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Team:Bielefeld-Germany/Results/Summary
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| + | '''Bisphenol A subproject''': We enabled ''E. coli'' to degrade BPA ''in vivo'' and improved the specific BPA degradation rate by creating a Fd<sub>bisd</sub>:CYP<sub>bisd</sub> fusion protein (BisdA | BisdB), changing the cytochrome P450 electron transport system from a putida-like bacterial class I type to a class V type. Furthermore, we could demonstrate that the fusion protein has a high specificity for BPA in comparison to similar bisphenols. Finally, we were greatly impressed by the fact that the fusion protein of FNR, BisdA and BisdB was not only capable to degrade BPA, but also displayed the highest maximal specific BPA degradation rate of all constructs. | ||
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| + | '''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<sup>-14</sup>). 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 biological active conformation of mRFP. Furthermore, we expressed and purified a fluorescent CspB fusion protein from ''C. halotolerans'' which has not been expressed in ''E. coli'' before. | ||
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| + | '''NAD<sup>+</sup> detection''': We were able to utilize NAD<sup>+</sup>-dependent DNA ligase from ''E. coli'' (LigA) for a highly sensitive and selective molecular beacon based bioassay detecting NAD<sup>+</sup> in nano molarity scale (limit of detection: 2 nM). The deadenylated form of LigA was able to ligate nicked DNA (a split target hybridized to a molecular beacon), resulting in an increase of fluorescence intensity. In relation to this, the initial velocity displayed a linear dependence on the employed NAD<sup>+</sup> concentrations as long as these remained the limiting factor for DNA ligation. Additionally, the NAD<sup>+</sup> bioassay has been successfully coupled to the NADH-dependent conversion of pyruvate to L-lactate by lactic acid dehydrogenase. | ||
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| + | <center><groupparts>iGEM011 Bielefeld-Germany</groupparts></center> | ||
Latest revision as of 00:34, 29 October 2011
Bisphenol A subproject: We enabled E. coli to degrade BPA in vivo and improved the specific BPA degradation rate by creating a Fdbisd:CYPbisd fusion protein (BisdA | BisdB), changing the cytochrome P450 electron transport system from a putida-like bacterial class I type to a class V type. Furthermore, we could demonstrate that the fusion protein has a high specificity for BPA in comparison to similar bisphenols. Finally, we were greatly impressed by the fact that the fusion protein of FNR, BisdA and BisdB was not only capable to degrade BPA, but also displayed the highest maximal specific BPA degradation rate of all constructs.
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 biological active conformation of mRFP. Furthermore, we expressed and purified a fluorescent CspB fusion protein from C. halotolerans which has not been expressed in E. coli before.
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 (limit of detection: 2 nM). The deadenylated form of LigA was able to ligate nicked DNA (a split target hybridized to a molecular beacon), resulting in an increase of fluorescence intensity. In relation 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 lactic acid dehydrogenase.
