9 2. DNA Ligase
- 1.10 3. DNA Polymerase
- 1.11 4. Taq Polymerase
- 1.12 5. Reverse Transcriptase
- 1.13 6. End-Modifying Enzymes
- 1.14 7. Vectors
- 1.15 A. Structure of a Plasmid Vector
- 1.16 B. Cosmid
- 1.17 C. Fosmid (F-based cosmid) vector
- 1.18 D. Shuttle Vector
- 1.19 E. Artificial Chromosome
- 1.20 F. Ti (Tumor Inducing) Plasmid
- 1.21 G. P element as Vector
- 1.22 I. Transcription vector
- 1.23 J. Host cell
Evolution is one of the Biology Topics that has been debated and studied for centuries, exploring the process by which species change over time.
Role of Recombinant DNA Technology to Improve Life – Introduction to Genetic Engineering and Its Applications
A gene is the fundamental physical and functional unit of heredity that carries information from one generation to the next. It is a specific sequence of nucleotides in DNA (in certain viruses also RNA) determining either the nucleotide sequence of a functional RNA (tRNA, rRNA, etc.) or the amino acid sequence of a functional polypeptide. Purposefully changing of structure of a gene in an organism is called genetic engineering. Jack Williamson used the term genetic engineering in his novel science fiction ‘Dragon’s Island’.
Recombinant DNA technology is the multitude of techniques for the in vitro recombination of two or more DNA molecules from different organisms. DNA having an inserted gene or DNA from an exogenous source is called recombinant DNA. Recombinant DNA allowed scientists to create a novel DNA sequence by ligating two or more nonhomologous DNA molecules, and genetic engineering was started with the production of recombinant DNA.
The desired DNA sequence (cloned DNA, insert DNA, target DNA, or foreign DNA) from a donor organism is extracted, enzymatically cleaved (cut, or digested), and joined (ligated) to another DNA entity (a cloning vector) to form a new, recombined DNA molecule (cloning vector-insert DNA construct, or DNA construct). This cloning vector-insert DNA construct is transferred into and maintained within a host cell. The introduction of DNA into a bacterial host cell is called transformation. Those host cells that take up the DNA construct (transformed cells) are identified and selected (separated, or isolated) from those that do not.
|1. Cloning||Production of many genotypically similar organisms, e.g., the production of Dolly, the sheep in 1996.|
|2. Glow in the dark cat||In 2007, scientists in South Korea developed such a cat by altering its DNA so that the furs of the cat glow in the dark. This cat produced several glowing cats.|
|3. Cows that pass out less gas||Cow generally produces much methane. Methane is a greenhouse gas responsible for global warming. With the help of genetic engineering such cows have been developed, that pass less methane gas.|
|4. Plants that reduce pollution||The scientists of Washington University have produced such popular plants that can absorb polluted water through their roots.|
|6. Environment friendly pig||With the help of genetical engineering such pigs could be developed that can metabolize more phosphorus and therefore, excrete less phosphorus.|
|7. Faster growing trees||Genetically engineered plants show more growth and therefore, they are very useful for forest development.|
|8. Better and long-lasting tomato||The genetically engineered tomatoes are larger in size and long-lasting.|
|9. Salmon showing higher growth||Scientists developed transgenic salmon fish that grow bigger than their natural brothers.|
|10. Pest resistant crop||Pests cause great damage to agricultural crops. But the newly developed Bt crops are pest resistant.|
|11. Banana vaccine||Such vaccine-producing banana trees could be produced and their produced vaccines may be stored in bananas. Bananas of such trees if taken by a man, may develop resistance to diseases like cholera and hepatitis.|
After a single recombinant DNA molecule, composed of a vector plus an inserted DNA fragment, is introduced into a host cell, the inserted DNA is replicated along with the vector, generating a large number of identical DNA molecules. The term gene cloning refers to the phenomenon of making many copies of a gene or replica of genes. If required, a DNA construct can be created so that the protein product encoded by the cloned DNA sequence is produced in the host cell. If a cloned gene is flanked by the properly positioned control sequences for transcription and translation, the host may also produce large quantities of the RNA and protein specified by that gene.
