We take content rights seriously. If you suspect this is your content, claim it here.
Available Formats
Download as PPTX, PDF, TXT or read online on Scribd
You are on page 1/ 50
MSc I Sem I
PSB104 RECOMBINANT DNA
TECHNOLOGY
Leela Chauhan Recombinant DNA Technology Vectors
In general, the word “cloning” means the creation of a perfect replica
Long before attempts were made to clone an entire organism, researchers learned how to reproduce desired regions or fragments of the genome, a process that is referred to as molecular cloning. Cloning small fragments of the genome allows for the manipulation and study of specific genes (and their protein products), or noncoding regions in isolation. A plasmid (also called a vector) is a small circular DNA molecule that replicates independently of the chromosomal DNA of microorganisms such as E. coli. In cloning, the plasmid molecules can be used to provide a “folder” in which to insert a desired DNA fragment. Plasmids are usually introduced into a bacterial host for proliferation. In the bacterial context, the fragment of DNA from the human genome (or the genome of another organism that is being studied) is referred to as foreign DNA, or a transgene, to differentiate it from the DNA of the bacterium, which is called the host DNA. Plasmids occur naturally in bacterial populations (such as Escherichia coli) and have genes that can contribute favorable traits to the organism, such as antibiotic resistance (the ability to be unaffected by antibiotics). Plasmids have been repurposed and engineered as vectors for molecular cloning and the large-scale production of important reagents, such as insulin and human growth hormone. An important feature of plasmid vectors is the ease with which a foreign DNA fragment can be introduced via the multiple cloning site (MCS). The MCS is a short DNA sequence containing multiple sites that can be cut with different commonly available restriction endonucleases Restriction endonucleases recognize specific DNA sequences and cut them in a predictable manner; they are naturally produced by bacteria as a defense mechanism against foreign DNA. Many restriction endonucleases make staggered cuts in the two strands of DNA, such that the cut ends have a 2- or 4-base single-stranded overhang. Because these overhangs are capable of annealing with complementary overhangs, these are called “sticky ends.” Addition of an enzyme called DNA ligase permanently joins the DNA fragments via phosphodiester bonds. In this way, any DNA fragment generated by restriction endonuclease cleavage can be spliced between the two ends of a plasmid DNA that has been cut with the same restriction endonuclease Recombinant DNA Molecules Plasmids with foreign DNA inserted into them are called recombinant DNA molecules because they are created artificially and do not occur in nature. They are also called chimeric molecules because the origin of different parts of the molecules can be traced back to different species of biological organisms or even to chemical synthesis. Proteins that are expressed from recombinant DNA molecules are called recombinant proteins. Not all recombinant plasmids are capable of expressing genes. The recombinant DNA may need to be moved into a different vector (or host) that is better designed for gene expression. Plasmids may also be engineered to express proteins only when stimulated by certain environmental factors, so that scientists can control the expression of the recombinant proteins. SV - 40 Simian virus 40 (SV40) belongs to polyomavirus family, the family of viruses that induce tumours in animals Simian virus 40 was first isolated from monkeys hence so named. SV40 was one of the first genetic elements to be studied by genetic engineering techniques and has been used extensively as a vector for transferring genes into eukaryotic cells. SV40 is a naked icosahedral virus with a diameter of 45 nm. Its capsid contains 72 protein subunits When the virus enters the host cell, its genome migrates to the nucleus. SV40 genome replicates inside the nucleus of the cell, but the capsid protein are synthesized in the cytoplasm and migrate inside the nucleus where, finally, the assembly of the virus take place. Vaccinia VV is a poxvirus that was clinically used as vaccine for smallpox. The genome of VV is completely sequenced and facilitates the creation of recombinant viral vectors that could carry up to 25 kb of foreign DNA VV is a very large, complex, and enveloped virus that contains a linear and dsDNA genome of about 180–190 kbp, with internal terminal repeats, and hairpin loops. The life cycle of the VV occurs in the cytoplasm and so it is a non integrative vector. An advantage of using vaccinia-derived vectors is that the vaccinia vectors may carry until 25 kb of foreign DNA without the need for viral deletions.27 Vaccinia vectors present other advantages as a broad host range that permits the infections of primary cultures and many different cell lines, cytoplasmic replication, or the fact that the viral genome does not splice its primary transcripts. Baculovirus Baculoviruses, which belong to the family Baculoviridae, are a group of DNA viruses that infect insects. These viruses are very small, rod-shaped structures, composing of a protein coat containing a small amount of DNA which encodes components for further viral reproduction. Baculoviruses use unique mechanisms to survive and proliferate in their environment. When baculoviruses are outside a host, waiting to be ingested, they survive within occlusion bodies. These are bodies of protein which envelop and protect the virions (the form of the virus when external to a host cell) until they are ingested by a host. The occlusion bodies are stable in the environment, but when they are ingested by an insect, the high pH of the insect gut causes the breakdown of the protein and the release of the virions. The virions enter cells and self-replicate, hijacking the machinery