Great Role of the Study and Use in Medicine of Marker and Reporter Genes - Term Paper Example

“Reporter Gene & Marker Gene in Biotechnology” The present era views genetic tailoring of the microorganisms through a novel class of tools for examining alterations in the condition of environment. Reporter genes as the name suggests, aided with multicolored fluorescent proteins helps in determining various chemical effects in the environment. These engineered microorganisms are exploited for various whole cell array formats on silicon chips and diverse optic fibers as they are capable of providing bioavailability of pollutants, the impact of these pollutants on the life and a variety of behavior patterns. It is due to their specificity, these biological tools are being used for sensing the minor alterations in our environment (Belkin, 2003). Studies are carried out with microorganisms as they are found in diverse habitats and potentially degrade all biological materials. This characteristic has been exploited to monitor pollutants and to study various aspects of environmental biotechnology encompassing pollutant clean-up, for controlling crop pathogens, for genetically engineered food products. In order to procure maximum benefit from these microorganisms they are being genetically modified and are therefore called genetically modified microorganisms (GMMs). It is therefore a system is devised to keep a constant track on the survival, growth pattern and various metabolic activities and for the utilization of these activities to study various environmental and genetic aspects. The system encompasses molecular and immunological methods, ELISA, flow cytometry, nucleic acid probing, molecular marking or tagging. A reporter gene encodes the phenotype that can be easily measured through the expression of the gene. This expression is doable when the reporter gene is fused with the suitable promoter target sequences. Expression of this gene can be perceived by the appearance of the reporter gene, since reporter is tagged with the gene of interest. The promoter is tagged with a marker gene which helps to identify the phenotype or the genotype of the transformed organism in a specific environment. Thus marker gene is a DNA sequence which is injected into the organism. For the expression of the reporter gene it is essential that is incorporated with the promoter sequences of the desired gene, as this site initiates the synthesis of RNA. Promoter has RNA polymerase binding site and thus can initiate the transformation process which is vital for the expression of reporter gene. It is imperative to understand the characteristics of reporter and marker gene to understand their utility in biotechnology. Characteristics of marker genes: it should be an indicator of an active microbial population, it should not have any background in the given environment and therefore enables easy selection in the population, the marker should not be very expensive but should be simple and should have broad range i.e. it should not have a host assortment, it should enable single cell detection, and should not undergo destruction while monitoring, it should be quantitative. Categories of markers: 1. Resistance markers: they display resistance to a particular drug, ion, metabolites and therefore provides uncomplicated and simple detection against the background and also in the population. E.g. heavy metal resistance markers like mercury (mer), nickel (ner), antibiotic resistance markers like kanamycin (neptII), tetracycline (tet), streptomycin, rifampicin (rif). 2. Metabolic markers: These are expressed as a result of metabolism of the organism, due to some action performed by the enzyme. The selection of the organism is done when a chromogenic substrate is added. E.g. β- galactosidase (lacZ); catechol 2, 3 dioxygenase (xylE); β-glucoronidase (gusA); levan sucrose (sacB). 3. Luminescence markers: they are capable of emitting light and hence provide easy selection of cells. They help in measuring cell activity. They require oxygen for bioluminescence. E.g. eukaryotic luciferase (luc) where exogenous luciferin substrate is essential for luminescence; bacterial or prokaryotic luciferase where aldehyde is required as the substrate (luxAB) while luxCDABE produce substrate endogenously. 4. Fluorescent markers: this method does not require any substrate. FACS (fluorescence activated cell sorting) is performed. E.g. Green Fluorescent Protein (GFP) obtained from Aequorea Victoria a jelly fish, it is also available as YFP and BFP i.e. yellow and blue fluorescent proteins. This has the advantage that it requires oxygen only during the folding of protein and does not require oxygen for fluorescence. β- glucuronidase (gusA) or β-galactosidase (lacZ). 5. Surface markers- they help in the appearance of specific surface epitopes and these epitopes are detected with the help of antibody detection technique. The color development is read with epifluorescence microscopy. 6. Nucleotide markers- some DNA sequence do not code for any protein but are inserted in the cell as markers. Characteristics of Promoter gene: reporter genes must be stable and should be desired, and should not be transferred or lost to other bacteria. The reporter must be sensitive and specific in responses to a greater extent. Factors playing vital role in selection of the reporter gene: 1. Energy requirements: some reporters like luxAB and luc do not display phenotypes in the absence of cellular energy. 2. Multi-copy number of plasmid in order to enhance the signal intensity. 