Team:Frankfurt/Project/Results
From 2013.igem.org
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plasmid backbone with ''HIS3'' | plasmid backbone with ''HIS3'' | ||
== Gap repair Cloning for Vector Assembly == __NOEDITSECTION__ | == Gap repair Cloning for Vector Assembly == __NOEDITSECTION__ | ||
- | 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 | + | <div style="text-align:block; direction: ltr; margin-left: 1em; width:920px"> |
+ | 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. | extract it. | ||
+ | </div> | ||
== Gas Chromatographic Analysis == __NOEDITSECTION__ | == 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. | |
{{:Team:Frankfurt/Footer}} | {{:Team:Frankfurt/Footer}} |
Revision as of 01:39, 5 October 2013
![iGEM Frankfurt Header](https://static.igem.org/mediawiki/2013/e/e7/IGEM-LOGO.png)
Contents |
DNA constructs for realisation of our idea
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
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:
- HMG-CoA-Reductase: The sequence was obtained from the Saccharomyces Genome Database and modified. This version was send to a company for de novo synthesis.
- FPP-Synthase: 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.
- GGPP-Synthase : The sequence was obtained from the Pubmed Database and modified. This version was send to a company for de novo synthesis.
construction of the insert:
pHXT7 HMG-CoA tHXT7 pPFK1 ERG20 tPFK2 pPGK1 GGPPS tCYC1
plasmid backbone with URA3
Steviol Production
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:
- bifunctional cyclase: The sequence was obtained from the Pubmed Database and modified. This version was send to a company for de novo synthesis.
- ent-kaurene oxidase: 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).
- ent-kaurenoic acid hydroxylase: 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).
construction of the insert:
pHXT7 CPS/KS tTAL1 pTPI1 KO tPDC1 pPGI1 KAH tCYC1
plasmid backbone with HIS3
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.