Recombinant DNA technology was developed from discoveries in molecular biology, nucleic acid enzymology, and the molecular genetics of both bacterial viruses (bacteriophages) and bacterial extrachromosomal DNA elements (plasmids). Recombinant DNA cloning procedure. DNA from a source organism is cleaved with a restriction endonuclease and inserted into a cloning vector. The cloning vector-insert (target) DNA construct is introduced into a host cell, and those cells that carry the construct are identified and grown. If required, the cloned gene can be expressed (transcribed and translated) in the host cell, and the protein (recombinant protein) can be harvested.
History of Recombinant DNA Technology
During the middle of the 20th century, when the nature and structural architecture of genetic material was revealed, scientists started thinking about genetic engineering. The discovery of restriction endonuclease during the 1960s by Warner Arber and Hamilton Smith made the path clear for genetic engineering.
To date, almost 6,000 different restriction endonuclease enzymes have been isolated, and more than 1000 are currently available for use in the laboratory. Four different classes of restriction endonuclease are recognized, each distinguished by a slightly different mode of action. Types I and III are rather complex and have only a limited role in genetic engineering, whereas type II restriction endo- & nucleases are the cutting enzymes that are so important in gene cloning. With the help of type II restriction endonuclease a desired gene from a large DNA molecule may be dissected out and inserted into another DNA molecule.
In 1970, Howard Temin and David Baltimore discovered an enzyme called reverse transcriptase in retroviruses whose genetic material is RNA. This RNA after its introduction in the host cell may produce a complementary DNA using reverse transcriptase. This complementary DNA is then integrated into the host genome leading to the multiplication of the viral genome in the host cell.
Paul Berg is regarded as the father of recombinant DNA technology. Berg founded recombinant DNA technology when he created a piece of recombinant DNA by joining together (splicing) DNA from the E. coli chromosome and DNA from a primate virus called SV40 (simian virus 40). Berg first isolated chromosomal DNA from E. coli and DNA from SV40. Then he cut both DNA samples with EcoRI, added E. coli DNA and viral DNA fragments to a reaction tube with the enzyme DNA ligase, and succeeded in creating a hybrid molecule of SV40 and E. coli DNA. The importance of this discovery was fully recognized when Paul Berg won the 1980 Nobel Prize in Chemistry for this experiment, which demonstrated that DNA could be cut from different sources with the same enzyme and that the restriction fragments could be joined to create a recombinant DNA molecule.
Herbert Boyer and Stanley Cohen isolated different fragments of DNA from animals, other bacteria, and viruses and, using restriction enzymes, ligated the fragments into a small plasmid from E. coli. This was the first recombinant DNA made. Finally, they transformed the engineered plasmid back into E. coli. The cells expressed the normal plasmid genes as well as those inserted into the plasmid artificially. This sparked the revolution in genetic engineering.
Nature of Genetic Engineering
The principal purpose of genetic engineering is to achieve of genetic alteration. Any organism that contains a foreign DNA sequence (gene or promoter) in its chromo¬some, its organellar genomes, or its plasmids, introduced by indirect or direct gene transfer techniques is called a genetically modified organism (GMO). In this technology, the genome of an organism is plan-wise modified using recombinant DNA technology. Such genetically modified organisms may express the gene obtained from exogenous sources and show improved characteristics like pest-resistant or high-yielding crops. The objective of genetic engineering is the cloning of a desired DNA or gene or the production of a desired product from a GMO. The steps of genetic engineering are,
- Identification of a gene of interest.
- Isolation of DNA from the organism which carries the desired gene.
- Isolation of desired genes from the DNA of the organism.
- Production of multiple copies of the gene using PCR.
- Insertion of the gene into some organism by a suitable vector to produce a transgenic organism.
- Employment of transgenic organism for production of desired product (gene or protein).
- Collection of the substance of interest from the transgenic organism.
The steps involved in fulfillment of the aims and objectives of genetic engineering in order to get a successful genetic engineering may be shown by the following flow chart.
The restriction enzyme EcoRI recognizes and cleaves the sequence 5′-GAATTC-3′. E. coli R strains, from which this enzyme is derived, protect their own DNA from fragmentation by also producing a specific methylase. EcoRI restriction enzyme is unable to cleave the methylated DNA.
Tools of Recombinant DNA Technology
The success of recombinant DNA technology depends on the discovery of several components chemical by nature. These components may be called helping tools of recombinant DNA technology. A description of several such tools used in DNA technology may be given below.
