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| | <h1>Designing a BioBrick Compatible Pichia Expression System</h1><br> | | <h1>Designing a BioBrick Compatible Pichia Expression System</h1><br> |
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| - | <center><font size="3"><b>Basic BioBrick Expression System Background </b></font></center>
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| - | <i><u><font size="4">• </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.
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| - | <i><u><font size="4">• </font>Why did we choose P. Pastoris?</u></i>
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| - | P. pastoris has a glycosylation pattern that is more compatible with the human immune system, when compared to the glycosylation pattern of Saccharomyces cerevisiae. P. pastoris is also known for its ability to grow in high densities using methanol as its only food source. Despite the usefulness of yeast species such as P. pastoris there are currently few items in the parts registry that are designed for use within yeast, and none that are specifically designed to be used with P. pastoris. Our team intends on producing pBB3G1 and pBB1Z1, two BioBrick compatible P. pastoris-E. coli shuttle vectors.
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| - | <i><u><font size="4">• </font>What are some benefits of this vector system?</u></i>
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| - | The pBB3G1 and pBB1Z1 vectors include features that increase its ease-of-use and versatility, such as: optional inducibility, optional product secretion, trans-kingdom conjugation (TKC), and episomal maintenance of the vector in the host organism. The vectors vary in their expression level. Constitutive expression is achieved in pBB1Z1 by the pGAP promoter. Methanol-induced expression is available in pBB3G1 by means of the pAOX1 promoter.
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| - | <i><u><font size="4">• </font> Why use Trans Kingdom Conjugation instead of traditional transformation methods?</u></i>
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| - | TKC involves the transfer of DNA from a bacterial cell to a eukaryotic cell, by means of conjugation. Harnessing the ability to shuttle plasmids between E. coli and P. pastoris through TKC would simplify transformation protocols. Currently, transformation methods (using shuttle plasmids cloned in E. coli) are a time consuming process, requiring isolation of the cloned plasmid and transformation into yeast. Transformation using TKC shortens the process by transferring the plasmid directly into the yeast cell. Utilizing TKC as a transformation protocol would translate to faster results in the laboratory and reduced costs in an industrial setting. Currently there are no BioBrick vectors in the parts registry that enable TKC between E. coli and P. pastoris.
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| - | <i><u><font size="4">• </font>How will TKC be achieved?</u></i>
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| - | TKC functionality is provided by the OriT<sup>P</sup> sequence which hosts the nick site that will be cleaved during the initiation of conjugation in order to linearize the plasmid, and ligated once transferred to the recipient. Importantly, the OriT<sup>P</sup> sequence -compared to other variations of OriT- does not require the presence of a helper plasmid within the recipient to complete the final ligation step of conjugal transfer.
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| - | <i><u><font size="4">• </font>Are there limitations to this system?</u></i>
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| - | One limitation to using P. pastoris as an expression system is that most shuttle plasmids must be integrated into the yeast chromosome. This results in lower expression of desired protein products, as well as lower transformation efficiency. We hope to improve the functionality of the pBB3G1/pBB1Z1 system by allowing the plasmid -once transferred to the yeast cell- to remain as an episomal plasmid. This is made possible by the inclusion of the PARS1 yeast autonomous replication sequence. This sequence ensures that the plasmid is maintained through several (~200) generations.
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| - | <i><u><font size="4">• </font>How will we screen the genes?</u></i>
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| - | Transformant selection is simplified by the inclusion of antibiotic resistance genes that are effective in both E. coli and P. pastoris. The backbones that will be submitted to the parts registry will include either Zeocin or Geneticin, however the resistance genes have been cloned into a KpnI site, and may be swapped for any selective marker specific to the user’s needs.
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| - | <p><b>Goal</b><br> To design a BioBrick compatible <i>E. coli</i>-<i>P. pastoris</i> shuttle vector.</p>
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| - | <p><b>Background</b><br>
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| - | <!--2013 edit <img style="width:500px;" src="http://i1158.photobucket.com/albums/p607/iGEM_MN/caffeine_synth_resize.png">
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| - | <p><b>Figure 1. Scheme showing proposed caffeine biosynthetic pathway in S. cerevisiae. </b></p>
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| - | <p><b>Methods</b><br>
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| - | <b>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.
