Difference between revisions of "Team:Minnesota/2A Tags"
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− | <font size="3">• </font> 2A peptide sequences are ~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). 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 (5). | + | <font size="3">• </font> 2A peptide sequences are ~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). 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> |
Revision as of 23:24, 18 September 2015