1. Restriction Endonuclease
In the bacterial cell, one type of enzyme can cut the foreign DNA (introduced into the cell) into pieces. These Restriction enzymes belong to the class of enzymes nucleases that break nucleic acids by clearing their phosphodiester bonds.
With the help of these enzymes, the bacteria may prevent the growth of the virus into the bacterial cell. Such enzymes are called restriction endonucleases. Commonly known as Molecular Scissors. The term ‘endonuclease’ applies to sequence-specific nucleases that break nucleic acid chains somewhere within the DNA, rather than at the ends of the molecule.
Various restriction endonucleases are available in different bacterial strains and each enzyme may recognize specific nucleotide sequences of DNA to cleave the DNA into pieces at this sequence of DNA. However, similar nucleotide sequence in the bacterial own DNA is not cleaved because the host genome remains methylated. From different species of bacteria, about 6000 restriction endonucleases have been isolated and more than 1000 are currently available for use in the laboratory.
Restriction enzymes belong to a larger class of enzymes called nucleases (hydrolytic enzymes that cleave nucleic acids; DNase cleaves DNA molecules whereas RNase cleaves RNA molecules). These are of two kinds; exonucleases and endonucleases. Exonucleases remove nucleotides from the ends (either 5’ or 3’) of the DNA whereas, endonucleases make cuts at specific positions within the DNA.
Discovery of DNA Technology
Warner Arber, Hamilton Smith, and Daniel Nathan discovered restriction endonuclease and for this, they were awarded with Nobel Prize in 1975.
Blunt End and Sticky End: Endonuclease cleaving of DNA produces two types of cleaving ends in the DNA. One type is known as blunt end and the other type is known as sticky end. Many restriction endonucleases make a simple double-stranded cut in the middle of the recognition sequence, which results in a blunt end or a flush end. PvuII and Alul are examples of blunt end cutters. Other restriction endonucleases cut DNA in a slightly different way. With these enzymes, the two DNA strands are not cut at exactly the same position but instead, the cleavage is staggered, usually by two or four nucleotides, so that the resulting DNA fragments have short single-stranded overhangs at each end. These are called sticky ends or cohesive ends, as base pairing between them can stick the DNA molecule back together again.
One important feature of sticky end enzymes is that restriction endonucleases with different recognition sequences may produce the same sticky ends. BamHI (recognition sequence GGATCC) and BgIII (AGATCT) are examples, both of which produce GATC sticky ends. The same sticky end is also produced by Sau3A, which recognizes only the tetranucleotide GATC.
Fragments of DNA produced by cleavage with either of these enzymes can be joined to each other by the DNA ligase, as each fragment carries a complementary sticky end. Also, two DNA molecules cleaved by the same restriction enzyme may produce such sticky ends that they may get complementary bases at the cut ends, and therefore, the pieces of DNA molecules may combine together by DNA ligase.
Naming of Restriction Endonucleases
The naming of restriction endonuclease follows a rule when the first letter of this name comes from the first letter of bacterial genera, the next two letters come from the name of the species and the rest of the name represents the strain of the bacterium. For example, EcoRI has come from E. coli RY 13, Hind III from Haemophilus influenzae Rd strain.
A List of Type II Restriction Endonucleases
|Enzyme||Recognition Site||Number of Bases||Ens Generated||Original Source of Enzyme|
|EcoRI||G/AATTC||6||5′ sticky||Escherichia coli RY13|
|BamHI||G/GATCC||6||5′ sticky||Bacillus amyloliquefaciens H|
|BgIII||A/GATCT||6||5′ sticky||Bacillus globigii|
|Pstl||CTGCA/G||6||3′ sticky||Providencia stuartii|
|Xmal||C/CCGGG||6||5′ sticky||Xanthomonas malvacearum|
|Acc65I||G/GTACC||6||5′ sticky||Acinetobacter calcoacetius 65|
|Kpnl||GGTAC/C||6||3′ sticky||Klebsiella pneumonia|
|Sau3A||/GATC||4||5′ sticky||Staphylococcus aureus 3A|
|Notl||GC/GGC-CGC||8||5′ sticky||Nocardia otitidis-caviarum|
|Pacl||TTAAT/TAA||8||3′ sticky||Pseuomonas alcaligenes|
Types of Restriction Endonucleases
Restriction endonuclease may be of four different types designated as type I, II, III, and IV. Only type II restriction enzymes are used in recombinant DNA technology. The nature and properties of different types of restriction endonuclease may be shown in the following table.