3. Oxygen requirement to detect reporter signals especially those encompassing enzymatic activities. One of the biggest drawbacks is their confinement only to aerobic organisms. 4. Managing the insertion site of the reporter gene so that it does not disrupts the cellular functions. 5. Background expression must be considered for the appropriate detection of reporter gene. 6. Physiological conditions like temperature, ionic strength, pH must be checked with respect to the environment for appropriate reporter gene expression. 7. Substrate requirement and its availability is also one of the major factors for considering the reporter gene selection. 8. Usage of codon should be specific 9. In order to study two parameters, it is essential to adopt dual reporters. 10. Host selection must be made that is able to grow in laboratory conditions. Application of target genes and promoters for the exploitation of reporter gene to understand physiology or particular genetic expression. 1. Metabolic activity can be understood with the constant expression of luciferase e.g. bacterial luxAB to measure FMNH2 to measure ATP within bacterial cells and emission is recorded from the metabolically active cells. 2. Growth rate is directly proportional to increase in biomass. This is studied by fusing reporter gene with cell division promoter gene. 3. Growth phase can be studied with the formation of secondary metabolites and hence expression of the reporter. 4. Biosensors: Huge population, rapid growth, cost effective and minimum maintenance of microorganisms aids in pollution monitoring, moreover their genome can be altered and tailored as per the requirement. Due to specificity of microorganisms, they are genetically engineered as environmental bioreceptors. In biosensors biological material is combined with an electronic device to detect minor or low level of alterations in the environment. There is a close interface between enzymes and substrates and therefore a exclusive biorecognition f two molecules can be analyzed. Biosensors provide the advantage that bioavailability, toxicity and genotoxicity could be studied well with the help of reporter gene. The stress sensing proteins heat shock proteins (hsp) are expressed in response to small changes in the microenvironment if any reporter gene is tagged with these hsps then its expression will be reported by the appearance of reporter gene product, making the detection easy. The drawback of this method encompass the non-specificity of chemical agent as microbial system could find out the pollutant but it is unable to tell the nature of chemical and to which class it belongs! 5. To assess environmental toxicity-Another approach is “lights off” or “lights on” assay where intensity of emitted light is directly co-related with the concentration of the chemical agent. It is used in various toxicity assays, to find out the concentration of the toxicant. This system is inappropriate to understand the nature of toxicants present in the sample. Genetically altered microorganisms most commonly used are Vibrio fischeri, Escherichia coli HB101 (harboring luxCDABE of V. fisheri) immobilized in polyvinyl alcohol. Pseudomonas fluorescens tagged with the plasmid, cyanobacteria Synechocystis PCC6803 encompassing luc from frirefly Photinus pyralis (Belkin, 2003). 6. It is evident that a single reporter system is not an efficient system to sense stress or any alteration in the environmental conditions, may it be chemical regulation, temperature regulation or pH regulation. It is therefore recommended to have a set of organisms with varied sensitivity responses for environmental pollutants (Belkin, 2003). 7. Detection of specific classes of pollutants-Bioluminescence is used as a reporter, GFP accumulation and phenol sensing are most prevalent ones. Microorganisms are used for the detection of specific pollutants because of their diverse nature, nutritional requirements and growth conditions. It is therefore a particular group of microorganisms can be used for the detection of a particular group of chemical agents present in the environment (Belkin, 2003). 8. Nutrient Bioavailability- Reporters are generated to sense the bioavailability of nutrients in a given environment. The cyanobacterial bioreporters are being created to sense the waste water nitrogen and phosphorus as they are the major basis of eutrophication in aquatic environments. Synechococcus sp with a glnA::lux fusion and Synechocystis sp. With nblA::lux fusion are the reporters that can sense available nitrogen. These organisms can potentially sense the nutrient bioavailability and this is indicated through the expression of the reporter gene (Belkin, 2003). 9. Bioluminescence or fluorescence- It is observed that bacterial reporter system encompassing bioluminescence genes are most widely used and it is finding its way for general use. It also possesses the advantage over the fluorescence as it is faster and more sensitive. The fact for its rapid reporting is due to its enzymatic activity whereas fluorescence is due to the presence of protein. One of the major advantages of using fluorescent proteins is its stability and is much more dependable in real-time monitoring and also in a situation where signal development is very feeble. 10. Dual labeling-Integration of two kinds of reporter genes in the same organism is dual labeling. Here, one reporter acts as responder to the analyte and other as an internal control. E.g. integration of GFP and YFP into the bacterium E. coli. The construct encompass two inducible promoters recA’::egfp and grpE’::dsRedExpress. The recA promoter is the imperative part of bacterial SOS and responds when any kind of DNA damage occurs while grpE is heat-shock protein which is activated against the stress induced by a variety of chemical agents which may have toxic effect on the cell. This amalgamation enables monitoring of genotoxicity as well as regular normal toxicity (Belkin, 2003). 