Team:Bielefeld-Germany/Nutshell
From 2011.igem.org
![](https://static.igem.org/mediawiki/2011/3/3c/Bielefeld-Germany2011-header-full.png)
The project
The development of sensitive and selective biosensors is an important research field in synthetic biology. Biosensors can be applied in a wide range of uses - from the detection of environmental toxics up to clinical diagnostics. Because cells have to sense their surroundings, there are a lot of natural systems that are similar to a biosensor. Prejudicial cellular biosensors often show negative side effects that complicate any practical application. Common problems are the limited use outside of a gene laboratory due to the use of genetically engineered cells, the low durability because of the usage of living cells and the appearance of undesired signals induced by endogenous metabolic pathways.
To solve these problems, the iGEM-Team Bielefeld 2011 aims to develope a cell-free bisphenol A (BPA) biosensor based on a coupled enzyme reaction fused to S-layer proteins for everyday use. Bisphenol A is a supposedly harmful substance which is used in the production of polycarbonate. To detect BPA it is degraded by a fusion protein under formation of NAD+ which is detected by an NAD+-dependent enzymatic reaction with a molecular beacon. Both enzymes are fused to S-layer proteins which build up well-defined nanosurfaces and are attached to the surface of beads. By providing these nanobiotechnological building blocks the system is expandable to other applications.
Subprojects
![](https://static.igem.org/mediawiki/2011/d/d7/IGEM_Bielefeld_Project.jpg)
Bisphenol A
In 2005, Sasaki et al. isolated a soil bacterium from the Sphingomonas genus which is able to degrade the environmental poison bisphenol A (BPA) with a unique rate and efficiency compared to other BPA degrading organisms. This strain was called Sphingomonas bisphenolicum AO1 and is able to completely decompose 120 mg BPA L-1 in about 6 hours. Three genes which are responsible for the first step of this effective BPA degradation by S. bisphenolicum AO1 were identified: a cytochrome P450 (bisdB), a ferredoxin (bisdA) and a ferredoxin-NADP+ oxidoreductase (FNR) (Sasaki et al., 2005b). The bisdAB genes from S. bisphenolicum AO1 were isolated, transformed into and expressed in E. coli and enabled this bacterium to degrade BPA, too (Sasaki et al., 2008). In addition, the BisdAB proteins from S. bisphenolicum AO1 were able to degrade BPA in a cell free system in which spinach reductase was added (Sasaki et al., 2005b). So we assume that the BisdAB proteins also work in a cell free system together with the ferredoxin-NAD(P)+ oxidoreductase from E. coli. The suggested reaction mechanism of the first BPA degradation step is shown in the project overview image above. More information on the background can be found here.NAD+ detection
Our selected NAD+ detection method displays a molecular beacon based approach. The ends of a single-stranded DNA molecule are labeled with a fluorophore as well as with an appropriate quencher. Both are in close proximity to each other due to a formed stem-loop, so that the detection of any fluorescence signal is prevented. Using two complementary targets hybridizing side-by-side with the hairpin enables NAD+-dependent DNA ligation by E. coli DNA ligase. Only after closing the gap between both hybridized targets the stem melts and the secondary structure gets broken down to a linearized probe-target hybrid. The immediate consequence is a disruption of the close proximity of the fluorophore and the quencher, so that an excitation with light is converted into a visible fluorescence signal. Hence, NAD+ concentration directly correlates with the emerging fluorescence signal. More information on the background can be found here.S-layer proteins
S-layers (crystalline bacterial surface layer) are crystal-like layers consisting of multiple protein monomers and can be found in various (archae-)bacteria. Especially their ability for self-assembly into distinct geometries is of scientific interest. At phase boundaries, in solutions and on a variety of surfaces they form different lattice structures. By modifying the characteristics of the S-layer through combination with functional groups and protein domains it is possible to realize various practical applications. Especially for the production of cell-free biosensors, functional fusion proteins are of great importance. Enzymes fused to immobilized S-layers showed a significantly longer durability and were more stable against physical and chemical treatment.
The iGEM-Team Bielefeld aims at the assembly, production and immobilization of S-layer fusion proteins for the detection of BPA by a coupled enzymatic reaction. The provision of various S-layers with different geometries offers the possibility for the scientific community to create functional nanobiotechnological surfaces with simple and standardized methods, quasi do it yourself nanobiotechnology.
Human Practice
The goals of our outreach are to awake the public awareness, start public discussions and participate in the outreach about iGEM. Also we want to promote the open source principle behind iGEM, arouse interest and hopefully prevented fear when facing the principles of synthetic biology. Therefore we organized and participated in various events. Check out our Human Practices section for more information.
Furthermore we provided a guide to do it yourself nanobiotechnology for fellow scientists, with detailed step by step instructions.
Achievments
With the BioBricks submitted by our team we enable a fast and selective BPA degradation in E.coli, a highly sensitive and selective NAD+ detection that facilitates a versatile NAD+ bioassay for future iGEM teams and the immobilization of S-layer fusion proteins, which implies the use of our S-layer proteins as nanobiotechnological building blocks.
As our approach is cell-free we can guarantee a high biosafety of our biosensor and were able to create a rather simple model for the BPA detection.
Check out our Achievenment page, if you want to know about further achievements of our team.