ZIAUDDIN UNIVERSITY Faculty of Engineering Science & Technology (ZUFEST), Department of Biomedical Engineering Lecture-3 Genetic manipulation strategies in environmental biotechnology Genetic Manipulation • Genetic engineering (GE), recombinant DNA technology, genetic manipulation/modification (GM) and gene splicing are the terms that apply to the direct manipulation of an organism’s genes. • GE uses the techniques of molecular cloning and transformation to alter the structure and characteristics of genes directly. • GE aims at isolating DNA fragments and recombining them. Genetic Manipulation Genes have been manipulated by man for a very long time. It has been proposed that the exchange of genetic information between organisms in nature is considerably more commonplace than is generally imagined and could explain the observed rates of evolution (Reanney 1976). Genetic Manipulation • Exchange involving a vector requires compatibility between the organism donating the genetic material, the vector involved, and the recipient organism. • Organisms which represent the ‘norm’, frequently being the most abundant members occurring in nature, are described as ‘wild type’. • Those with DNA which differs are described as ‘mutant’. Alteration can be by the normal processes of evolution which constantly produces mutants, a process which may be accelerated artificially, or by deliberate reconstruction of the genome. Varied Applications • Isolation of a particular gene, gene part or region of a genome. • Production of a particular RNA and protein molecules in quantities. • Improvement in the production of biochemicals and commercially important organic chemicals. • Production of varieties of plants having particular desirable characteristics. • Correction of genetic defects in higher organisms • Creation of organisms with economically important features. Training: Manipulation of Bacteria Without Genetic Engineering A general procedure is to take a sample of bacteria from the site of contamination from which a pure culture is obtained in the laboratory and identified, using standard microbiology techniques. The ‘training’ may be required either to improve the bacterium’s tolerance to the pollutant or to increase the capabilities of pathways already existing in the bacterium to include the ability to degrade the pollutant, or a combination of both. Training: Manipulation of Bacteria Without Genetic Engineering Tolerance may be increased by culturing in growth medium containing increasing concentrations of the pollutant so that, over successive generations, the microbe becomes more able to withstand the toxic effects of the contaminant. Improving the microbe’s ability to degrade a contaminant, sometimes referred to as catabolic expansion. Training: Manipulation of Bacteria Without Genetic Engineering Under laboratory conditions where cultures of bacteria are isolated from each other to prevent crosscontamination, mutations are most likely to occur as a result of an error in DNA replication. An increased rate of error may be forced upon the organism, speeding up the rate of mutation, by including a mutagen in the growth medium. Training: Manipulation of Bacteria Without Genetic Engineering A mutagen is a chemical which increases the rate of error in DNA replication, often by causing a very limited amount of damage to the DNA such that the DNA polymerase, is unable to determine the correct base to add in to the growing nucleotide chain. If the error in the nascent strand cannot be recognized and corrected, the fault becomes permanent and is handed on through the generations. Manipulation of Bacteria by Genetic Engineering Genetic manipulation by the deliberate introduction of defined genes into a specified organism is a very powerful technique. The techniques have produced some exciting hybrids in all areas of research, both microscopic; bacteria and fungi, usually described as recombinants, and macroscopic; principally higher plants and animals, commonly described as transgenics. Basic Principles of Genetic Engineering There are endless permutations of the basic cloning procedures but they all share some fundamental requirements. These are: 1. Enzymes, solutions and equipment necessary to perform the procedures 2. Desired piece of DNA to be transferred 3. A cloning vector 4. Recipient cell which may be a whole organism. 5. Marker genes to ensure transfer Enzymes, solutions and equipment • Isolation of DNA and purified from contaminating material • To insert the DNA into the vector, ends must be prepared. • Done by restriction endonucleases and staggered ends are produced. Enzymes, solutions and equipment Preparation of the vector is dictated by the type of end prepared for the insert DNA: flush or ‘sticky’. • If it is flush, it does not much matter how that was achieved so long as the vector receiving it is also flush. • If it is sticky, the appropriate sticky end must be prepared on the vector by a suitable restriction endonuclease. Enzymes, solutions and equipment The prepared insert, or ‘foreign’ DNA is incubated with the prepared vector in an aqueous solution containing various salts required. Ligase is an enzyme, the function of which is to make the bond between the free phosphate on a nucleotide base and the neighboring ribose sugar, thus ‘repairing’ the DNA to make a complete covalently linked chain. DNA for transfer • A piece of double-stranded DNA which contains the coding sequence for a gene. • Obtained from a number of sources, for example, genomic DNA, a cDNA library, a product of a polymerase chain reaction (PCR) or a piece of DNA chemically produced on a DNA synthesizer machine. • Another source is DNA copy of an RNA virus as in the replicative form of RNA viruses. Genomic Libraries Genomic DNA, in this context, is material which has been isolated directly from an organism, purified and cut up into pieces of a size suitable to be inserted into a cloning vector. These pieces may either be ligated in total mixture, into a suitable vector to produce a genomic library, Genomic libraries are very useful, as they may be amplified, and