11. Immobilization and integration into biosensors- Storage and upholding becomes extremely easy when the cells are immobilized in the alginate solution or in agar. The process has the advantage that cells can be used again and again without any alteration (Belkin, 2003). 12. Whole-cell arrays- The present decade witnessed the chip assays, the microarray. Microarray enables us not only to characterize but also to quantify biomolecules. In this process a large number of reactive molecules are fixed in the grid and then they are rendered to multi-component analyte mixture. Alteration in the site can be monitored with the fluorescence. The technique has been used widely in the development of medicine, biology, toxicology, for drug screening, drug designing, genotoxicity assays etc (Belkin, 2003). Future of reporter genes: in environmental microbiology and to study the expression of genes for the field of medical science, environmental studies and to understand the development of the organisms. Improvements desired in reporter activities- To design a reporter that can be activate in anaerobic environment and for Archaebacteria. Construction of reporters for the organisms that are beyond the scope of laboratory culture. Augmentation in reporter expression is desired. Elimination of substrate addition for reporter activities e.g. luciferin for eukaryotic luciferase reporters. Maximum returns in the form of activities with minimum cost input. Compatible software to study the images Use of reporters can well be studies with the help of the following case studies: Case I: Pseudomonas fluorescens with phenazine production genes: Development of biocontrol agent active against “Take All” disease of wheat. Take All is a soil borne fungal disease of wheat, therefore gene bearing the expression of antifungal agent, PCA (Phenazine Carboxylic Acids) can be genetically induced in bacterial cells and this bacterial culture is allowed to colonize in rhizosphere. Antifungal gene activity can be reported through the expression of reporter gene. Case II: Use of bacterial biosensor to monitor TNT degradation in soils contaminated with TNT and heavy metals. It is observed that laboratory conditions differ from the practical conditions and therefore the results may be false positive. It is therefore essential that complete analysis of microbial population in the rhizosphere and also in the soil contaminated with TNT and heavy metals should be made. Proper sampling from different parts of the given area of land or from different part of the rhizosphere is made. The synergetic and antagonistic approaches must be considered in order o avoid false positive results which leads to failure of any technology. The case studies are imperative in the sense that they are the indicators of the use of reporter genes for the assessment of environment of the given area. Such constructs are very valuable in this epoch of industrialization. Many similar constructs can be made to monitor the prevalent pollutants in the environment. Conclusion With the advent of modifications in the marker and reporter genes through genetic engineering, detection of pollutants has become more realistic under a given set of conditions. These conditions may vary in practicality. Reporter genes are very helpful in performing screening of organisms but it is still a challenge for the scientists across the world to implement these genetically engineered tools without drawbacks in environmental variability. It is evident that reporter and marker genes not only mark the examination of stress or environmental pollutants but they are being constantly exploited for the regular drug designing where impact on live cells can be monitored. They are being used in high-throughput screening to get the desired traits. This is one part of the story and adds to the advantages in the field of medical and industrial upgradation to monitor genetic modifications either of the drug itself or the impact of alteration of genome. The expression of gene at various levels of the development not only makes one understand the genetics of diseases but also enable us to appreciate the development and life cycle of the organism in details. On the contrary, genetic engineering and marker gene encompassing antibiotic resistance is producing strains in the environment which are producing deleterious consequences on the living beings on the planet. It is essential to understand that AIDS, Swine flu are big examples where the virus is of animal origin and it is human endeavor that paved the way for its survival within human systems! One must comprehend that tools in genetic engineering be used for the betterment of life. It is desired to have a complete understanding of the technology and when we discard the undesired species we should take utmost care not to create havoc in the environment but must follow proper disposal systems in order to avoid the birth of another dreaded microbe to shake the human survival! References: Belkin, S., 2003. Microbial whole-cell sensing systems of environmental pollutants. Current Opinion in Microbiology. 6, 206- 212. Reporter Genes For Monitoring Microbial Cell Activity and/or The Environment: An Opinion. Sponsored by the European Commission Biotechnology Programme, DGXII. Read More