Team:Minnesota/Project/Pichia Expression System
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- | < | + | <b><font size="4">Basic Background </font></b> |
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- | <i><u><font size=" | + | <i><u><font size="3">• </font>What's the idea behind this expression system?</u></i> |
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Laboratory and industrial projects that involve the high volume production of a protein often select E. coli as an expression system due to its rapid growth. However, bacterial expression systems are not always a viable option. In the case where proper folding of the protein of interest requires post-translational modification (such as the addition of disulfide bonds or glycosylation,) a eukaryote must be used. Although several well-defined eukaryotic options exist, yeast is often selected for its ease of use in the laboratory. One yeast species in particular, Pichia pastoris, has gained popularity as an expression system for recombinant human proteins. | Laboratory and industrial projects that involve the high volume production of a protein often select E. coli as an expression system due to its rapid growth. However, bacterial expression systems are not always a viable option. In the case where proper folding of the protein of interest requires post-translational modification (such as the addition of disulfide bonds or glycosylation,) a eukaryote must be used. Although several well-defined eukaryotic options exist, yeast is often selected for its ease of use in the laboratory. One yeast species in particular, Pichia pastoris, has gained popularity as an expression system for recombinant human proteins. | ||
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- | <p><b> | + | <p><b><font size="3">Methods</font></b><br> |
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- | + | <b><font size="4">• </font>Vector Design</b><br> | |
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Sequences for AOX1, GAP, G418, and Zeocin were obtained from pPICZ, pPICZα, pGAP from Invitrogen. The Wintergreen odor generator (BBa_J45700) was selected as a reporter gene, as well as yeast green fluorescent protein (GFP). In order to improve upon an existing part in the Registry of Standard Biological Parts, we codon-optimized the Wintergreen odor generator for expression in P. pastoris using the Codon Optimization tool provided by IDT. Kozak sequences were added for each open reading frame to be expressed in P. pastoris. The basal vectors pMNBB-ICI and pMNBB-CCI (please refer to Vector Nomenclature, and Figure 1) were assembled from the above sequences in Clone Manager 9. | Sequences for AOX1, GAP, G418, and Zeocin were obtained from pPICZ, pPICZα, pGAP from Invitrogen. The Wintergreen odor generator (BBa_J45700) was selected as a reporter gene, as well as yeast green fluorescent protein (GFP). In order to improve upon an existing part in the Registry of Standard Biological Parts, we codon-optimized the Wintergreen odor generator for expression in P. pastoris using the Codon Optimization tool provided by IDT. Kozak sequences were added for each open reading frame to be expressed in P. pastoris. The basal vectors pMNBB-ICI and pMNBB-CCI (please refer to Vector Nomenclature, and Figure 1) were assembled from the above sequences in Clone Manager 9. | ||
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+ | <img src="http://i791.photobucket.com/albums/yy194/GopheriGEM/basalvectorspMNBB_zps0c246be7.png"> | ||
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+ | <b><i>Figure 1. Basal vectors of the pMNBB P. pastoris expression system. Note that the basal vectors do not contain the oriT sequence that enables conjugation, nor the PARS1 sequence that confers episomal maintenance. Restriction enzymes presented here are entirely unique to ensure compatibility with the BioBrick™ platform.</i></b> | ||
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+ | <img src="http://i791.photobucket.com/albums/yy194/GopheriGEM/pMNBB-ICIandpMNBB-ITI_zpscb577f1e.png"> | ||
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+ | <i><b>Figure 2. Creating the conjugal variant of pMNBB-IXX. (A.) As seen in pMNBB-ICI, the pAOX1 promoter does not contain the OriT sequence. (B.) Addition of the OriT sequence within the AOX1 promoter enables conjugal transfer. | ||
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The project was designed to yield two basal P. pastoris expression vectors, as well as two variations upon the basal vectors, as listed in Table 1. | The project was designed to yield two basal P. pastoris expression vectors, as well as two variations upon the basal vectors, as listed in Table 1. | ||
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- | Vector Assembly | + | <b><font size="4">• </font>Vector Assembly</b> |
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We began assembling the vectors by amplifying the individual fragments by PCR using Phusion HF polymerase (New England Biolabs). Next, we attempted several transformations of E. coli C2566 with Gibson assemblies of the vectors. Unfortunately the transformations resulted in either no growth, or growth of unidentifiable colonies, as determined by Go-Green Taq (Promega) colony screens. We believe that the lack of growth may be due to vector not circularizing, or in the case of the unidentifiable colonies that appeared on the pMNBB-CCI plates, limited selectivity imparted by our Zeocin media (25ug/mL). | We began assembling the vectors by amplifying the individual fragments by PCR using Phusion HF polymerase (New England Biolabs). Next, we attempted several transformations of E. coli C2566 with Gibson assemblies of the vectors. Unfortunately the transformations resulted in either no growth, or growth of unidentifiable colonies, as determined by Go-Green Taq (Promega) colony screens. We believe that the lack of growth may be due to vector not circularizing, or in the case of the unidentifiable colonies that appeared on the pMNBB-CCI plates, limited selectivity imparted by our Zeocin media (25ug/mL). | ||
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- | <b>Results</b> | + | <b><font size="4">• </font>Results</b> |
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First we investigated the possibility that the vectors were not circularizing. In order to simplify the Gibson assembly we began overlapping fragments, reducing the pieces that would need to join in a successful Gibson assembly. The initial two piece overlaps were successful, however overlaps beyond two pieces were problematic. Gibson assemblies in which the fragment number had been reduced by using two piece overlaps produced the same results as the single piece Gibson assemblies, either no growth, or growth of unidentified colonies. | First we investigated the possibility that the vectors were not circularizing. In order to simplify the Gibson assembly we began overlapping fragments, reducing the pieces that would need to join in a successful Gibson assembly. The initial two piece overlaps were successful, however overlaps beyond two pieces were problematic. Gibson assemblies in which the fragment number had been reduced by using two piece overlaps produced the same results as the single piece Gibson assemblies, either no growth, or growth of unidentified colonies. | ||
We decided to try an alternate PCR protocol (Shevchuk et al. 2004). Finally we were able to string together the five fragments of pMNBB-ICI. | We decided to try an alternate PCR protocol (Shevchuk et al. 2004). Finally we were able to string together the five fragments of pMNBB-ICI. | ||
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- | <b>Conclusions</b> | + | <b><font size="4">• </font>Conclusions</b> |
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Unfortunately we have not yet been able to assemble either vector in its entirety. The basal inducible vector, pMNBB-ICI, has been visualized in its linear state on a gel. We will continue to work on assembling both vectors so that they may be available for future teams to improve upon and characterize. If the pMNBB system functions as expected, it will certainly be a very useful tool not only for iGEM teams, but for academic research, and industrial processes. | Unfortunately we have not yet been able to assemble either vector in its entirety. The basal inducible vector, pMNBB-ICI, has been visualized in its linear state on a gel. We will continue to work on assembling both vectors so that they may be available for future teams to improve upon and characterize. If the pMNBB system functions as expected, it will certainly be a very useful tool not only for iGEM teams, but for academic research, and industrial processes. | ||
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Latest revision as of 02:17, 28 September 2013