Team:TU Munich/Project/Overview
From 2012.igem.org
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Caffeine is a purine-alkaloid and its biosynthesis is known from coffee plants and tea plants.(3) The molecule acts as a competitive antagonist on adenosine receptors and therefore increases indirectly neurotransmitter concentrations resulting in warding of drowsiness and restoring of alertness. | Caffeine is a purine-alkaloid and its biosynthesis is known from coffee plants and tea plants.(3) The molecule acts as a competitive antagonist on adenosine receptors and therefore increases indirectly neurotransmitter concentrations resulting in warding of drowsiness and restoring of alertness. | ||
- | The idea is to perform a heterologous gene expression of the three enzymes 7-methylxanthosine synthase, N-methyl nucleosidase and caffeine synthase required for caffeine biosynthesis in ''Saccharomyces cerevisiae''. | + | The idea is to perform a heterologous gene expression of the three enzymes 7-methylxanthosine synthase (CaXMT1), N-methyl nucleosidase (CaMXMT1) and caffeine synthase (CaDXMT1) required for caffeine biosynthesis in ''Saccharomyces cerevisiae''. |
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[[file:TUM12_experiment_overwiew_caffeine.png|500px|thumb|right| Explanations on the figure]] | [[file:TUM12_experiment_overwiew_caffeine.png|500px|thumb|right| Explanations on the figure]] | ||
<center>'''Experimental results:'''</center><br> | <center>'''Experimental results:'''</center><br> | ||
- | + | Successful cloning of the three enzymes CaXMT1, CaMXMT1 and CaDXMT1 into pTUM102 and pSB1C3. | |
+ | |||
<center>'''Conclusion and outlook:'''</center><br> | <center>'''Conclusion and outlook:'''</center><br> |
Revision as of 22:46, 24 September 2012
Contents |
Overview
Vision
Vector Design
To be able to test and quantify the expression of desired enzymes in yeast we designed an expression vector which is compatible to the iGEM RFC25 standard based on the commercially available pYES2 vector from Invitrogen.
50-100 words on results
Regulation of Genexpression
By developing inducible promoters and placing them upstream of our biosynthetic pathways we create the possibility to make S. cerevisiae dynamically respond to concentration changes in its medium as well as to external stimuli.
An optimal inducing substance needs to be inexpensive, nontoxic and fully controllable in its application. Only substances with these characteristics allow to precisely regulate a system temporally, spatially and quantitatively.
Constitutive promoters
When all enzymes are expressed under control of promoters with the same strength, the enzyme with the lowest kinetic rates will cause a bottleneck in the pathway. Hence a multitude of promoters of different strengths is needed.
50-100 words on results
Ethanol-inducible promoter
50-100 words on results
Light-switchable promoter
The idea behind a lightswitchable system is to create a gene expression system which can be induced and deactivated by light of a certain wavelengths.
This system is extremely attractive, as induction does not require the addition of a specific substance. This makes induction cheap, fast, precise and also compatible to the bavarian purity law.
50-100 words on results
Biosynthesis pathways
50-100 words introduction
Limonene
Limonene is a cyclic terpene and a major constituent of several citrus oils. D-Limonene has been used as a component of flavorings and fragrances. It is formed from geranyl pyrophosphate by limonene synthase.
We will produce the flavoring substance limonene by expressing limonene synthase in S. cerevisiae, which naturally synthesizes the educt geranyl pyrophosphate.
50-100 words on results
Thaumatin
Thaumatin is a natural α+β-protein which is synthesized by the katamfe plant (Thaumatococcus daniellii). It is said to be 2.000 to 100.000 times sweeter than sucrose on molar basis, but the sweetness builds slow and lasts long. It has been approved as a sweetener by the European Union (E957).
Our aim is to have S. cerevisiae secrete functional Thaumatin by expressing Preprothaumatin – a principle which has been proven by Edens et al. in 1984.(1)
50-100 words on results
Caffeine
Caffeine is a purine-alkaloid and its biosynthesis is known from coffee plants and tea plants.(3) The molecule acts as a competitive antagonist on adenosine receptors and therefore increases indirectly neurotransmitter concentrations resulting in warding of drowsiness and restoring of alertness.
The idea is to perform a heterologous gene expression of the three enzymes 7-methylxanthosine synthase (CaXMT1), N-methyl nucleosidase (CaMXMT1) and caffeine synthase (CaDXMT1) required for caffeine biosynthesis in Saccharomyces cerevisiae.
Successful cloning of the three enzymes CaXMT1, CaMXMT1 and CaDXMT1 into pTUM102 and pSB1C3.
belongs to ongoing results
Xantohumol
Xanthohumol is known as a putative cancer chemopreventive agent, due to its antioxidant activities (Miranda et al., 2000).(2) Our goal is a heterologous gene expression of all enzymes required for xanthohumol biosynthesis in S. cerevisiae.
The pathway for the production of this plant secondary metabolite is composed of five steps, starting with the conversion of phenylalanine and followed by four further enzymatic reactions.
50-100 words on results
Genome integration
As we can't obey the letter of the German Purity Law (there is a zero tolerance policy concerning transgenic ingredients), we try our best to meet the spirit. Thus, it is unacceptable for us to work with antibiotics to keep up the selective environment. Since we can't work with auxotrophies in beer either, we have to make sure the yeasts don't get rid of the biobricks. The most promising way to accomplish a long lasting presence of our constructs is to achieve genome integration.
50-100 words on results
Brewing our SynBio Beer
Businessplan for our TUM Brew
50 - 70 words introduction
50-100 words on results
Our RFC for Standardization of BioBrick part descriptions
50 - 70 words introduction
50-100 words on results
References
- [1] Synthesis and processing of the plant protein thaumatin in yeast, Luppo Edens, Isaäc Bom, Adrianus M. Ledeboer, Jan Maat, Marjolein Y. Toonen, Chris Visser, C. Theo Verrips. Cell Volume 37, Issue 2, June 1984, Pages 629–633
- [2] C.L Miranda, J.F Stevens, V Ivanov, M McCall, B Frei, M.L Deinzer, D.R Buhler, Antioxidant and prooxidant actions of prenylated and nonprenylated chalcones and flavanones in vitro, J. Agric. Food Chem., 48 (2000), pp. 3876–3884
- [3] Insertion of Coffein, Römpp Online. Version 3.19
- [4] Use of the KlADH4 Promoter for Ethanol-Dependent Production of Recombinant Human Serum Albumin in Kluyveromyces lactis. Michele Saliola, Cristina Mazzoni, Nicola Solimando, Alessandra Crisà, Claudio Falcone, Gerard Jung, and Reinhard Fleer. Appl Environ Microbiol. 1999 January; 65(1): 53–60.
- [5] Development of a genetic switch based on a photosensitive protein signals the beginning of light-based biological engineering. Andrew R. Mendelsohn Nature Biotechnology, Vol. 20, October 2002, Pages 985-987
- [6] Molecular analysis of UAS, a cis element cotaining stress response elements responsible for ethanol induction in the KlADH4 of Kluyveromyces lactis. Cristina Mazzoni, Francesca Santori, Michele Saliola and Claudio Falcone Research in Microbiology, Vol. 151, 2000, Pages 19-28