Team:Frankfurt/Project/Results

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Revision as of 01:42, 5 October 2013

Team: iGEM Frankfurt - 2012.igem.org

Gap repair Cloning for Vector Assembly

Gap repair means the homologue recombination in yeast in order to assemble desired DNA fragments. It is easier and more effective than cloning via restriction and ligation. This method was used to assemble the mevalonate overexpression plasmid in year 2012. Since in last year the KAH fragment, which is crucial for the second plasmid, was not able to be constructed the reconstruction of the steviol plasmid via gap repair was done this year. We were able to grow cultures containig the plasmid on selective media with the corresponding auxotrphy, but unfortunately we were to short on time to extract it.

Gas Chromatographic Analysis

Since the first plasmid created last year leads to a higher amount of geranylgeranylpyrophosphate, is was one of our aims for this year to prove that this actually worked out the way it was intended. For this reason we used gas chromatography coupled with mass spectrometry - but sadly didn't get the results in time to present them here.

iGEM-Team Frankfurt 2013

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 {{:Team:Frankfurt/NavBarTemp/Header}}{{:Team:Frankfurt/NavBarTemp/NavCss}}
-== DNA constructs for realisation of our idea == __NOEDITSECTION__
-The DNA constructs for steviol production in yeast were designed in year 2012 and could be reused to
-achieve this years objectives.
-===Mevalonate Pathway Overexpression=== __NOEDITSECTION__
-The vector for improving the MVA pathway flow contains three genes: A trunctated version of the HMG-CoA-Reductase, the native gene of the Farnesylpyrophosphate Synthase (ERG20) and the GGPP Synthase gene from ''Sulfolobius acileratius''. The sources of the physical DNA were as following: <br> <br>
-* <big> HMG-CoA-Reductase: </big> The sequence was obtained from the Saccharomyces Genome Database and modified. This version was send to a company for ''de novo'' synthesis.
-* <big> FPP-Synthase: </big> The gene was obtained from genomic DNA of the strain CEN.PK2. By using primers which contained overlaps with the pre- and suffix sequences it was amplified by PCR.
-* <big> GGPP-Synthase </big>: The sequence was obtained from the Pubmed Database and modified. This version was send to a company for ''de novo'' synthesis.
-<br>
-construction of the insert:
-pHXT7 ''HMG-CoA'' tHXT7  pPFK1 ''ERG20'' tPFK2 pPGK1 ''GGPPS'' tCYC1<br>
-plasmid backbone with ''URA3''
-===Steviol Production=== __NOEDITSECTION__
-The vector for steviol production also contains three genes: The bifunctional cyclase from ''Gibberella fujikuroi'' (CPS/KS), the ''ent''-kaurene oxidase from ''Gibberella fujikuroi'' (KO) and the ''ent''-kaurenoic acid hydroxylase from ''Stevia rebaudiana'' (KAH). The sources of the physical DNA were as following: <br> <br>
-* <big> bifunctional cyclase: </big> The sequence was obtained from the Pubmed Database and modified. This version was send to a company for ''de novo'' synthesis.
-* <big> ''ent''-kaurene oxidase: </big> The sequence was obtained from the Pubmed Database and modified. This version was send to a company for ''de novo'' synthesis (4 x 500 bp for Gibson Assembly).
-* <big> ''ent''-kaurenoic acid hydroxylase: </big> The sequence was obtained from the Pubmed Database and modified. This version was send to a company for ''de novo'' synthesis (4 x 500 bp for Gibson Assembly).
-<br>
-construction of the insert:
-pHXT7 ''CPS/KS'' tTAL1  pTPI1 ''KO'' tPDC1 pPGI1 ''KAH'' tCYC1<br>
-plasmid backbone with ''HIS3''
 == Gap repair Cloning for Vector Assembly == __NOEDITSECTION__
 <div style="text-align:block; direction: ltr; margin-left: 1em; width:920px">
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 extract it.
-</div>
 == Gas Chromatographic Analysis == __NOEDITSECTION__
 Since the first plasmid created last year leads to a higher amount of geranylgeranylpyrophosphate, is was one of our aims for this year to prove that this actually worked out the way it was intended. For this reason we used gas chromatography coupled with mass spectrometry - but sadly didn't get the results in time to present them here.
+</div>
 {{:Team:Frankfurt/Footer}}