Cutting Sites of Restriction Endonucleases
The sequence of bases in the DNA that is recognized by a restriction endonuclease is known as the restriction sequence. This restriction site is a sequence of 4-8 bases. Sometimes two or more such enzymes may recognize the same restriction sequence. These enzymes are called isos- rhizomes. The restriction sites usually contain twofold symmetry and the sites having such twofold symmetry are palindromes that have similar sequences in the reverse direction. A verbal palindrome, for example, ‘radar’, reads the same from left to right as from right to left. The two strands of DNA lie in opposite directions.
When one writes restriction site ‘palindrome’, GAATTC, he is looking at the sequence of the ‘top’ strand, which runs 5′ to 3′ when read from left to right – so one should write the sequence as 5’GAATTC3′. On the ‘bottom’ (complementary) strand, one has to read from right to left to see the 5′ to 3′ sequence, which would also be 5’GAATTC3′. Different restriction enzymes isolated from different sources often recognize the same DNA sequence, although they may cleave the DNA differently. Such enzymes are called isoschizomers of each other.
2. DNA Ligase
This enzyme helps in ligating the DNA fragments produced by restriction endonuclease. Ligases join nucleic acid molecules together with the help of ATP molecules.
They join DNA molecules together by synthesizing phosphodiester bonds between nucleotides at the ends of two different molecules, or at the two ends of a single molecule. That is why DNA Ligase is commonly known as Molecular glue. Breaking and joining DNA using restriction enzymes and DNA ligase. Linear DNA (insert) and a closed-circular plasmid DNA (vector) each contain the recognition site for BamHI and EcoRI. Mixing the DNA fragments with compatible ends together in the presence of DNA ligase can result in the formation of vector-insert hybrid DNA molecules.
3. DNA Polymerase
DNA polymerases are hydrolytic enzymes that synthesize new polynucleotides complementary to an existing DNA or RNA template. They make copies of DNA molecules.
4. Taq Polymerase
A bacterium named Thermus aquaticus that lives in hot springs uses this enzyme for its DNA replication. This enzyme is heat stable and remains active even at 94°C. In PCR, Taq polymerase is used for DNA replication. This enzyme has an optimum working temperature of 720C.
5. Reverse Transcriptase
The reverse transcriptase is an RNA-dependent DNA polymerase and so makes DNA copies of RNA rather than DNA templates. Reverse transcriptases are involved in the replication cycles of retroviruses, including the human immunodeficiency viruses that cause acquired immune deficiency syndrome, or AIDS, These viruses (HIV) have RNA genomes that are copied into DNA after infection of the host. In the test tube, reverse transcriptase can be used to make DNA copies of mRNA molecules. These copies are called complementary DNAs (cDNAs). Their synthesis is important in some types of gene cloning and in techniques used to map the regions of a genome that specify particular mRNAs.
6. End-Modifying Enzymes
End-modifying enzymes remove or add chemical groups, make changes to the ends of DNA molecules, and provide one means of labeling DNA molecules with radioactive and other markers. Terminal deoxynucleotidyl transferase, obtained from calf thymus tissue, is one example of an end-modification enzyme. It is a template-independent DNA polymerase because it is able to synthesize a new DNA polynucleotide without base-pairing of the incoming nucleotides to an existing strand of DNA or RNA. Terminal deoxynucleotidal transferase (TdT) catalyzes the addition of deoxynucleotides to the 3′-ends of DNA molecules. Besides the above-mentioned enzymes recombinant DNA technology used also some other enzymes in the technology. A list of several such enzymes has been shown in the following table.
Vector is used for introducing foreign DNA into a cell. It is usually a self-replicating small circular DNA that has the capability to bind with foreign DNA. With the help of a vector, a desired DNA may be introduced into a host cell, and inside the host cell, the gene or foreign DNA may be amplified. This is known as cloning and the vector is called a cloning vector. Several cloning vectors used in recombinant DNA technology are plasmid, cosmid, bacteriophage, artificial chromosome, etc. All the vectors have several common features making them ideal tools used in cloning such as,
Characteristics of Vectors
- Small size, making them easy to manipulate once they are isolated.
