Introduction to Vectors
In order to study a DNA fragment (e.g., a gene), it needs to be amplified and eventually purified. These tasks are accomplished by cloning the DNA into a vector. A vector is generally a small, circular DNA molecule that replicates inside a bacterium such as Escherichia coli (can be a virus).
Properties of Good Vectors
1. Replicate autonomously (relaxed control so that it can generate multiple copies of itself in a single host)
2. Easy to isolate and purify.
3. Easily introduce into the suitable host cells.
4. Should have suitable marker genes that allow easy detection and selection of transformed host cell.
5. Should integrate itself or carry insert DNA to the genome of the host.
6. The cells transformed with the vectors containing the DNA insert (Chimaeric vector) should be identifiable or selectable from those transformed by the vector molecules only.
7. Should contain unique target sites for as many restriction enzymes as possible into which DNA insert can be integrated without disrupting essential functions.
8. For expression the vector should contain at least suitable control elements e.g. promoter, operator and ribosome binding sites.
NOTE: vector should have coevolved with their specific natural host species. Therefore choice of vectors depends upon the host species into which DNA to be inserted. And most naturally occurring vectors don’t have all the required functions therefore useful vectors have been created by joining segments performing specific functions called Modules.
Cloning and Expression Vectors
Vectors used for propagation of insert DNA in a suitable host are called Cloning vectors.
Vectors designed for the expression of or production of the protein specified by the DNA insert is termed as Expression vectors.
Properties of a GOOD HOST
1. Easy to transform
2. Support replication of recombinant DNA
3. Lack active restriction enzymes e.g. E coli K12 substrain HB101
4. Free from element that interfere with the replication of recombinant DNA
5. Have no methylasae (cause methylation and producing resistant to useful restriction enzymes)
6. Deficient in normal recombination function so that the DNA insert is not altered b y recombination events.
E coli as good host (supports the vectors plasmid, bacteriophage, cosmids, phasmids and shuttle vectors)
Six different types of cloning vectors:
1. Plasmid cloning vectors
2. Phage l cloning vectors
3. Cosmid cloning vectors
4. Shuttle vectors
5. Yeast artificial chromosomes (YACs)
6. Bacterial artificial chromosomes (BACs)
Three most commonly used types of vectors:
1) Plasmid vectors (e.g., pUC plasmids);
2) Bacteriophage vectors (e.g., phage l); and
3) Phagemid vectors (e.g., pBlueScriptTM).
Plasmid
A plasmid is an extra-chromosomal DNA molecule separate from the chromosomal DNA which is capable of replicating independently of the chromosomal DNA. In many cases, it is circular and double-stranded. Plasmids usually occur naturally in bacteria, but are sometimes found in eukaryotic organisms (e.g., the 2-micrometre-ring in Saccharomyces cerevisiae).
Plasmid size varies from 1 to over 200 kilobase pairs (kbp). The number of identical plasmids within a single cell can be zero, one, or even thousands under some circumstances. Plasmids can be considered to be part of the mobilome, since they are often associated with conjugation, a mechanism of horizontal gene transfer.
The term plasmid was first introduced by the American molecular biologist Joshua Lederberg in 1952.
Plasmids can be considered to be independent life-forms similar to viruses, since both are capable of autonomous replication in suitable (host) environments. However the plasmid-host relationship tends to be more symbiotic than parasitic (although this can also occur for viruses, for example with Endoviruses) since plasmids can endow their hosts with useful packages of DNA to assist mutual survival in times of severe stress. For example, plasmids can convey antibiotic resistance to host bacteria, who may then survive along with their life-saving guests who are carried along into future host generations.
• 1 Vectors
• 2 Gene Therapy
• 3 Types
• 4 Plasmid DNA extraction
• 5 Conformations
• 6 Simulation of plasmids
Vectors
There are two types of plasmid integration into a host bacteria: Non-integrating plasmids replicate as in the top instance; whereas episomes, the lower example, integrate into the host chromosome.
Plasmids used in genetic engineering are called vectors. Plasmids serve as important tools in genetics and biotechnology labs, where they are commonly used to multiply (make many copies of) or express particular genes.[2] Many plasmids are commercially available for such uses.
