it s gonna be sweet - main page - 2012.igem.org2012.igem.org/files/presentation/frankfurt.pdf ·...
TRANSCRIPT
It‘s gonna be sweet
Team Frankfurt
Advantages of „Stevia“
Stevia = composite of several steviol glycosides
anticariogenic
antidiabetic
no impact on blood sugar
no delay of satiety
few calories
pH stable, heat stable
protective effect on vitamin C
sweet diterpenes have certain advantages compared to both
sugar and other sweeteners
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Rebaudioside A
Rebaudioside A = steviol glycoside of the stevia plant
best taste
highest sweetness (300 × saccharose)
least bitterness of all steviol glycosides
extraction:
stevia plants are dried and a composition (including rebaudioside A)
of steviol glycosides are isolated
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Saccharomyces cerevisiae – a model organism
Why do we use S.cerevisiae?
two required enzymes are localized in the ER
established food additive producer
closer related to Stevia than E.coli is
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Metabolic pathway
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Metabolic pathway
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Metabolic pathway
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Metabolic pathway
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Gap repair – homologue recombination in yeast
Homologous Sequences:
At least 40 bp long
Free ends will be
recombined
Similiar to double strand
break repair
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Gap repair – example from our project
Required Components
linearized plasmid
DNA fragments containing
appropriate homology to the
neighbouring fragments
In vivo assembly in yeast
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Yeast BioBrick Assembly (RFC proposal)
Aim:
Getting biobricks compatible to gap
repair cloning
construction of two standard
primers for gene amplification
Primers contain homologue overlaps
to terminator/promoter-fragments
Gap repair cloning is possible
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construction of standard primers that bind prefix of promoter and
have a homologue overlap to the respective terminator
possibility to combine every promoter every terminator
Aim:
Variability of the terminator/promoter-fragment
Yeast BioBrick Assembly (RFC proposal)
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Results of the project Assembly of the mevalonate pathway overexpression plasmid and
transformation in yeast
Assembly of the steviol synthesis plasmid
Creation of BioBricks of genes
• HMG-CoA-R: BBa_K849000
• FPPS: BBa_K849001
• Bifunctional Cyclase: BBa_K849003
• KO: BBa_K849004
Geranylgeranyl pyrophosphate detection
Yeast BioBrick Assembly (new BioBrick RFC proposal)
Human practice & Ethics
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Acknowledgements
Martin, Tamara, Corinna, Virginia, Charlotte, Andre, Marco Sebastian, Christian, Leonard, Wagner, Jana (from left to right)
Team Frankfurt 2012 thanks :
Prof. E. Boles (instructor, lab support)
Boles group (lab support)
Prof. J. Soppa (lab space)
Prof. K. Fendler (financial support)
Prof. M. Schubert-Zsilavecs (university support)
Prof. B. Tudzynski (provision of plasmids)
Prof. H. Bode (analytical support)
Prof. H. Schwalbe (recomendation)
Christina Wagner (financial management)
Prof. E. Bamberg (financial support)
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References
Pictures: Slide 1: CC-BY Ethel Aardvark: http://en.wikipedia.org/wiki/File:Stevia_rebaudiana_foliage.jpg. 04.10.2012
Slide 2: http://www.sugarbeater.com/joomla/images/stories/white%20powder%20stevia.jpg. 04.10.2012
Slide 4: http://130.91.8.212/images/S_cerevisiae.jpg. 04.10.2012
http://de.wikipedia.org/wiki/Datei:Nucleus_ER.svg. 04.10.2012
Key References: • J.E. Brandle, P.G. Telmer, 2007. Steviol glycoside biosynthesis. Phytochemistry. 68, 1855–1863.
• Tania V. Humphrey, Alex S. Richman, Rima Menassa, Jim E. Brandle, 2011. Spatial organisation of four enzymes from Stevia rebaudiana that are involved in steviol glycoside synthesis. Plant Molecular Biology. 61, 47–62.
• Pia Dahm, 2011. Metabolic engineering der Taxolbiosynthese in Saccharomyces cerevisiae.
• K. A. Donald, R. Y. Hampton, I. B. Fritz, 1997. Effects of overproduction of the catalytic domain of 3-hydroxy- 3methylglutaryl coenzyme A reductase on squalen synthesis in Saccharomyces cerevisiae. Applied and Environmental Microbiology. 63 (9), 3341.
• Kenro Tokuhiro, Masayoshi Muramatsu, Chikara Ohto, Toshiya Kawaguchi, Shusei Obata, Nobuhiko Muramoto, Masana Hirai, Haruo Takahashi, Akihiko Kondo, Eiji Sakuradani, Sakayu Shimizu, 2009. Overproduction of Geranylgeraniol by Metabolically Engineered Saccharomyces cerevisiae. Applied and Evironmental Microbiology. 75 (17), 5536. DOI: 10.1128/AEM.00277-09.
• Juan Rico, Ester Pardo, Margarita Orejas, 2010. Enhanced Production of a Plant Monoterpene by Overexpression of the 3-Hydroxy-Methylglutaryl Coenzyme A Reductase Catalytic Domain in Saccharomyces cerevisiae. Applied and Environmental Biology. 76 (19), 6449.
• Udo Kienle, 2007. Stevia rebaudiana – Natürliche Süße im Behördendschungel. Journal Culinaire No. 5.
• Shao-bing Hua, Mengsheng Qiu, Eva Chan, Li Zhu, Ying Luo, 1997. Minimum Length of Sequence Homology Required for in Vivo Cloning by Homologous Recombination in Yeast. Plasmid. 38 (2), 91-96.
• Tomonobu Toyomasu, Hiroshi Kawaide, Atsuko Ishizaki, Shoko Shinoda, Minoru Otsuka, Wataru Mitsuhashi, Takeshi Sassa, 2000. Cloning of a Full-length cDNA Encoding ent-Kaurene Synthase from Gibberella fujikuroi: Functional Analysis of a Bifunctional Diterpene Cyclase. Bioscience, Biotechnology and Biochemistry. 64 (3), 660- 664.
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