- Can be used to transfer gene(s) easily from cell to cell (usually by transformation).
- Easy to isolate from the host organism.
- Easy to detect and select because of the presence of marker gene(s) (e.g., genes conferring antibiotic resistance).
- Must contain a replication origin.
- Multiple copies in a cell, help in obtaining large amounts of DNA.
- Clustered restriction sites (polylinker) to allow insertion of cloned DNA.
- Method to detect the presence of inserted DNA (e.g., alpha complementation).
Different Restriction Endonucleases and Their Nature
|Type||Nature of Composition||Mode of Action||Example|
|I||Composed of many protein sub-units.||DNA is cleaved at the site far away from the recognition site.||Hsd M|
|II||Associated with cluster of protein subunit. More used in molecular Biology.||Usually, DNA is cleaved at the site of recognition. However, some enzymes bring about cleavage at the nearer position of the recognition site.||Bcg I, BP II|
|III||Having changeable character.||Cleavage occurs outside the restriction site. It acts on the DNA that contains two restriction sites.||Eco P15|
|IV||Need Mg2+ for activity.||Usually cuts methylated DNA.||Mcr Bc|
A. Structure of a Plasmid Vector
In certain cases, the bacterial cell contains one or more extrachromosomal small circular double-stranded autonomously replicating DNA which is known as a plasmid. The size of a plasmid may vary from 1 kb – 500 kb and in a bacterial cell there may be 1-4 plasmids. However, in certain cases, a bacterial cell may contain 10-100 plasmids. Plasmid DNA constitutes about 0.5-5% of the total DNA content of a bacterial cell. Typically, a plasmid contains one replication origin, a marker gene, and other genes like tra (required for bacterial conjugation), etc. In most of the cases marker gene confers antibiotic resistance to a bacterial strain and because of this a plasmid-containing bacterium may easily be recognized. When treated with the antibiotic, only bacteria with the plasmid-encoded resistance gene will survive. Other bacteria will die. Other traits have been exploited to detect plasmids.
Owing to the presence of replication origin, the plasmid is self-replicating. Plasmids vary in their copy number. Some plasmids exist in just one or a few copies in their host cells, whereas others exist in multiple copies. Such multicopy plasmids are in general more useful as the amount of plasmid DNA is higher, making them easier to isolate and purify. The type of origin of replication controls the copy number since this region on the plasmid determines how often DNA polymerase binds and induces replication.
The bacterial plasmid used for gene cloning is called a plasmid cloning vector. To make an efficient cloning vector, a bacterial plasmid is made to carry several unique restriction enzyme sites. Usually, these sites are grouped in one location called the multiple cloning site (MCS) or polylinker. This allows researchers to open the cloning vector at one site without disrupting any of the vector’s replication genes. Fragments of foreign DNA are digested with enzymes matching those in the polylinker. Ligase connects the vector and insert.
Except this, the plasmid vector also contains ampicillin resistance, a marker gene and because of this, the bacteria with this plasmid vector can grow in a medium containing ampicillin. Some vectors have ways to detect whether or not they contain an insert. The simplest way to do this is insertional inactivation of an antibiotic gene. Here, the vector has two different antibiotic resistance genes. The foreign DNA is inserted into one of the antibiotic-resistant genes. Thus, the host bacteria will be resistant to one antibiotic and sensitive to the other.
Sometimes, a lacZ gene is inserted into the plasmid. The gene lacZ produces beta-galactosidase enzyme and if in the medium X-gal is present, the bacterium with beta-galactosidase enzyme cleaves X-gal to produce a blue colour. Within the lacZ gene a restriction site is present and cleaving at this site with a suitable enzyme helps to insert foreign DNA into the plasmid. Such a cloning plasmid vector with an exogenous DNA fragment inserted into its lacZ cannot produce functional beta-galactosidase anymore. Thus, the plasmid vector carrying lac Z and ampR is helpful for gene cloning.