The gene to be replicated is inserted into copies of a plasmid containing genes that make cells resistant to particular antibiotics and a multiple cloning site (MCS, or polylinker), which is a short region containing several commonly used restriction sites allowing the easy insertion of DNA fragments at this location. Next, the plasmids are inserted into bacteria by a process called transformation. Then, the bacteria are exposed to the particular antibiotics. Only bacteria which take up copies of the plasmid survive the antibiotic, since the plasmid makes them resistant. In particular, the protecting genes are expressed (used to make a protein) and the expressed protein breaks down the antibiotics. In this way the antibiotics act as a filter to select only the modified bacteria. Now these bacteria can be grown in large amounts, harvested and lysed (often using the alkaline lysis method) to isolate the plasmid of interest.
Another major use of plasmids is to make large amounts of proteins. In this case, researchers grow bacteria containing a plasmid harboring the gene of interest. Just as the bacteria produces proteins to confer its antibiotic resistance, it can also be induced to produce large amounts of proteins from the inserted gene. This is a cheap and easy way of mass-producing a gene or the protein it then codes for, for example, insulin or even antibiotics.
However, a plasmid can only contain inserts of about 1-10 kbp. To clone longer lengths of DNA, lambda phage with lysogeny genes deleted, cosmids, bacterial artificial chromosomes or yeast artificial chromosomes could be used.
One way of grouping plasmids is by their ability to transfer to other bacteria. Conjugative plasmids contain so-called tra-genes, which perform the complex process of conjugation, the transfer of plasmids to another bacterium (Fig. 4). Non-conjugative plasmids are incapable of initiating conjugation, hence they can only be transferred with the assistance of conjugative plasmids, by 'accident'. An intermediate class of plasmids are mobilizable, and carry only a subset of the genes required for transfer. They can 'parasitize' a conjugative plasmid, transferring at high frequency only in its presence. Plasmids are now being used to manipulate DNA and may possibly be a tool for curing many diseases.
It is possible for plasmids of different types to coexist in a single cell. Seven different plasmids have been found in E. coli. But related plasmids are often incompatible, in the sense that only one of them survives in the cell line, due to the regulation of vital plasmid functions. Therefore, plasmids can be assigned into compatibility groups.
Another way to classify plasmids is by function. There are five main classes:
• Fertility-F-plasmids, which contain tra-genes. They are capable of conjugation.
• Resistance-(R)plasmids, which contain genes that can build a resistance against antibiotics or poisons. Historically known as R-factors, before the nature of plasmids was understood.
• Col-plasmids, which contain genes that code for (determine the production of) bacteriocins, proteins that can kill other bacteria.
• Degradative plasmids, which enable the digestion of unusual substances, e.g., toluene or salicylic acid.
• Virulence plasmids, which turn the bacterium into a pathogen.
Plasmids can belong to more than one of these functional groups.
Plasmids that exist only as one or a few copies in each bacterium are, upon cell division, in danger of being lost in one of the segregating bacteria. Such single-copy plasmids have systems which attempt to actively distribute a copy to both daughter cells.
Some plasmids include an addiction system or "postsegregational killing system (PSK)", such as the hok/sok (host killing/suppressor of killing) system of plasmid R1 in Escherichia coli.[5] They produce both a long-lived poison and a short-lived antidote. Daughter cells that retain a copy of the plasmid survive, while a daughter cell that fails to inherit the plasmid dies or suffers a reduced growth-rate because of the lingering poison from the parent cell.
Conformations
Plasmid DNA may appear in one of five conformations, which (for a given size) run at different speeds in a gel during electrophoresis. The conformations are listed below in order of electrophoretic mobility (speed for a given applied voltage) from slowest to fastest:
• "Nicked Open-Circular" DNA has one strand cut.
• "Relaxed Circular" DNA is fully intact with both strands uncut, but has been enzymatically "relaxed" (supercoils removed). You can model this by letting a twisted extension cord unwind and relax and then plugging it into itself.
• "Linear" DNA has free ends, either because both strands have been cut, or because the DNA was linear in vivo. You can model this with an electrical extension cord that is not plugged into itself.
• "Supercoiled" (or "Covalently Closed-Circular") DNA is fully intact with both strands uncut, and with a twist built in, resulting in a compact form. You can model this by twisting an extension cord and then plugging it into itself.
• "Supercoiled Denatured" DNA is like supercoiled DNA, but has unpaired regions that make it slightly less compact; this can result from excessive alkalinity during plasmid preparation. You can model this by twisting a badly frayed extension cord and then plugging it into itself.
The rate of migration for small linear fragments is directly proportional to the voltage applied at low voltages. At higher voltages, larger fragments migrate at continually increasing yet different rates. Therefore the resolution of a gel decreases with increased voltage.