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| - | After settling on the two genes, the sequences were taken from NCBI and finalized for synthetic synthesis (XMT1 accession number DQ422954 and DXMT1 accession number DQ422955). A program was designed to optimize these plant genes for yeast use. Because of the similarity of the two genes (~88% similarity) the program was also designed to identify regions of homology between the two genes and adjust the codon usage such that homologous recombination will not occur at the nucleotide level while the overall protein sequence would be retained. The codons were chosen in descending order of complementary tRNA abundance. Any BioBrick cut sites found within the gene sequences were also removed.</p>
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| - | <p>To synthesize the overall plasmid backbone, PCR primers were designed to amplify five desired fragments (with 25-30 bp overlap) from different plasmids, which could be joined by Gibson Assembly:<br>
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| - | 1. BioBrick destination plasmid pSB1C3 containing the MCS and rep (pMB1).<br>
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| - | 2. BioBrick destination plasmid pSB1C3 containing the Chloramphenical resistance gene. <br>
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| - | 3. 2u Gibson Extract provided by the Schmidt-Dannert lab (University of Minnesota) containing the 2u ORI for yeast. <br>
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| - | 4. pkT127 provided by the Schmidt-Dannert lab containing G418 resistance genes and tTEF1.<br>
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| - | 5. ATCC plasmid provided by the Schmidt-Dannert lab containing the ADH1 promoter to drive the G418 R gene.</p>
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| - | <p><b>Parts List</b><br>
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| - | BBa_K814000 dehydroquinate synthase (DHQS) generator<br>
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| - | BBa_K814001 ATP-grasp (ATPG) generator<br>
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| - | BBa_K814002 dehydroquinate O-methyltrasferase (O-MT) generator<br>
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| - | BBa_K814003 shinorine non-ribosomal peptide synthase (NRPS) generator<br>
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| - | BBa_K814004 mycosporine-glycine biosynthetic pathway<br>
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| - | BBa_K814005 shinorine biosynthetic pathway<br>
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| - | BBa_K814006 negative control for mycosporine-like amino acid biosynthesis<br>
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| - | BBa_K814007 ScyA (acetolactate synthase) generator<br>
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| - | BBa_K814008 ScyB (leucine dehydrogenase) generator<br>
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| - | BBa_K814009 ScyC generator<br>
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| - | BBa_K814010 partial scytonemin biosynthetic pathway, scyCB<br>
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| - | BBa_K814011 scytonemin biosynthetic pathway, scyBAC<br>
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| - | BBa_K814012 XMT1 protein generator<br>
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| - | BBa_K814013 DXMT1 protein generator
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| - | </p>
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| - | <!-- 2013 edit
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| - | <img src="http://i1158.photobucket.com/albums/p607/iGEM_MN/colorarrowscopy.jpg">
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| - | <p><b>Figure 2:</b> Pieces for Gibson assembly of our novel, shuttle backbone. The individual pieces relate to the described components enumerated above. 1. Purple arrow; 2. Orange arrow; 3. Green arrow; 4. Blue arrow; 5. Light green arrow.</p>
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| - | <img src="http://i1158.photobucket.com/albums/p607/iGEM_MN/cells.jpg
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| - | <p><b>Figure 3. Creation of a shuttle vector designed for BioBrick components.</b> A), Overnight culture of the pSB1C3 shipping vector showing RFP expression (a blank LB tube is shown as a control on the right). B), RFP expression in the pGHMM2012 shuttle vector. Cell pellets clearly indicate expression of RFP from this plasmid. C), PCR screening for caffeine biosynthetic components into pGHMM2012. The panels from left to right are positive clones for Xmt, Dxmt and controls for each of these components.</p>
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| - | <img style="width:500px;" src="http://i1158.photobucket.com/albums/p607/iGEM_MN/growth_curve_resize.png">
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| - | <p><b>Figure 4. Investigation of caffeine toxicity in yeast. </b>The yeast were resilient in the presence of caffeine and there was no significant decrease in growth overtime at each different concentration. For reference, the concentration of caffeine in coffee is roughly 600mg/L.</p>
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| - | <img src="http://i1158.photobucket.com/albums/p607/iGEM_MN/hplccaffeine-1_resize.jpg">
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| - | <p><b>Figure 5. HPLC Data. </b>description.</p>
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| - | <p><b>Conclusions</b><br>
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| - | We were able to design and synthesize a novel yeast- E. coli hybrid plasmid which was optimized to prevent homologous recombination in our two synthetic genes: XMT1 and DXMT1. </p>
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| - | <p>If the HPLC results can confirm the presence of caffeine in the yeast cultures, we would confirm the presence of the two target proteins by SDS-PAGE, possibly even performing antibody detection if there is difficulty confirming the presence.</p>
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| - | <p>If the HPLC results cannot confirm the presence of caffeine, the first step would be to sequence our genes and align them with the sequences submitted to IDT for the gBlocks. We could also measure the production of the intermediates compared to the cultures to test whether caffeine synthesis is being arrested in the pathway.</p>
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