Difference between revisions of "Part:BBa K123000:Experience"

 
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<partinfo>BBa_K123000 AddReview 4</partinfo>
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<I>Bielefeld-Germany 2011</I>
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'''Wrong sequence in the parts.igem!'''
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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 sent to the registry.
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'''Bisphenol A degradation with BioBricks <partinfo>K123000</partinfo> and <partinfo>K123001</partinfo>'''
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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:
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[[Image:Bielefeld-Germany2011_K123000K123001char.jpg|650px|thumb| '''Figure 1: 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).
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Revision as of 14:25, 23 August 2011

This experience page is provided so that any user may enter their experience using this part.
Please enter how you used this part and how it worked out.

Applications of BBa_K123000

User Reviews

UNIQd0e621ff585a3138-partinfo-00000000-QINU UNIQd0e621ff585a3138-partinfo-00000001-QINU

••••

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 sent to the registry.


Bisphenol A degradation with BioBricks BBa_K123000 and BBa_K123001 The bisphenol A degradation with the BioBricks BBa_K123000 and BBa_K123001 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 (BBa_J23110). With this promoter upstream of a polycistronic bisdAB gene we were able to completely degrade 120 mg L-1 BPA in about 30 - 33 h. By fusing BBa_K123000 and BBa_K123001 together we could improve the BPA degradation of E. coli even further, so 120 mg L-1 BPA can be degraded in 21 - 24 h. This data is shown in the following figure:

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Figure 1: 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 BBa_J23110 with RBS BBa_B0034. 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).

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).

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