At a specified, low voltage, the migration rate of small linear DNA fragments is a function of their length. Large linear fragments (over 20kb or so) migrate at a certain fixed rate regardless of length. This is because the molecules 'reptate', with the bulk of the molecule following the leading end through the gel matrix. Restriction digests are frequently used to analyse purified plasmids. These enzymes specifically break the DNA at certain short sequences. The resulting linear fragments form 'bands' after gel electrophoresis. It is possible to purify certain fragments by cutting the bands out of the gel and dissolving the gel to release the DNA fragments.
Because of its tight conformation, supercoiled DNA migrates faster through a gel than linear or open-circular DNA.
In order to study a DNA fragment (e.g., a gene), it needs to be amplified and eventually purified. These tasks are accomplished by cloning the DNA into a vector. A vector is generally a small, circular DNA molecule that replicates inside a bacterium such as Escherichia coli (can be a virus).
Properties of Good Vectors
1. Replicate autonomously (relaxed control so that it can generate multiple copies of itself in a single host)
2. Easy to isolate and purify.
3. Easily introduce into the suitable host cells.
4. Should have suitable marker genes that allow easy detection and selection of transformed host cell.
5. Should integrate itself or carry insert DNA to the genome of the host.
6. The cells transformed with the vectors containing the DNA insert (Chimaeric vector) should be identifiable or selectable from those transformed by the vector molecules only.
7. Should contain unique target sites for as many restriction enzymes as possible into which DNA insert can be integrated without disrupting essential functions.
8. For expression the vector should contain at least suitable control elements e.g. promoter, operator and ribosome binding sites.
NOTE: vector should have coevolved with their specific natural host species. Therefore choice of vectors depends upon the host species into which DNA to be inserted. And most naturally occurring vectors don’t have all the required functions therefore useful vectors have been created by joining segments performing specific functions called Modules.
Cloning and Expression Vectors
Vectors used for propagation of insert DNA in a suitable host are called Cloning vectors.
Vectors designed for the expression of or production of the protein specified by the DNA insert is termed as Expression vectors.
Properties of a GOOD HOST
1. Easy to transform
2. Support replication of recombinant DNA
3. Lack active restriction enzymes e.g. E coli K12 substrain HB101
4. Free from element that interfere with the replication of recombinant DNA
5. Have no methylasae (cause methylation and producing resistant to useful restriction enzymes)
6. Deficient in normal recombination function so that the DNA insert is not altered b y recombination events.
E coli as good host (supports the vectors plasmid, bacteriophage, cosmids, phasmids and shuttle vectors)
Six different types of cloning vectors:
1. Plasmid cloning vectors
2. Phage l cloning vectors
3. Cosmid cloning vectors
4. Shuttle vectors
5. Yeast artificial chromosomes (YACs)
6. Bacterial artificial chromosomes (BACs)
Three most commonly used types of vectors:
1) Plasmid vectors (e.g., pUC plasmids);
2) Bacteriophage vectors (e.g., phage l); and
3) Phagemid vectors (e.g., pBlueScriptTM).
Plasmid
A plasmid is an extra-chromosomal DNA molecule separate from the chromosomal DNA which is capable of replicating independently of the chromosomal DNA. In many cases, it is circular and double-stranded. Plasmids usually occur naturally in bacteria, but are sometimes found in eukaryotic organisms (e.g., the 2-micrometre-ring in Saccharomyces cerevisiae).
Plasmid size varies from 1 to over 200 kilobase pairs (kbp). The number of identical plasmids within a single cell can be zero, one, or even thousands under some circumstances. Plasmids can be considered to be part of the mobilome, since they are often associated with conjugation, a mechanism of horizontal gene transfer.
The term plasmid was first introduced by the American molecular biologist Joshua Lederberg in 1952.
Plasmids can be considered to be independent life-forms similar to viruses, since both are capable of autonomous replication in suitable (host) environments. However the plasmid-host relationship tends to be more symbiotic than parasitic (although this can also occur for viruses, for example with Endoviruses) since plasmids can endow their hosts with useful packages of DNA to assist mutual survival in times of severe stress. For example, plasmids can convey antibiotic resistance to host bacteria, who may then survive along with their life-saving guests who are carried along into future host generations.
• 1 Vectors
• 2 Gene Therapy
• 3 Types
• 4 Plasmid DNA extraction
• 5 Conformations
• 6 Simulation of plasmids
Vectors
There are two types of plasmid integration into a host bacteria: Non-integrating plasmids replicate as in the top instance; whereas episomes, the lower example, integrate into the host chromosome.
