The impact of biotechnology is being felt increasingly in modern days across several sectors and disciplines. Biotechnology can be defined as any technological application that makes use of biological systems or living things with an aim of making or altering products or given processes for specified purposes (Thieman & Palladino, 2009). It is an applied field in biology which is involves modification of living organisms by human desires and intentions. Biotechnology borrows heavily from the various pure biological sciences. These include; microbiology, genetics, biochemistry, animal cell culture, cell biology, embryology, and molecular biology (Talbot, 2001). The knowledge and methods used in biotechnology also comes from other fields beside biological sciences. They include information technology, chemical and bioprocess engineering, as well as biorobotics.
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Biotechnology has brought about a number of transformations within the sphere of living organisms. Trichoderma reesei has emerged as an industrially useful cellulolytic filamentous and is also a mesophilic fungus (Sparks, 2006). It is characterized by the ability to secret cellulolytic enzymes like the cellulases and hemicellulases in large quantities. Many researchers in this field have emerged and are trying to establish the possibility of developing the Trichoderma reesei as a host which can be used to produce low cost enzymes. The enzymes can be used in turn to convert the cellulose in plant biomass materials into industrially valuable bioproducts. Sugars and bioethanol are some of the bioproducts that are produced in the process. With the developments in biochemistry, Trichoderma reesei is advancing to become a commercially viable alternative in the process of cellulose hydrolysis. Biochemists have already established important strains of Trichoderma reesei which are gaining ground in industrial applications (Thieman & Palladino, 2009). In 2008, biotechnologists released a genome of the Trichoderma reesei which is 33Mb with a total of seven chromosomes. Most of the transformations of Trichoderma reesei are DNA mediated
Transformation in virtually all fungi is now possible with the use of exogenous deoxyribonucleic acid, especially the ones that can be grown in culture (Talbot, 2001). Two major methods have been used in maintaining the transformation process. The use of an autonomously replicating plasmid can be used for transforming DNA. Alternatively, the DNA can be integrated into the chromosomes. Transformation in fungi is useful as far as gene cloning and gene function analysis is concerned (Talbot, 2001). Markers are essentially in ensuring meaningful transformation in fungi. Several markers have been identified for the different fungi. Most useful markers can be selected and counter-selected. The use of marker genes in the transformation of fungi helps in visualizing hyphae on roots, nematodes and in soil as well as in the observation of interactions in situ. One of the most common marker for fungi and other organisms is the gene for jellyfish green fluorescent protein (gfp) (Thieman & Palladino, 2009). Fungi can generally be transformed by the use of either nutritional markers which match an auxotrophic requirement or dominant, selectable antibiotic resistance markers. A gene (bar) coding for phosphinothricin acetyltransferase has been separated from S. hygroscopicus. It is usually used as a selective marker during the transformation of higher plants and a number of filamentous fungi (Talbot, 2001).
There a number of differences between genetically encoded and chemical fluorescent markers. Genetically encoded fluorescent markers, for instance the green fluorescent protein (Aquorea Victoria) can detect localization of cell proteins as well as organelles in living organisms and cells (Sparks, 2006). The changes in the spectral properties of the proteins are useful when tracking all enzyme activities. Chemical fluorescent markers, like calcium are visible, making them useful when it comes to receiving and enhancing sensitivity of specific responses to specified stimulus.
Numerous variants of Discosoma sp. Red Fluorescent Protein, commercially known as DsRed, have been developed over the past decade. The variants include; DsRed2, DsRed-Express, and DsRed1-E5- which is the Fluorescent Timer. Most researchers have made attempts to understand the DsRed fluorophore in terms of its structure, and the light absorbing and emitting characteristics (Bevison & Glick, 2002). The DsRed1-E5 is a variant that contains two amino acid substitutions (V105 and S197T). These acid substitutions serve to increase the fluorescence intensity of DsRed1-E5 giving it a unique spectral property. The variant changes in color as the protein ages (Bevison & Glick, 2002). Once synthesized, the DsRed1-E5 starts emitting fluorescence which is green in color. With time the wavelengths of the fluorescence shifts to the longer regions, causing a change in color. The protein becomes bright red when it is mature. It is the predictable color change that can be used to monitor the on and off phases of gene expression (Talbot, 2001). The DsRed1-E5 enables the detection of the green and red emissions using fluorescence microscopy and flow cytometry. The change in color with time facilitates the tracking of not only up-regulation, but also down-regulation in the process of gene expression (Bevison & Glick, 2002).
The paper has discussed Trichoderma reesei as an industrially useful cellulolytic filamentous fungus. Some of the numerous uses of T. reesei have been highlighted. The types of transformation markers available for fungi have identified. Moreover, the difference between genetically encoded and chemical fluorescent markers has been pointed out. The paper has also explained what DsRed1-E5 is and its uses.
Bevison, B. J. & Glick, B. S. (2002). Rapidly maturing variants of the Discosoma red fluorescent protein (DsRed). Journal of National Biotechnology, 20 (3): 83–9.
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Sparks, D. L. (2006). Advances in biotechnology: genetically encoded and chemical fluorescent markers. Academic Press.
Talbot, N. J. (2001). Molecular and cellular biology of filamentous fungi: a practical approach. Oxford University Press.
Thieman, W. J. & Palladino, M. A. (2009). Introduction to Biotechnology (2nd ed). San Francisco, CA: Pearson.