Difference between revisions of "Team:Minnesota/2A Tags"

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<i><font size="3">• </font> &nbsp; &nbsp; &nbsp; 2A peptide sequences are ~20 amino acids in length and can easily be places 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).</i>
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<font size="3">• </font> &nbsp; &nbsp; &nbsp; 2A peptide sequences are ~20 amino acids in length and can easily be places 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).
 
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Revision as of 23:04, 18 September 2015

Team:Minnesota/Project/Insulin - 2015.igem.org

 

Team:Minnesota/Project/Insulin

From 2015.igem.org

Team:Minnesota - Main Style Template Team:Minnesota - Template

Polycistronic Eukaryotic Gene Expression


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 the industrially relevant yeast, Pichia pastoris, 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 P. pastoris has its own unique problems. Unlike in E. coli, 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 challenged, 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).

Viral 2A Tags: Basic Background



      2A peptide sequences are ~20 amino acids in length and can easily be places 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).
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Why are we expressing human Insulin?
      The ability to produce recombinant human Insulin cheaply has long been a lucrative goal. There are millions of people worldwide who are dependent on Insulin derived from production methods that make the product expensive -and further yet- potentially dangerous.Our team thinks that the current production methods for human Insulin are inefficient and can be optimized by being expressed in Pichia pastoris.