Plasmids used in genetic engineering are called vectors. Plasmids serve as important tools in genetics and biotechnology labs, where they are commonly used to multiply (make many copies of) or express particular genes.[2] Many plasmids are commercially available for such uses.
The gene to be replicated is inserted into copies of a plasmid containing genes that make cells resistant to particular antibiotics and a multiple cloning site (MCS, or polylinker), which is a short region containing several commonly used restriction sites allowing the easy insertion of DNA fragments at this location. Next, the plasmids are inserted into bacteria by a process called transformation. Then, the bacteria are exposed to the particular antibiotics. Only bacteria which take up copies of the plasmid survive the antibiotic, since the plasmid makes them resistant. In particular, the protecting genes are expressed (used to make a protein) and the expressed protein breaks down the antibiotics. In this way the antibiotics act as a filter to select only the modified bacteria. Now these bacteria can be grown in large amounts, harvested and lysed (often using the alkaline lysis method) to isolate the plasmid of interest.
Another major use of plasmids is to make large amounts of proteins. In this case, researchers grow bacteria containing a plasmid harboring the gene of interest. Just as the bacteria produces proteins to confer its antibiotic resistance, it can also be induced to produce large amounts of proteins from the inserted gene. This is a cheap and easy way of mass-producing a gene or the protein it then codes for, for example, insulin or even antibiotics.
However, a plasmid can only contain inserts of about 1-10 kbp. To clone longer lengths of DNA, lambda phage with lysogeny genes deleted, cosmids, bacterial artificial chromosomes or yeast artificial chromosomes could be used.
One way of grouping plasmids is by their ability to transfer to other bacteria. Conjugative plasmids contain so-called tra-genes, which perform the complex process of conjugation, the transfer of plasmids to another bacterium (Fig. 4). Non-conjugative plasmids are incapable of initiating conjugation, hence they can only be transferred with the assistance of conjugative plasmids, by 'accident'. An intermediate class of plasmids are mobilizable, and carry only a subset of the genes required for transfer. They can 'parasitize' a conjugative plasmid, transferring at high frequency only in its presence. Plasmids are now being used to manipulate DNA and may possibly be a tool for curing many diseases.
It is possible for plasmids of different types to coexist in a single cell. Seven different plasmids have been found in E. coli. But related plasmids are often incompatible, in the sense that only one of them survives in the cell line, due to the regulation of vital plasmid functions. Therefore, plasmids can be assigned into compatibility groups.
Another way to classify plasmids is by function. There are five main classes:
• Fertility-F-plasmids, which contain tra-genes. They are capable of conjugation.
• Resistance-(R)plasmids, which contain genes that can build a resistance against antibiotics or poisons. Historically known as R-factors, before the nature of plasmids was understood.
• Col-plasmids, which contain genes that code for (determine the production of) bacteriocins, proteins that can kill other bacteria.
• Degradative plasmids, which enable the digestion of unusual substances, e.g., toluene or salicylic acid.
• Virulence plasmids, which turn the bacterium into a pathogen.
Plasmids can belong to more than one of these functional groups.
Plasmids that exist only as one or a few copies in each bacterium are, upon cell division, in danger of being lost in one of the segregating bacteria. Such single-copy plasmids have systems which attempt to actively distribute a copy to both daughter cells.
Some plasmids include an addiction system or "postsegregational killing system (PSK)", such as the hok/sok (host killing/suppressor of killing) system of plasmid R1 in Escherichia coli.[5] They produce both a long-lived poison and a short-lived antidote. Daughter cells that retain a copy of the plasmid survive, while a daughter cell that fails to inherit the plasmid dies or suffers a reduced growth-rate because of the lingering poison from the parent cell.
Conformations
Plasmid DNA may appear in one of five conformations, which (for a given size) run at different speeds in a gel during electrophoresis. The conformations are listed below in order of electrophoretic mobility (speed for a given applied voltage) from slowest to fastest:
• "Nicked Open-Circular" DNA has one strand cut.
• "Relaxed Circular" DNA is fully intact with both strands uncut, but has been enzymatically "relaxed" (supercoils removed). You can model this by letting a twisted extension cord unwind and relax and then plugging it into itself.
• "Linear" DNA has free ends, either because both strands have been cut, or because the DNA was linear in vivo. You can model this with an electrical extension cord that is not plugged into itself.
• "Supercoiled" (or "Covalently Closed-Circular") DNA is fully intact with both strands uncut, and with a twist built in, resulting in a compact form. You can model this by twisting an extension cord and then plugging it into itself.
