Difference between revisions of "Team:Minnesota/2A Tags"
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− | <font size="3">• </font> 2A peptide | + | <font size="3">• </font> The 2A peptide sequence, also known as a cisacting hydrolase element, is ~20 amino acids in length and can easily be placed between genes of interest. When translated, the sequence causes the ribosome to skip over a peptide bond and allow the translation of multiple discrete polypeptides from a single mRNA molecule, leaving an 18 amino acid sequence on the C-terminus of the upstream protein and a proline residue on the N-terminus of the downstream protein (5). 2A viral sequences are particularly promising due to their small size (~60-70 nucleotides) and high cleavage rate which has been found to form 1:1 molar ratios of gene product in biscistronic sequences (3). These features are especially impressive when compared to internal ribosome entry sites (IRES), another popular method for creating polycistronic sequences in eukaryotes. IRES require large sequences (~500 nucleotides), which can be problematic when using size restricted vectors and can experience up to a 10-fold decrease in expression levels for downstream gene products (5). The 2A tag sequence can be used to achieve ribosomal “skip” at the transcript C terminus of the upstream gene, allowing a discrete protein to be produced from that sequence and polycistronic expression in a eukaryotic organism. However, despite the attractiveness of using 2A sequences to create large multi-enzyme polycistronic sequences, little work has been done beyond simple bicistronic insertions and preliminary investigations suggest that the gene order within larger polycistronic sequences can affect the overall efficiency of larger pathways such that genes further downstream of the translational start site have lower levels of translation in 2A polycistronic sequences, and that the differences in relative molar amounts in enzyme result in the changes of product production (5). Understanding how the order of genes in longer polycistronic sequences affects translation rates is important for optimizing engineered metabolic pathways and limiting the buildup of potentially toxic intermediates. |
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<i><u><font size="3">• </font>Experimental Approach</u></i> | <i><u><font size="3">• </font>Experimental Approach</u></i> | ||
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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%> | ||
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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