Part:BBa_K123000:Experience

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Applications of BBa_K123000

User Reviews

UNIQ9cb44506b4ada70b-partinfo-00000000-QINU UNIQ9cb44506b4ada70b-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 sent to the registry.

Fig. 1: Alignment of the DNA sequence of BBa_K123000 entered into the parts.igem (K123000 registry) with our sequencing results (K123000 real) for this BioBrick (made with Clonemanager).

Fig. 2: Alignment of the aminoacid sequence (translated in silico) of BBa_K123000 entered into the parts.igem (K123000 registry) with our sequencing results (K123000 real) for this BioBrick (made with Clonemanager).

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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