Template:Team:TU Munich/Overview

Vector Design

To be able to test and quantify the expression of our biosynthetic pathways in yeast in yeast we designed an expression vector which is compatible to the iGEM RFC25 standard based on the commercially available pYES2 vector from Invitrogen.

Ethanol Inducible Promoter

The KlADH4-Promoter from the yeast ''Kluyveromyces lactis'' regulates the expression of a mitochondrial alcohol dehydrogenase in an ethanol-dependent way.

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.

Constitutive Promoter

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.

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)

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.

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.

Caffeine

Caffeine is a purine-alkaloid and its biosynthesis is known from coffee plants and tea plants.(3) It can block specific receptors in the hypothalamus in a competitive manner, which leads to decreased neurotransmitter-release and therefore decreased neuron activity.

The biosynthetic pathway of caffeine starts with xanthosine, which is a natural component of the purine-metabolism of all organism and involves a total of 3 enzymes.

Xanthohumol

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.

Human Practice

Our project envisions genetically modified organisms in the scope of food production. In Germany genetic engineering is a highly sensitive topic. Keeping in mind that Germany has a long tradition of brewing beer it is even more important to inform the general public about synthetic biology and our project.

RFC

iGEM’s core idea is standardization []: genetic elements are modified to easily combine them. Similar to electrical engineers biologists shall be enabled to construct large and more complex systems with fewer difficulties []. A central element of this idea is the Parts Registry. Scientists have access to a huge variety of standardized genetic element, the BioBricks. This collection is constantly growing. Thereby it contributes to the successful diffusion and the acceptance of synthetic biology and it is the basis for and of great benefit to all researchers, supporters and participants of the iGEM competition.

Yet over time our team has come to the conclusion that iGEM’s core idea, standardization, is not fully implemented in the Parts Registry. Accessing the Registry frequently to plan a project using BioBricks, one very quickly realizes that the part descriptions are often unstructured. There are excellent examples of well-structured and organized descriptions. Unfortunately a defined standard is missing.

Modeling

To be able to predict the behavior of a given biological system, one has to create a mathematical model of the system. The model is usually generated according to the Law of Mass Action and then simplified by assuming certain reactions to be fast. This model then could e.g. facilitate optimizations of bio-synthetic pathways by regulating the relative expression levels of the involved enzymes.

Until now modeling was only carried for the GAL1p as large amounts of data were produced shortly before the wiki freeze and hence could not be processed in time