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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 |
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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). |
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| + | <b>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. |
| + | 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. |
| + | <br><br> |
| + | <b>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. |
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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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- | <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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