of the host cell to produce DNA and protein, forming more viruses. After replication has occurred, the final step is the production of a protein called polyhedrin. This protein crystallises in the cell and surrounds the virions, forming an occlusion body ready for dispersal. The virions produced by an occlusion body are called occlusion- derived virions (ODV). Another type of virion that the baculoviruses produce are called budding virions (BV) which are responsible for the cell to cell, systemic infection within insects. Retroviral vectors Retrovirus vectors are RNA-based vectors belonging to the family of retroviridae. The genome of the retrovirus vector is a 7 to 10 kb single stranded RNA containing long terminal repeats (LTR) Retroviruses enter the cells via cell fusion of the envelope protein with the cell membrane. Retroviral vector-mediated gene transfer has been central to the development of gene therapy. Retroviruses have several distinct advantages over other vectors, especially when permanent gene transfer is the preferred outcome. The most important advantage that retroviral vectors offer is their ability to transform their single stranded RNA genome into a double stranded DNA molecule that stably integrates into the target cell genome. This means that retroviral vectors can be used to permanently modify the host cell nuclear genome BACTERIAL ARTIFICIAL CHROMOSOME A bacterial artificial chromosome (BAC) is an engineered DNA molecule used to clone DNA sequences in bacterial cells (for example, E. coli). BACs are often used in connection with DNA sequencing. Segments of an organism's DNA, ranging from 100,000 to about 300,000 base pairs, can be inserted into BACs. The BACs, with their inserted DNA, are then taken up by bacterial cells. As the bacterial cells grow and divide, they amplify the BAC DNA, which can then be isolated and used in sequencing DNA. A large piece of DNA can be engineered in a fashion that allows it be propagated as a circular artificial chromosome in bacteria--so-called bacterial artificial chromosome, or BAC. Each BAC is a DNA clone containing roughly 100 to 300 thousand base pairs of cloned DNA. Because the BAC is much smaller than the endogenous bacterial chromosome, it is straightforward to purify the BAC DNA away from the rest of the bacteria cell's DNA, and thus have the cloned DNA in a purified form. This and other powerful features of BACs have made them extremely useful for mapping and sequencing mammalian genomes. The BAC is based on a plasmid in Escherichia coli that is termed the F (for fertility) plasmid. The F plasmid (or F factor) contains information that makes possible the process called conjugation . A BAC contains the conjugation promoting genetic information as well as stretch of DNA that is destined for incorporation into the bacterium. The foreign DNA (e.g., portion of human genome) is flanked by sequences that mark the boundaries of the insert. The sequences are referred to as sequence tag connectors. When the BAC becomes incorporated into the genome of Escherichia coli the sequence tag connectors act as markers to identify the inserted foreign DNA. The most dramatic recent example of the power of BACs is their use by The Institute for Genomic Research (TIGR ) in the technique of shotgun cloning that was employed in the sequence determination of the human genome YEAST ARTIFICIAL CHROMOSOME
Yeast artificial chromosome (YAC) is a human-engineered DNA
molecule used to clone DNA sequences in yeast cells. YACs are often used in connection with the mapping and sequencing of genomes. Segments of an organism's DNA, up to one million base pairs in length, can be inserted into YACs. The YACs, with their inserted DNA, are then taken up by yeast cells. As the yeast cells grow and divide, they amplify the YAC DNA, which can be isolated and used for DNA mapping and sequencing. YAC vectors allows the insertion of DNA fragments up to 100-500 kb. A YAC vector includes TEL: telomere (TEL) is located at each end of chromosome. It protects the linear DNA from degradation by nucleases. CEN: The centromere which is the attachment site for mitotic spindle ARS: Replication origin sequences which are specific DNA sequences that allows the DNA replication machinery to assemble on the DNA and move at the replication forks. A and B: YAC contains two selectable markers that allow the easy isolation of yeast cells that have taken up the artificial chromosome. URA3-involved in uracil biosynthesis TRP1 -involved in tryptophan biosynthesis Recognition site for the two restriction enzymes EcoRI and BamHI. Ampr – an ampicillin resistance gene allows positive selection by ampicillin resistance in bacteria A YAC is built using an initial circular DNA plasmid, which is typically cut into a linear DNA molecule using restriction enzymes; DNA ligase is then used to ligate a DNA sequence or gene of interest into the linearized DNA, forming a single large, circular piece of DNA Advantages Yeast expression vectors, such as YACs, YIps (yeast integrating plasmids), and YEps (yeast episomal plasmids) can be used to express eukaryotic proteins that require posttranslational modification. YACs vectors are able to insert large fragments of DNA and can be utilized to clone and assemble the entire genomes of an organism. the inserted sequences can be cloned and physically mapped using a process called chromosome walking Study expression of entire mammalian genes Not used too much anymore, but important for historical reasons. One of the very early ways that scientists--geneticists--manipulated large pieces of DNA...very important to be able to isolate and reproduce large pieces of DNA for the purposes of isolating disease genes. So what scientists did was they co-opted the parts of a yeast chromosome, which are important for packing that DNA, and replicating the DNA, and they took out all the yeast sequences in the middle and they put in human sequences or some other sequence, and then stuck it back in the yeast cell, and the yeast cell didn't know the difference and replicated the human DNA instead, and so served as a Trojan horse for the genetics researchers to be able to replicate, and therefore study, the human and other organism DNA that they were interested in. Disadvantages YACs are significantly less stable than BACs, producing “chimeric effects” Difficult to isolate YACs from host yeast genome Have low copy number Slow growth rate Poor transformation efficiency HIGH AND LOW COPY NUMBER OF PLASMIDS
