Difference between revisions of "Part:BBa K123000:Experience"
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The bisphenol A degradation with the BioBricks <partinfo>K123000</partinfo> and <partinfo>K123001</partinfo> works in ''E. coli'' KRX in general. Because Sasaki ''et al.'' (2008) reported problems with protein folding in ''E. coli'' which seem to avoid a complete BPA degradation, we did not use the strong T7 promoter for expressing these BioBricks but a medium strong constitutive promoter (<partinfo>J23110</partinfo>). With this promoter upstream of a polycistronic ''bisdAB'' gene we were able to completely degrade 120 mg L<sup>-1</sup> BPA in about 30 - 33 h. By fusing <partinfo>K123000</partinfo> and <partinfo>K123001</partinfo> together we could improve the BPA degradation of ''E. coli'' even further, so 120 mg L<sup>-1</sup> BPA can be degraded in 21 - 24 h. This data is shown in the following figure: | The bisphenol A degradation with the BioBricks <partinfo>K123000</partinfo> and <partinfo>K123001</partinfo> works in ''E. coli'' KRX in general. Because Sasaki ''et al.'' (2008) reported problems with protein folding in ''E. coli'' which seem to avoid a complete BPA degradation, we did not use the strong T7 promoter for expressing these BioBricks but a medium strong constitutive promoter (<partinfo>J23110</partinfo>). With this promoter upstream of a polycistronic ''bisdAB'' gene we were able to completely degrade 120 mg L<sup>-1</sup> BPA in about 30 - 33 h. By fusing <partinfo>K123000</partinfo> and <partinfo>K123001</partinfo> together we could improve the BPA degradation of ''E. coli'' even further, so 120 mg L<sup>-1</sup> BPA can be degraded in 21 - 24 h. This data is shown in the following figure: | ||
− | [[Image:Bielefeld-Germany2011_K123000K123001char.jpg|650px|thumb| '''Figure | + | [[Image:Bielefeld-Germany2011_K123000K123001char.jpg|650px|thumb| '''Figure 3: BPA degradation by ''E. coli'' KRX carrying genes for BisdA and BisdB (only ''bisdA'' (black), polycistronic ''bisdAB'' (red) and fusion protein between BisdA and BisdB (green)) behind the medium strong constitutive promoter <partinfo>J23110</partinfo> with RBS <partinfo>B0034</partinfo>. Cultivations were carried out at 30 °C in LB + Amp + BPA medium for 24 h and 36 h, respectively, with automatic sampling every three hours in 300 mL shaking flasks without baffles with silicon plugs. At least three biological replicates were analysed (three for ''bisdA'' alone, seven for ''bisdAB'' polycistronic and five for the fusion protein). ''']] |
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+ | We also carried out these cultivations at different temperatures and BPA concentrations, but the chosen conditions (30 °C and 120 mg L<sup>-1</sup> BPA) seem to be the best. Higher BPA concentrations have an effect on the growth of ''E. coli'' and higher temperature leeds to a worse BPA degradation (probably due to misfolding of the enzymes). These data on the effect of the temperature on the BPA degradation is shown in fig. 4. | ||
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+ | [[Image:Bielefeld-Germany_temperatureBPAdegrad.jpg|650px|thumb| '''Figure 4: BPA degradation by ''E. coli'' KRX carrying genes for BisdA and BisdB (polycistronic ''bisdAB'' (black) and fusion protein between BisdA and BisdB (striped)) behind the medium strong constitutive promoter <partinfo>J23110</partinfo> with RBS <partinfo>B0034</partinfo>. Cultivations were carried out at different temperatures in LB + Amp + BPA medium (starting concentration 120 mg L<sup>-1</sup> BPA) for 24 h in 300 mL shaking flasks without baffles with silicon plugs. Samples were taken at the end of the cultivation. Three biological replicates were analysed. ''']] | ||
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Revision as of 14:53, 30 August 2011
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UNIQ02ed2be3f4989510-partinfo-00000000-QINU UNIQ02ed2be3f4989510-partinfo-00000001-QINU
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Bielefeld-Germany 2011 |
Wrong sequence in the parts.igem! Sequence is not entered into the parts.igem correctly. This BioBrick was probably synthesized in the Freiburg assembly standard 25 because it has the accordant restriction sites and it was codon optimized for E. coli but the original sequence from Sphingomonas bisphenolicum was entered to the registry because amino acid sequence of the real sequence and the sequence that was entered is identical (translated in silico).
Error creating thumbnail: Invalid thumbnail parameters We also carried out these cultivations at different temperatures and BPA concentrations, but the chosen conditions (30 °C and 120 mg L-1 BPA) seem to be the best. Higher BPA concentrations have an effect on the growth of E. coli and higher temperature leeds to a worse BPA degradation (probably due to misfolding of the enzymes). These data on the effect of the temperature on the BPA degradation is shown in fig. 4. Error creating thumbnail: Invalid thumbnail parameters |