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From 2012.igem.org

Approach

The conventional methods in sewage treatment plants are unable to treat waste water sufficiently because the most frequently used micro contaminants like estrogen, Bisphenol A, Dicolfenac etc. are very difficult to break down. The goal of Bielefeld’s iGEM team is to develop a biological filtersystem using immobilized laccases to purify municipal and industrial wastewater from synthetic estrogens and other aromatic compounds. Laccases are copper-containing oxidase enzymes found in many organisms, and one of their properties is the ability to break down a wide range of aromatic and phenolic compounds. For this purpose, genes of various bacterial and eukaryotic laccases should be isolated and expressed in Escherichia coli and Pichia pastoris. The choice of the expressionsystem depends on the glycosylation status of the enzyme.

Isolation and Generating of new BioBricks

The first step for Step of our project is to isolate the specific gene sequences and to generate new BioBricks for the iGEM competition. The Laccases of the following organisms are isolated:

bacterial laccases:

1. Escherichia Coli
   File:Bielefeld2012 Approach Isolation Production Purification.jpg

   First steps of our Project
2. Baccilus Halodurans
3. Baccilus Pumilus
4. Streptomyces griseus
5. Streptomyces lavendulae
6. Thermus thermophilus
7. Xanthomonas campestris

Eukaryota laccases:

1. Arabidopsis thaliana
2. Tramestes versicolor
3. Trametes villosa

For more information about the Organisms click here.

These BioBricks are used to design new plasmids and vector-systems. Plasmids, which are used for production in E.coli, includes a T7-Promotor, a ribosomal-binding-sequence ,a His-Tag-Sequence at the end of the Protein an a Chloramphenicol resistent.

The genetically modified organisms are used to produce different laccases.

The team is aiming to manufacture this enzyme economically and safely with the help of methods from synthetic biology. It should also be possible to extend the concept to other, in part poisonous and carcinogenic pollutants in drinking and waste water, as well as into industrial application, such as in paper and textile industries or even for bioremediation of contaminated soil.