accessed almost limitlessly, to look for a specific DNA sequence thus reducing the amount of work involved in any one experiment. cDNA libraries In eukaryotes, the first product of transcription from DNA is not messenger RNA (mRNA) but heterogeneous nuclear RNA (hnRNA). This is mRNA prior to the removal of all the noncoding sections, or introns, which are discarded during the processing to produce the mature mRNA. Complementary DNA (cDNA) is DNA which has been artificially made using the mature mRNA as a template, which is then used as the template for the second strand. Thus the synthetic DNA product is simply a DNA version of the mRNA and so should overcome the problems of expression outlined above Polymerase chain reaction • The polymerase chain reaction (PCR) is a powerful technique which amplifies a piece of DNA of which only a very few copies are available. • The process is repeated by a constant cycling of denaturation of double-stranded DNA at elevated temperature to approximately 95 ◦C, followed by cooling to approximately 60 ◦C to allow annealing of the probe and complementary strand synthesis. Cloning vectors • A cloning vector is frequently a plasmid or a bacteriophage (bacterial virus) which must be fairly small and fully sequenced, able to replicate itself when reintroduced into a host cell, thus producing large amounts of the recombinant DNA for further manipulation. • It must carry on it ‘selector marker’ genes. These are different from the reporter genes which are indicators of genomic integrity and activity. • A common design of a cloning vector is one which carries two genes coding for antibiotic resistance. The ‘foreign’ gene is inserted within one of the genes so that it is no longer functional therefore it is possible to discriminate Cloning vectors Standard cloning vectors normally carry only selector marker genes required for plasmid construction. To make the manipulations easier, these genes normally contain a multicloning site (MCS) which is a cluster of sites for restriction enzymes constructed in such a way to preserve the function of the gene. Cloning vectors This is pGEM (Promega 1996) which has a MCS in the s-gal gene. This codes for s-galactosidase from the E. coli lac operon, which has the capacity to hydrolyse x-gal, a colourless liquid, to produce free galactose and ‘x’ which results in a blue pigment to the colony. Thus the screening for successful insertion into the MCS is a simple scoring of blue (negative) or white (possibly positive) colonies. The success of the experiment can be determined quickly as this cloning vector also has sequences at either side of the MCS which allows for rapid DNA sequencing. Expression vectors These are similar to the vectors described above but in addition have the required signals located before and after the ‘foreign’ gene which direct the host cell to translate the product of transcription into a protein. Reporter genes • There are many such genes in common use and these usually code for an enzyme. • The most common is β-galactosidase. This enzyme, supplied with the appropriate reagents, may also catalyze a color change by its activity. • Other reporter genes produce enzymes which can cause the emission of light such as the luciferase isolated from fireflies, or whose activity is easy and quick to assay like the bacterial β glucuronidase (GUS), which is probably the most frequently used reporter gene in transgenic plants. Analysis of Recombinants I. The design of the plasmid was such that insertion of ‘foreign’ DNA allows for a colour test, or causes a change in antibiotic sensitivity, either to resistance (positive selection) or sensitivity (negative selection). This constitutes the first step in screening. II. The second stage is usually to probe for the desired gene using molecules which will recognise it and to which is attached some sort of tag, usually radioactive or one able to produce a colour change. III. The next stage is normally to analyse the DNA isolated from possible recombinants, firstly by checking the size of the molecule or pieces thereof, or by sequencing the DNA. Analysis of Recombinants DNA sequencing has become a standard part of recombinant analysis procedure. However, if a large number of samples are to be analysed it is usually quicker and cheaper to scan them by a procedure described as a Southern blot. Recombinant Bacteria • Genetic engineering of micro-organisms for use in environmental biotechnology has tended to focus on the expansion of metabolic pathways either to modify the existent metabolic capability or to introduce new pathways. • This has various applications, from the improved degradation of contaminants, to the production of enzymes for industry, thus making a process less damaging to the environment. • One such experimental example taken from ‘clean technology’ with potential for the manufacturing industry, is a strain of Eschericia coli into which was engineered some 15 genes originating from Pseudomonas. These were introduced to construct a pathway able to produce indigo for the dyeing of denim Recombinant Yeast • Yeast, being unicellular eukaryotes, has become popular for cloning and expressing eukaryotic genes. • These are fairly simple to propagate, some species being amenable to culture in much the same way as bacteria. • There are several types of plasmid vector available for genetic engineering, some of which have been constructed to allow replication in both bacteria and yeast Recombinant Virus • The insect virus, Baculovirus, has been shown to be the method of choice for the overexpression of genes in many applications of molecular biology • The viral genome is large relative to bacterial plasmids and so DNA manipulations are normally carried out on a plasmid maintained in Eschericia coli. • One example of interest to environmental biotechnology is the replacement of p10, one of the two major Baculovirus proteins, polyhedrin being the other, by the gene for a scorpion neurotoxin, with a view to improving the insecticidal qualities of the virus Transgenic Plants • Currently, genetic engineering in agribiotechnology