The plasmid copy number is the number of copies of a given plasmid in
a cell. To ensure survival and thus the continued propagation of the plasmid, they must regulate their copy number. If a plasmid has too high of a copy number, they may excessively burden their host by occupying too much cellular machinery and using too much energy. On the other hand, too low of a copy number may result in the plasmid not being present in all of their host's progeny. Plasmids may be either high copy number plasmids or low copy number plasmids; the regulation mechanisms between these two types are often significantly different. Biotechnology applications may involve engineering plasmids to allow a very high copy number. High copy number plasmids, also called relaxed plasmids, require a system to ensure that replication is inhibited once the number of plasmids in the cell reaches a certain threshold. Relaxed plasmids are generally regulated through one of two mechanisms: antisense RNA or iteron binding groups Glyphosate tolerance Glyphosate-tolerant crops Glyphosate herbicide kills plants by blocking the EPSPS enzyme, an enzyme involved in the biosynthesis of aromatic amino acids, vitamins and many secondary plant metabolites. There are several ways by which crops can be modified to be glyphosate- tolerant. One strategy is to incorporate a soil bacterium gene that produces a glyphosate tolerant form of EPSPS. Another way is to incorporate a different soil bacterium gene that produces a glyphosate degrading enzyme. Glyphosate herbicide-resistant crop plants, introduced commercially in 1994, now represent approximately 85% of the land area devoted to transgenic crops. Herbicide resistance in commercial glyphosate- resistant crops is due to expression of a variant form of a bacterial 5- enolpyruvylshikimate-3-phosphate synthase with a significantly decreased binding affinity for glyphosate at the target site of the enzyme. As a result of widespread and recurrent glyphosate use, often as the only herbicide used for weed management, increasing numbers of weedy species have evolved resistance to glyphosate. Weed resistance is most often due to changes in herbicide translocation patterns, presumed to be through the activity of an as yet unidentified membrane transporter in plants. Search identified a single non-target gene that, when overexpressed in E. coli and Pseudomonas, confers high-level glyphosate resistance. The gene, yhhS, encodes a predicted membrane transporter of the major facilitator superfamily involved in drug efflux. An alternative mode of glyphosate resistance in E. coli is due to reduced accumulation of glyphosate in cells that overexpress this membrane transporter and discuss the implications for potential alternative resistance mechanisms in other organisms such as plants TSR often involves mutations in genes encoding the protein targets of herbicides, affecting the binding of the herbicide either at or near catalytic domains or in regions affecting access to them. Detoxification of glyphosate Detoxification of the glyphosate molecule is another strategy that has been employed to confer glyphosate resistance. Soil microorganisms can metabolizes glyphosate by two different routes i) cleavage of the carbon- phosphorus bond, resulting in the formation of phosphate and sarcosine (the C-P lyase pathway) e.g., by Pseudomonas sp. PG2982; ii) oxidative cleavage of the C-N bond on the carboxyl side catalyzed by glyphosate oxidoreductase (GOX), resulting in the formation of aminomethylphosphonic acid (AMPA) and glyoxylate (the AMPA pathway). Neither of these mechanisms has been shown to occur in higher plants to a significant degree. The C-P lyase pathway requires an unknown number of genes and the activity has not been reconstituted in vitro, casting doubt on the ability to create the activity in transgenic plants. The AMPA pathway appears to be the predominant route for degradation of glyphosate in soil by a number of Gram-positive and Gram- negative bacteria. Most recently, a glycine oxidase from B. subtilis was also demonstrated to metabolize glyphosate into AMPA and glyoxylate, but using a reaction mechanism different from GOX. Nif genes Transfer of nif genes Nitrogen is one of the most important nutrients for plant growth. To enhance crop productivity, chemical nitrogen fertilizer is commonly applied in agriculture. Biological nitrogen fixation, the conversion of atmospheric N2 to NH3, is an important source of nitrogen input in agriculture and represents a promising substitute for chemical nitrogen fertilizers.). Thus, many biologists hope to reconstitute a nitrogenase biosynthetic pathway in a eukaryotic host, with the final aim of developing N2‐fixing cereal crops. With the advent of synthetic biology and a deep understanding of the fundamental genetic determinants necessary to sustain nitrogen fixation in bacteria, much progress has been made toward this goal. Transfer of native and refactored nif (nitrogen fixation) genes to non‐diazotrophs has been attempted in model bacteria, yeast, and plants. Specifically, nif genes from Klebsiella oxytoca, Azotobacter vinelandii, and Paenibacillus polymyxa have been successfully transferred and expressed in Escherichia coli, Saccharomyces cerevisiae, and even in the tobacco plant. These advances have laid the groundwork to enable cereal crops to “fix” nitrogen themselves to sustain their growth and yield.