Team:TU Munich/Project/Overview

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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).
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).
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Our aim is to have ''S. cerevisiae'' secrete functional thaumatin by expressing Prepro-Thaumatin – a principle which has been proven by [[http://www.ncbi.nlm.nih.gov/pubmed/6327079 Edens et al., 1984]].
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Our aim is to have ''S. cerevisiae'' secrete functional thaumatin by expressing preprothaumatin – a principle which has been proven by [[http://www.ncbi.nlm.nih.gov/pubmed/6327079 Edens et al., 1984]].
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[[file:TUM12_experiment_overwiew_Thaumati.png|500px|thumb|right|]]
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<center>'''Experimental results:'''</center><br>
<center>'''Experimental results:'''</center><br>
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The BioBrick for Prepro-Thaumatin [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801080 BBa_K801080] as well as a expression cassette [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801080 K801080] were successfully cloned, expressed in yeast, purified using a ion exchange chromatography (see figure C) and detected in the SDS-PAGE. Therefore, the expression of Thaumatin in yeast could be demonstrated and functionality of the BioBrick is confirmed.
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The BioBrick for preprothaumatin [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801080 BBa_K801080] as well as a expression cassette [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801080 K801080] were successfully cloned, expressed in yeast, purified using a ion exchange chromatography (see figure C) and detected in the SDS-PAGE. Therefore, the expression of thaumatin in yeast could be demonstrated and functionality of the BioBrick is confirmed.
<center>'''Conclusion and outlook:'''</center><br>
<center>'''Conclusion and outlook:'''</center><br>
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A proof of principle for the expression of thaumatin was achieved. Further goals would include to increase the expression of the thaumatin and to investigate the secretion.
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A proof of principle for the expression of thaumatin was achieved. Further goals are the increase of the expression of thaumatin and the investigation of the secretion.
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Revision as of 23:55, 26 September 2012


Contents

Overview


Vision


We, TU Munich’s 2012 iGEM team, strive to catalyze the diffusion process of knowledge about genetic engineering and synthetic biology among the general public. Using the example of iGEM’s first and finest SynBio Beer we involve, interest and inspire people to reconsider preconceived ideas and encourage them to openly engage in a broad discussion weighing pros and cons of genetic engineering in foodstuff. We sketch a future where new technology can be applied in a meaningful way to complement traditional foods or beverages.

Biosynthesis pathways



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.


Explanations on the figure
Experimental results:

(+)-Limonene synthase 1 [BBa_K801065] and (+)-limonene synthase 1 with yeast consensus sequence [BBa_K801060] were successfully cloned into our new yeast expression vector pTUM100. Expression of recombinant limonene synthase in Saccharomyces cerevisiae was proven by western blotting. The functionality of the enzyme was verified by in vivo and in vitro detection of limonene (GC-MS).

Furthermore, we established gene constructs of limonene synthase coding sequence with different yeast specific promotors and terminators [BBa_K801062], [BBa_K801063], [BBa_K801064].

Last but not least, we have brewed iGEM's first SynBio beer containing limonene.

Conclusion and outlook:

We have achieved functional expression of Citrus limon limonene synthase in yeast. We

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., 1984].


TUM12 experiment overwiew Thaumati.png
Experimental results:

The BioBrick for preprothaumatin BBa_K801080 as well as a expression cassette K801080 were successfully cloned, expressed in yeast, purified using a ion exchange chromatography (see figure C) and detected in the SDS-PAGE. Therefore, the expression of thaumatin in yeast could be demonstrated and functionality of the BioBrick is confirmed.

Conclusion and outlook:

A proof of principle for the expression of thaumatin was achieved. Further goals are the increase of the expression of thaumatin and the investigation of the secretion.

Caffeine

Caffeine is a purine-alkaloid and its biosynthesis is known from coffee plants and tea plants. 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.



TUM12 experiment overwiew caffeine.png
Experimental results:


Conclusion and outlook:

The homologue expression of the three required enzymes for caffeine synthesis in Saccharomyces cerevisiae INVSc1 transformed with pTUM102_CaXMT1, pTUM102_CaMXMT1 and pTUM102_CaDXMT1 succeeded. Further testing of caffeine production using crude extracts from lysed transformed yeast cells and working on genome integration of the expression cassette consisting of all three enzymes flanked by promoter and terminator for caffeine production during expression are in progress.


Xantohumol

Xanthohumol is known as a putative cancer chemopreventive agent, due to its antioxidant activities [Miranda et al., 2000]. 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.


Explanations on the figure
Experimental results:

The whole biosynthetic pathway for the production of Xantohumol was converted into BioBricks. Except for APT each of the enzymes were cloned in two versions one having the proposed consensus sequence for more efficient expression in yeast chassis and another for usage of these BioBricks in other chassis. All BioBricks were sequenced. Sequences can be found in the registry of standard biological parts: BBa_K801090 BBa_K801091 BBa_K801092 BBa_K801093 BBa_K801094 BBa_K801095 BBa_K801096 BBa_K801097 BBa_K801098


Conclusion and outlook:

The construction of the Xantohumol pathway was achieved whereas the expression and characterization has to be postponed after the European Jamboree or might be an interesting task for next year's iGEM teams.