is focusing on genetic modifications to improve crop plants with respect to quality, nutritional value, and resistance to damage by pests and diseases. • Other avenues aim to increase tolerance to extreme environmental conditions, to make plants better suited for their role in pollutant assimilation, degradation or dispersion by phytoremediation, or to modify plants to produce materials which lead to the reduction of environmental pollution. Transformation of plants There are two practical problems associated with genetic engineering of plants which make them more difficult to manipulate than bacteria. Firstly they have rigid cell walls and secondly they lack the plasmids which simplify so much of genetic engineering in prokaryotes. The first problem is overcome by the use of specialised techniques for transformation, and the second by performing all the manipulations in bacteria and then transferring the final product into the plant. Transformation of plants • The most popular method of transforming plants is by the Ti plasmid but there are at least two other methods also in use. • The first is a direct method where DNA is affixed to microscopic bullets which are fired directly into plant tissue. An example of this technology is the introduction into sugarcane, of genes able to inactivate toxins produced by the bacterium, Xanthomonas albilineans, causing leaf scald disease • The second is by protoplast fusion which is a process whereby the plant cell wall is removed leaving the cell surrounded only by the much more fragile membrane. This is made permeable to small fragments of DNA and then the cells allowed to recover and grow into plants. Examples of developments in plant GE The purpose of these examples is to illustrate the potential plant genetic engineering could bring to future practical applications in the field of environmental biotechnology. Broad range protection A general strategy to protect plants from various viruses, fungi and oxidative damage is by a range of agents, has been proposed using tobacco plants as a model. The transgenics express the iron-binding protein, ferritin, in their cells which appears to afford them far-ranging protection Examples of developments in plant GE Resistance to herbicides • ‘Glyphosate’, one of the most widely used herbicides, is an analogue of phosphoenol pyruvate and shows herbicidal activity because it inhibits the enzyme 5-enolpyruvylshikimate3-phosphate synthase. • The gene coding for this enzyme has been identified, isolated and inserted into a number of plants including petunias. In this case, the gene was expressed behind a CaMV promoter and introduced using A. tumefaciens, leading to very high levels of enzyme expression. • As a consequence, the recombinant plants showed significant resistance to the effects of glyphosate Examples of developments in plant GE Improved resistance to pests • Plants have an inbuilt defence mechanism protecting them from attack by insects but the damage caused by the pests may still be sufficient to reduce the commercial potential of the crop. • Plants are being engineered to have an increased self-defence against pests. • With a view to increasing resistance to sustained attack, the genes coding for the δ-endotoxin of the bacterium, Bacillus thuringiensis (Bt). • Examples are of synthetic B. thuringiensis δ-endotoxin genes transferred, in the first case, by A. tumefaciens into Chinese cabbage (Cho et al. 2001) and in the second, by biolistic bombardment into maize Examples of developments in plant GE Improved resistance to disease • Bacteria communicate with each other by way of small diffusible molecules such as the N-acylhomoserine lactones (AHLs) of Gram negative organisms described as ‘quorum sensing’, they are able to detect when a critical minimum number of organisms is present, before reacting. • These responses are diverse and include the exchange of plasmids and production of antibiotics and other biologically active molecules. • Plants are susceptible to bacterial pathogens such as Erwinia carotovora, which produces enzymes capable of degrading its cell walls. The synthesis of these enzymes is under the control of AHLs. • The rationale behind using AHLs for plant protection is to make transgenic plants, tobacco in this case, which express this signal themselves. Examples of developments in plant GE Improved tolerance • The example is of bacterial rather than plant modification but impinges on interaction between the two. Pseudomonas syringae produces a protein which promotes the formation of ice crystals just below 0 ◦C thus increasing the risk of frost damage. • They transferred it to the bacterium Eschericia coli to simplify the genetic manipulations. the mutants were able to compete with the wild type and protect this particularly susceptible crop against frost damage. • Salt tolerance in tomatoes has been established by introducing genes involved in Na+/H+ antiport, the transport of sodium and hydrogen ions in opposite directions across a membrane. • Improved tolerance to drought, salt and freezing in Arabidopsis has been achieved by overexpressing a protein which induces the stress response genes. Examples of developments in plant GE Improved plants for phytoremediation • The genetic modification of a poplar to enable mercury to be removed from the soil and converted to a form able to be released to the atmosphere. This process is termed ‘phytovolatilisation’ • A bacterial gene encoding pentaerythritol tetranitrate reductase, an enzyme involved in the degradation of explosives, has been transferred into tobacco plants. The transgenics have been shown to express the correct enzyme to determine their ability to degrade TNT. Examples of developments in plant GE New products from plants • The rape plant, Arabidopsis thalia has become a popular choice for the production of recombinant species. • One such recombinant is a rape plant, the fatty acid composition in the seed of which has been modified. It now produces triacylglycerols containing elevated levels of trierucinic acid suitable for use in the polymer industry Thank You