With the help of restriction endonuclease, the plasmid DNA is cleaved along its restriction site and then a foreign DNA is ligated with it with the help of DNA ligase before its introduction into the bacterial cell. By converting a bacterial cell into a competent one the external recombinant plasmid vector (containing an inserted DNA fragment) may be introduced into the competent bacterial cell. If an E. coli cell is kept in 100 mM CaCl2 solution for 30 minutes, the cell becomes competent and at this stage, the cell can uptake plasmid from the external medium.
pBR322: An ideal plasmid vector should have the capacity to uptake DNA of at least 15 kb size. The first cloning vector was pSC101 obtained from E. coli cell. This plasmid vector contained a restriction endonuclease EcoRI cutting site and a tetracyclin-resistant gene. However, widely used plasmids were constructed in the latter periods. One such constructed plasmid vector is pBR322.
The name “pBR322” conforms with the standard rules for vector nomenclature:
- “p” indicates that this is a plasmid.
- “BR” identifies the laboratory in which the vector was originally constructed (BR stands for Bolivar and Rodriguez, the two researchers who developed pBR322).
- “322” distinguishes this plasmid from others developed in the same laboratory (there are also plasmids called pBR325, pBR327, pBR328, etc.). The plasmi pBR322 was the first widely used plasmid vector. pBR322 is a small plasmid (4363 bp) that was constructed using components from naturally occurring bacterial plasmids and other DNA fragments.pBR322 contains the following components.
- Origin of replication: pBR322 carries the ColEl replication origin and rop gene to ensure a reasonably high plasmid copy number (15-20 copies per cell), which can be increased 200-fold by chloramphenicol amplification.
- Antibiotic resistance genes: pBR322 carries two genes that can be used as selectable markers: the ampicillin resistance gene (termed bla or, more commonly, AMPR) and the tetracycline resistance gene (termed tet or TETR). They help in the identification and elimination of non-recombinants and selectively permit the growth of recombinants.
- Cloning sites: The plasmid carries a number of unique restriction enzyme recognition sites. Some of these are located in one or other of the antibiotic-resistance genes. For example, sites for PstI, PvuI, and SacI are found within AMPR, and sites for BamHI and Hind III are located within TETR.
The antibiotic resistance genes in pBR322 allow for the direct selection of recombinants in a process called insertional inactivation. For example, if one wants to clone a DNA fragment into the BamHI site of pBR322, then the insert DNA will interrupt the gene responsible for tetracycline resistance, but the gene for ampicillin resistance will not be altered. Transformed cells are first grown on bacterial plates containing ampicillin to kill all the cells that do not contain a plasmid. Those cells that grow on & ampicillin are then replica plated onto a medium containing both ampicillin and tetracycline. Those cells that grow in the presence of ampicillin, but die under tetracycline selection, contain plasmids that have foreign DNA inserts. In other words, the insertion of a foreign DNA fragment into an antibiotic resistance gene inactivates the gene product and leads to antibiotic sensitivity.
pUC19: Another cloning vector was pUC19 made at the University of California (“p” indicates that this is a plasmid and “UC” denotes the University of California). pUC8 was one of the most popular E. coli cloning vectors. pUC8 contains a mutation, within the origin of replication, which results in the plasmid having a copy number of 500-700 even before amplification. This has significantly increased the yield of cloned DNA obtainable from E. coli cells transformed with recombinant pUC8 plasmids.
The plasmid also contains the ampicillin resistance gene (ampR) and the lacZ’ gene, which encodes the first 63 amino acids of lacZ – the α-peptide. Embedded within the coding sequence of lacZ’ are the recognition sites for a number of restriction enzymes. This multiple cloning site (or polylinker) is used to clone DNA fragments. The presence of insert DNA will disrupt the function of the lacZ a-peptide and is used for screening (alpha complementation).
The pUC19 plasmid vector has a short portion of the β-galactosidase gene (the alpha fragment), and the bacterial chromosome has the rest of the gene. If both gene fragments are transcribed and then translated into proteins, the partial proteins combine to form functional β-galactosidase. If DNA is inserted into the plasmid-borne gene segment, the encoded subunit is not made and β-galactosidase is not produced. When β-galactosidase is expressed, the bacteria can degrade X-gal, which turns the bacterial colony blue. If a piece of DNA is inserted into the alpha fragment gene, the bacteria cannot split X-gal and they stay white.