• "Supercoiled Denatured" DNA is like supercoiled DNA, but has unpaired regions that make it slightly less compact; this can result from excessive alkalinity during plasmid preparation. You can model this by twisting a badly frayed extension cord and then plugging it into itself.
The rate of migration for small linear fragments is directly proportional to the voltage applied at low voltages. At higher voltages, larger fragments migrate at continually increasing yet different rates. Therefore the resolution of a gel decreases with increased voltage.
At a specified, low voltage, the migration rate of small linear DNA fragments is a function of their length. Large linear fragments (over 20kb or so) migrate at a certain fixed rate regardless of length. This is because the molecules 'reptate', with the bulk of the molecule following the leading end through the gel matrix. Restriction digests are frequently used to analyse purified plasmids. These enzymes specifically break the DNA at certain short sequences. The resulting linear fragments form 'bands' after gel electrophoresis. It is possible to purify certain fragments by cutting the bands out of the gel and dissolving the gel to release the DNA fragments.
Because of its tight conformation, supercoiled DNA migrates faster through a gel than linear or open-circular DNA.
Essential Features of Plasmid Vectors
Replication
Replication of plasmid DNA is carried by the same enzymes that replicate the E. coli bacterial chromosome. The number of plasmids per cell range from 1 to a few thousand. The control of plasmid copy number exists near the plasmid's origin of replication. Although there is normally only one origin per plasmid, only one is active when there are more. Derived from the pMB1 plasmid (or the very similar ColE1 replicon), the origin of replication relies upon host genes (for DNA polymerases [DNA polymerizartion], DNA-dependent RNA polymerase [primase function] and dnaB, dnaC, dnaD and dnaZ). As these gene products are fairly stable, protein synthesis can be stopped (such as through the addition of antibiotics) while plasmid replication continues. Unidirectional replication is primed by an RNA primer RNA II that is, in turn, negatively regulated by RNA I plus the Rop protein. Mutations that weaken this interaction result in high copy number plasmids. These genes also control plasmid incompatibility such that only one version of a set of related plasmids can persist in a cell and its descendants. In the wild, plasmids are transmitted to new hosts via conjugation. However, this ability has been deleted in most vectors.
Selectable Markers (Antibiotic resistance)
Ampicillin inhibits synthesis of bacterial cell wall amp resistance depends upon production of an enzyme that catalyzes beta-lactam ring degradation in the periplasmic space.
Tetracycline binds to 30S subunit of the ribosome to prevent ribosome translocation tet resistance produces a protein that prevents tetracycline from entering the cell.
Chloramphenicol binds to the 50S subunit of the ribosome to prevent protein synthesis cat (chloramphenicol resistance) produces an enzyme system component that converts chloramphenicol to a form that cannot bind the ribosome.
Kanamycin (and the closely related neomycin) are aminoglycosides that bind sub-components of the ribosome and prevent protein synthesis. kan (and neo) resistance depend upon the synthesis of an aminoglycoside phosphotransferase located in the periplasmic space that inhibits their transport into the cell.
Some features of pUC19:
• High copy number in E. coli, ~100 copies/cell, provides high yield.
• Selectable marker is ampR. Ampicillin in growth medium prevents growth of all other E. coli.
• Cluster of restriction sites called a polylinker occurs in the lacZ (b-galactosidase) gene.
• Cloned DNA disrupts reading frame and b-galactosidase production.
• Add X-gal to medium; turns blue in presence of b-galactosidase.
• Plaque growth: blue = no inserted DNA and white = inserted DNA.
• Some % of digested vectors will reanneal with no insert. Remove 5’ phosphates with alkaline phosphatase to prevent recircularization (this also eliminates some blue plaques).
• Plasmids are transformed into E. coli by chemical incubation or electroporation (electrical shock disrupts the cell membrane).
• Cloned inserts >5-10 kb typically are unstable; good for <10kb. href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEh8Ucdl7o3q_caX9HDVuUvYdGhTxvFW_OjOXoSVXO9Oiycu_-FdwZtX65zs4dJs0t1NGX97uP1s5QuKpIDoOmzF-n-0KOZf-g6jyxmAmbsZOIFVgEQMSIvR-Piuu8WGF8e2D-B_ygaLIG5F/s1600/13.JPG">
1 comments:
Thank you so much ! I really appreciate it.
Preclinical CMC
CHO commercial license
Host Cell Line
Biologics CDMO
Post a Comment