Difference between revisions of "Team:Minnesota/2A Tags"
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Our team developed a <a href="https://2015.igem.org/Team:Minnesota/Modeling">Mathematical Model </a> to estimate gene order for optimal biosynthetic production using 2A sequences and gladly present it as an open-source community tool to streamline future applications. We also attempted to demonstrate the utility of this technology in yeast by expressing genes to produce compounds in the beta carotenoid pathway. | Our team developed a <a href="https://2015.igem.org/Team:Minnesota/Modeling">Mathematical Model </a> to estimate gene order for optimal biosynthetic production using 2A sequences and gladly present it as an open-source community tool to streamline future applications. We also attempted to demonstrate the utility of this technology in yeast by expressing genes to produce compounds in the beta carotenoid pathway. | ||
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<img src="https://static.igem.org/mediawiki/2015/f/f1/PMN500.png" width=96% height=86%> | <img src="https://static.igem.org/mediawiki/2015/f/f1/PMN500.png" width=96% height=86%> | ||
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+ | The genes of interest illustrated above <i>(crtEBI)</i> were synthesized via IDT and cloned into a <i> pESC-URA</i> plasmid as shown via homologous recombination in <i>S. cerevisiae</i>, and screened by induction of the galactose promoter. Since <i>S.cerevisiae</i> is capable of producing the precursor farnesyl diphosphate, successful transformants expressing all 3 genes would turn red in the presence of galactose due to the lycopene produced while the unsuccessful ones would remain white. | ||
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+ | <img src="https://lh6.googleusercontent.com/647DkU0hioAWE7U4i3RvLqEyiAsF4oe-COiOIhjYp1LKvf-QXdmXcQt0lPhzVZOroyXYBy5_VZhTNw=w2536-h1335" width=96% height=86%> | ||
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+ | We could then simultaneously inoculate <i>S. cerevisiae</i> in order to harvest the plasmid and for transformation into <i>E. coli </i>. Once a successful transformation had occurred, we could again harvest the plasmid, digest it for our insert, and ligate it into a linearized shipping vector <i>(pSB1C3)</i>. Testing is currently in progress. | ||
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+ | The efficiency of the 2A sequences is also to be tested by inserting a yeast enhanced GFP gene between each of the crt genes. We would then grow the yeast, read the GFP fluorescence using a plate reader, and normalize the readings based on OD600. | ||
+ | Ideally, we plan to test the model by reorganizing the genes and quantifying the amount of lycopene that was produced using high-purity liquid chromatography, and use the data to enforce model validity. | ||
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+ | <a href="https://2015.igem.org/Team:Minnesota/Practices">Click here to learn more about our software development, education, and public engagement work! </a> | ||
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<font size="1.5">References | <font size="1.5">References | ||
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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 | (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- | + | (5) Geier, M.; Fauland, P.; Vogl, T.; Glieder, A., Compact multi-enzyme pathways in P. pastoris. Chem. Commun. 2014, 51, 1643- |
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Latest revision as of 02:11, 19 September 2015