The advantage of using pUC19 is that identification of recombinant cells can be achieved by a single-step process, by plating onto agar medium containing ampicillin plus X-gal. With pBR322 selection of recombinants is a two-step procedure, requiring replica plating from one antibiotic medium to another. A cloning experiment with pUC19 can therefore be carried out in half the time needed with pBR322. Another advantage of pUC19 lies with the clustering of the restriction sites, which allows a DNA fragment with two different sticky ends (say EcoRI at one end and BamHI at the other) to be cloned without resorting to additional manipulations such as linker attachment.
Long DNA fragments can be cloned using a cosmid vector which is a hybrid between a lambda phage DNA molecule and a bacterial plasmid. A cosmid contains an origin of replication, bacterial gene(s) specifying antibiotic resistance, and the bacteriophage lambda cohesive ends (cos sites) that enable the in vitro packaging of recombinant DNA molecules with a minimum size of 38kb, and a maximum size of 52kb. A cos site (cohesive site; cohesive end site) is a 12bp long, cohesive, single-stranded projection at each 5′ end of double-stranded, linearized λ-DNA molecules. Cosmids are used to clone large DNA fragments (e.g., for the construction of eukaryotic gene libraries) and are usually present as 20-70 copies per cell. pLR5 is an example of cosmid.
C. Fosmid (F-based cosmid) vector
This F-factor-based cosmid cloning vector is designed in the form of an F-plasmid and it contains an origin sequence and polylinkers. Such a vector may integrate about 40 kb of DNA within it and this vector is less in number within the host cell.
D. Shuttle Vector
This type of vector may function in both eukaryotes and prokaryotes. A shuttle vector (bifunctional vector) is a plasmid cloning vector containing two different origins of replication, which allow its selection and autonomous replication in two different organisms (e.g. S. cerevisiae and E. coli, or A. tumefaciens and E. coll). For example, yeast episomal plasmid variant pJDB219 replicates in both E. coli and Saccharomyces cerevisiae.
E. Artificial Chromosome
An artificial chromosome (mini chromosome) is a plasmid shuttle vector that is engineered to replicate autonomously both in organism A (prokaryote, e.g. E.coli) and organism B (eukaryote, e.g. Saccharomyces cerevisiae), and functions in a eukaryotic host as a chromosome (i.e. replicates autonomously, segregates in mitosis and meiosis). Bacterial artificial chromosome (BAC), yeast artificial chromosome (YAC), and human artificial chromosome (FLAC) are three cloning vectors produced artificially. These vectors contained marker genes and replication origin. The artificial chromosome may insert larger DNA fragments for introduction into the host cell.
BAC is produced in the form of F-plasmid and it is larger than a plasmid. BAC allows to clone DNA fragments of more than 300kb (average size: 150kb). The BAC is composed of the E. coli plasmid pMBO 131, carrying a chloramphenicol resistance gene, Hind III and Bam HI cloning sites, a bacteriophage lambda cos site, and a lox P site.
YAC is a high-capacity 11.5kb plasmid cloning vector, replicating both in E. coli and yeast. Such a chromosomal vector contains a centromere and each arm of the chromosome contains a marker gene. Larger DNA fragments (0.2-2.0 Mb) may be cloned with this type of vector.
HAC was constructed in 1997 and it is about 1/10 – 1/15th of a human chromosome. It is a high-capacity cloning vector, assembled de novo and containing human centromere DNA and telomere repeats that can be introduced and maintained in human cells as a mitotically stable autonomous mini chromosome. HACs are equipped with selectable marker genes, functioning in mammalian cells. HAC helps in introducing larger DNA fragments and it is suitable for gene therapy.
F. Ti (Tumor Inducing) Plasmid
Plants do not contain any naturally occurring plasmid DNA molecules. However, the tumour-inducing (Ti) plasmid of the soil microorganism Agrobacterium tumefaciens, has been used extensively as a suitable vector to introduce genes into plant cells.
Gram-negative soil bacterium Agrobacterium tumefaciens is a bacterium that has evolved a natural genetic engineering system; it contains a segment of DNA that is transferred from the bacterium to plant cells and is responsible for crown gall disease. The name refers to the galls or tumors that often form at the crown (junction between the root and the stem) of infected gymnosperms and dicotyledonous angiosperms. After the infection of a wound site by A. tumefaciens, two key events occur:
- The plant cells begin to proliferate and form tumors
- They begin to synthesize an arginine derivative called an opine.