Vector Design


pTUM100

Designing an expression vector for yeast which is compatible to the iGEM cloning principles and standards was the main aim of this subproject. Based on the commercially available pYES2 vector we created a vectors containing inducible and constitutive promoters in order to establish efficient possibilities to clone and express our enzymes.


Explanations on the figure: Figfure A shows the new multiple cloning site containing the RFC 10/25 restriction sides and the DNA sequence coding for the Strep-tag II. Figure B gives an overview of all important functional elements located on the vector backbone. Upstream to the new MCS lies a T7 promoter primer binding site allowing easy forward sequencing of integrated gene constructs using the standard T7 primer. The URA 3 gene is a prototrophy marker used for the selection of transfected cells. Figure C to E present the successfully designed BioBricks: pTUM100 simply contains the new MCS, the transcription terminator and the further elements required for cloning and transfection. pTUM102 to pTUM104 contain in addition the constitutive promoters pTef1, pTef2 and ADH. On pTUM104 is the galactose inducible promoter pGAL1 located.
Experimental results:

Using the pYES vector from Invitrogen we first deleted five forbidden restriction sites in the vector back bone via side directed mutagenesis. Furthermore the original multiple cloning site was replaced for a multiple cloning site compatible to the RFC 10/25 cloning standards. To allow easy extraction and purification of proteins for in vitro applications the new multiple cloning site allows to express proteins with a Strep-tag II. Exclusion of the galactose inducible promoter provided a powerful basis vector for the integration of user-defined promoters. This way the pTUM100 vector gives a valuable contribution to our and to further protein expression and promoter characterization experiments in the yeast Saccharomyces cerevisiae. Moreover we used the pTUM100 to integrate the three constitutive promoters Tef1, Tef2 and ADH which come all with different promoter intensities.

Outlook and conclusion:

The galactose inducible expression system was a great aid for the majority of all subprojects. Especially the opportunity to purify and detect (via Western blot) proteins using the Strep-tag II did facilitate our laboratory practice and accelerated our work progress. To cover even more demands we are planning to design a second vector template containing a His-tag.

All BioBricks were sequenced. Sequences can be found in the registry of standard biological parts under the following entries: pTUM100: BBa_K801000, BBa_K801001, BBa_K801002, BBa_K801003 and BBa_K801004


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.


Ethanol-inducible promoter

The KlADH4-Promoter from the yeast Kluyveromyces lactis regulates the expression of a mitochondrial alcohol dehydrogenase in an ethanol-dependent way.
Explanations on the figure
Experimental results:

50-100 words on results

Conclusion and outlook:

Light-switchable promoter

The idea behind a light-switchable 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.


Principle of light-dependent switching of gene-expression.
Experimental results:

50-100 words on results

Conclusion and outlook:

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.


Plasmid backbone used for integration of our expression cassettes
Experimental results:

First experiments to characterize the function of the yeast integration system were performed and the used selection marker was maintained in the yeast cultur although the selection pressure was switched off. This indicates that first integrations were achieved.

Conclusion and outlook:

Maintaining the plasmids harboring our expression cassettes in the yeast cells during the brewing process is best possible using genome integration. This becomes increasingly interesting, when a yeast strain with different expression cassettes is to be created. Because this is intendet for the next step of our project the integration of our expression cassettes becomes increasingly important.

Brewing our SynBio Beer


Contrary to popular opinion, the chief ingredient of beer is not YPD but gyle, a carefully prepared mixture of malt, hop, barley and water. Although the name S. cerevisiae suggests that it is used in the beer brewing process, in fact the yeast employed in brewing have strongly adapted to gyle, as they are reutilized in every succeeding brewing cycle. Hence some investigation on how our yeast performs in gyle was necessary.



Picture of the first SynBio Beer brewed during the iGEM competition in 2012
Experimental results:

Our experiments show that the growth of several different yeast strains is not impaired in gyle!

Expression assays proved the necessity of genome integration for a proper SynBio Beer.

Conclusion and outlook:

As soon as cloning of integration vectors containing expression cassettes for our biosynthetic pathways are finished, we will repeat the brewing experiments.

References


  • [Edens et al., 1984] Edens, L., Bom, I., Ledeboer, A. M., Maat, J., Toonen, M. Y., Visser, C., and Verrips, C. T. (1984). Synthesis and processing of the plant protein thaumatin in yeast. Cell, 37(2):629–33.
  • [Miranda et al., 2000] Miranda, C. L., Stevens, J. F., Ivanov, V., McCall, M., Frei, B., Deinzer, M. L., and Buhler, D. R. (2000). Antioxidant and prooxidant actions of prenylated and nonprenylated chalcones and flavanones in vitro. J Agric Food Chem, 48(9):3876–84.