Difference between revisions of "Team:Minnesota/2A Tags"
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<h1>Polycistronic Eukaryotic Gene Expression</h1><br> | <h1>Polycistronic Eukaryotic Gene Expression</h1><br> | ||
− | Over the last several decades, the metabolic engineering of microbes to produce medicines, biofuels, food additives, and enzymes has grown to a multi-billion dollar industry. The use of engineered biosynthetic pathways has many advantages over more conventional organic methods including cost efficiency, relative environmental friendliness, chiral specificity, and the ability to produce products that would otherwise be difficult to synthesize with conventional organic chemistry techniques (6). While much of current metabolic engineering is being done in bacteria such as Escherichia coli due to its high growth rate and ease to work with, eukaryotic systems, such as <i>Saccharomyces cerevisiae</i> the industrially relevant yeast, <i>Pichia pastoris</i>, are also attractive and have received much attention due to their ability to glycosylate proteins and form disulfide bonds (1) within proteins which may otherwise be misfolded and inactive if expressed in a prokaryotic system. However, expressing novel and complex metabolic pathways in a eukaryotic system such as <i>S. cerevisiae</i> has its own unique problems. Unlike in <i>E. coli</i>, where it is possible to organize a large number of coding sequences into a single operon with conventional methods, each gene in a eukaryotic system requires its own promoter, transcription factors, and ribosome binding site. Coordinating multiple unique promoters and the absence of sufficient antibiotic selective markers provides a significant | + | Over the last several decades, the metabolic engineering of microbes to produce medicines, biofuels, food additives, and enzymes has grown to a multi-billion dollar industry. The use of engineered biosynthetic pathways has many advantages over more conventional organic methods including cost efficiency, relative environmental friendliness, chiral specificity, and the ability to produce products that would otherwise be difficult to synthesize with conventional organic chemistry techniques (6). While much of current metabolic engineering is being done in bacteria such as Escherichia coli due to its high growth rate and ease to work with, eukaryotic systems, such as <i>Saccharomyces cerevisiae</i> the industrially relevant yeast, <i>Pichia pastoris</i>, are also attractive and have received much attention due to their ability to glycosylate proteins and form disulfide bonds (1) within proteins which may otherwise be misfolded and inactive if expressed in a prokaryotic system. However, expressing novel and complex metabolic pathways in a eukaryotic system such as <i>S. cerevisiae</i> has its own unique problems. Unlike in <i>E. coli</i>, where it is possible to organize a large number of coding sequences into a single operon with conventional methods, each gene in a eukaryotic system requires its own promoter, transcription factors, and ribosome binding site. Coordinating multiple unique promoters and the absence of sufficient antibiotic selective markers provides a significant challenge, and the use of several identical promoters poses the risk of interference, recombination, and suppression between the promoters (2, 4, 5, 8). To circumnavigate this problem, polycistronic sequences can be recreated in eukaryotes using viral 2A sequences (7). |
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+ | <font size="3">References | ||
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+ | (1) Cregg, J. M.; Tolstorukov, I.; Kusari, A.; Sunga, J.; Madden, K.; Chappell, T., Expression in the yeast Pichia pastoris. Meth. Enzymol. 2009, 463:169-89 | ||
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+ | (2) Cullen, B. R.; Lomedico, P. T.; Ju, G., Transcriptional interference in avian retroviruses - implications for the promoter insertion model of leukaemogenesis. Nature. 1984, 307(5948): 241-245 | ||
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+ | (3) Donnelly, M. L. L.; Hughes, L. E.; Luke, G. A.; Mendoza, H.; ten Dam, E.; Gani, D.; Ryan, M. D., The “cleavage” activities of foot-and-mouth disease virus 2A site-directed mutants and naturally occuring “2A-like” sequences. Journal General Virology, 2001, 82: 1027-1041 | ||
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+ | (4) Emmerman, M.; Temin, H. M., Comparison of promoter suppression in avian and murine retrovirus vectors. Nucleic Acids Res. 1986, 14(23): 9381-9396 | ||
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+ | (5) Geier, M.; Fauland, P.; Vogl, T.; Glieder, A., Compact multi-enzyme pathways in P. pastoris. Chem. Commun. 2014, 51, 1643-1646. | ||
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+ | (6) Keasling, J. D.; Mendoza, A.; Baran, P. S., Synthesis: A constructive debate. Nature. 2012, 492: 188-189 | ||
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+ | (7) Ryan, M. D.; Drew, J., Foot-and-mouth disease virus 2A oligopeptide mediated cleabage of an artificial polyprotein. EMBO J. 1994, 13(4): 928-933 | ||
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+ | (8) Zhu, T.; Guo, M.; Sun, C.; Qian, J.; Zhuang, Y.; Chu, J.; Zhang S., A systematical investigation on the genetic stability of multi-copy Pichia pastoris strains. Biotechnol Lett. 2009, 31(5): 679-84 | ||
+ | </font> | ||
<i><u><font size="3">• </font>Why are we expressing human Insulin?</u></i> | <i><u><font size="3">• </font>Why are we expressing human Insulin?</u></i> |
Revision as of 23:10, 18 September 2015