The opines produced by the infected plant, e.g., nopaline, which is formed through the reductive condensation of arginine and α-ketoglutarate, can be used by Agrobacterium cells as their sole source of carbon and energy.
Other soil bacteria are unable to metabolize opines, and thus these molecules serve to promote the growth of more Agrobacterium cells and may provide a selective advantage to Agrobacterium over competition from other microorganisms present in the soil. The opine synthesized is used as an energy source by the infecting bacteria.
The ability of Agrobacterium tumefaciens to cause crown gall disease is associated with the presence of the Ti (tumor-inducing) plasmid within the bacterial cell. This plasmid carried within the bacterium is responsible for its ability to cause crown gall disease. The tumour-inducing (Ti) plasmid of Agrobacterium tumefaciens is large (-200 kbp) and carries a number of genes that are required for the infection process. After infection, part of the Ti plasmid called the T-DNA, becomes integrated into the plant genome at an apparently random position through non-homologous recombination.
T-DNA, approximately 23 kbp in size, contains not only the genes responsible for the cancerous properties of the transformed cells (e.g., those controlling the production of the plant hormones auxin and cytokinin to stimulate cell division and growth) but also those responsible for the synthesis of opines, which are amino acid derivatives. If T-DNA is replaced by the desired gene in the plasmid, the gene of interest may be introduced into the plant cell. The T-DNA segment contains both a transgene and a selective marker or reporter gene.
In practice, Agrobacterium is used to transfer genes of interest into plants using tissue culture. Either protoplasts or a piece of callus are cultured with Agrobacterium harboring a Ti plasmid with modified T-DNA. After coculture, the plant cells are harvested and incubated with the herbicide or antibiotic used as the selectable marker. This kills all the cells that were not transformed with T-DNA or failed to express the genes on the T-DNA. The transformed plant cells are then induced to produce shoot and root tissue by altering the hormone conditions in the medium. The small transgenic plants can then be screened for transgene expression levels.
G. P element as Vector
P elements are DNA-only transposons in Drosophila species. P elements move by a conservative (cut-and-paste) transposition mechanism. The transposition activity of P elements is strictly limited to germ-line cells. P elements can be used as gene transfer vectors to carry DNA fragments of interest into the Drosophila germ line. Any foreign DNA can be cloned into a P element which in turn can be inserted into a plasmid. After microinjection of this plasmid into Drosophila embryos the P element together with the foreign DNA can transpose into germline chromosomal DNA leading to the formation of a transgenic Drosophila.
H. Expression vector
Transgene is any gene that originates from one and has been transferred to a second organism (cell). A transgenic animal is an animal whose nuclear genome contains foreign DNA (e.g. genes) that has been transferred by transfection or direct gene transfer. Following the entry of the vector with transgene in a host cell, the transgene may be expressed there. An expression vector is specifically constructed so as to achieve efficient transcription of the cloned DNA fragment and translation of its mRNA (expression).
Transgene may be transcribed and mRNA produced from the transgene may be translated in the host cell by the expression vector. An expression vector contains sequences upstream of the cloned gene that control transcription and translation of the cloned gene. An expression vector should contain a strong inducible promoter, a multiple cloning site for the insertion of target genes, and a transcriptional terminator. Additionally, a ribosome binding site (RBS) is included to promote efficient translation. Examples, are pGEX-3X plasmid, and Baculovirus.
I. Transcription vector
Sometimes transgene tagged with vector after entry within the host cell can only be transcribed without its expression. Transcription vectors may only be used for the production of copies of a gene. So this type of vector is also called a cloning vector.
J. Host cell
A host cell is required for in vivo cloning of genes along with the vectors. The vector introduces the gene into the host cell and in the host cell, the vector multiplies increasing the number of genes. Among the prokaryotes, E. coli is the most popular host cell but sometimes Bacillus subtilis is also used as the host cell. Among the eukaryotes, Saccharomyces cerevisiae is mostly used as the host cell. In some cases, mammalian cells are also used as a eukaryotic host. Aside from the components mentioned above some instruments are also used in gene technology. Several such tools are a gene gun (for introducing genes in the cell), a PCR machine (for in vitro gene cloning), and an electrophoresis apparatus (for separating DNA fragments and proteins of variable sizes).