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From 2011.igem.org

Bisphenol A subproject: We enabled E. coli to degrade BPA in vivo and improved the specific BPA degradation rate by creating a Fd_bisd:CYP_bisd fusion protein (BisdA | BisdB), changing the cytochrome P450 electron transport system from a putida-like bacterial class I type to a class V type.

S-layer subproject: Four different S-layer BioBricks with different lattice structures were created and sent to the partsregistry. The behaviour of these genes when expressed in E. coli were characterized and purification strategies for the expressed proteins were developed. Two purified fluorescent S-layer fusion proteins from different organisms were immobilized on beads, leading to a highly significant fluorescence enhancement of these beads (p < 10^-14). Furthermore regarding the other two S-layers (CspB from Corynebacterium glutamicum and Corynebacterium halotolerans) we discovered that while expression with a lipid anchor resulted in an integration into the cell membrane, the expression with a TAT-sequence resulted in a secretion into the medium. We also detected that those S-layers seem to stabilize the biologically active conformation of mRFP. Furthermore we expressed and purified a fluorescent CspB fusion protein from C. halotolerans which has never been expressed in E. coli until now.

NAD⁺ detection: We were able to utilize NAD⁺-dependent DNA ligase from E. coli (LigA) for a highly sensitive and selective molecular beacon based bioassay detecting NAD⁺ in nano molarity scale. The deadenylated form of LigA could ligate nicked DNA (a split target hybridized to a molecular beacon) resulting in an increase of fluorescence intensity which was still measurable in presence of 2 nM NAD⁺. Relating to this, the initial velocity displayed a linear dependence on the employed NAD⁺ concentrations as long as these remained the limiting factor for DNA ligation. Additionally, the NAD⁺ bioassay has been successfully coupled to the NADH-dependent conversion of pyruvate to L-lactate by lactate dehydrogenase.

<groupparts>iGEM011 Bielefeld-Germany</groupparts>