Difference between revisions of "Part:BBa K2940010"

Revision as of 15:49, 21 October 2019

Immobilised dye-degrading peroxidase: Dyp (without signal peptides)+ Cytochrome b5

This part is designed for the DyP-Cytb5 expression. So the signal peptides were removed from the original coding sequence (NC_000964.3) to make sure DyP can anchor with Cytb5. The strong constitute promoter and RBS combination (BBa_K608002) were inserted prior to each start codon. This part is related to the ghost shells project, which aims at immobilising DyP from Bacillus subtilis to the inner surface of the cytosolic membrane of Escherichia coli with the help of the C-terminal membrane anchor protein Cytb5. Then, the expression of Lysis protein E from PhiX174 can cause the formation of a pore in the cell wall of E. coli, which results in the release of the cytosol. Theoretically, the empty cellular envelopes with immoblised DyP can be used for degradation.

Usage and Biology

Dye-decolorising peroxidase (DyP) has been recognised for its capacity to catalyse extracellularly the H2O2-dependent oxidation of various dye molecules, including anthraquinone-based dyes and azo dyes [1]. The DyP chosen here is from Bacillus subtilis (BsDyP) and recent study showed this peroxidase can maintain activity in an environment at high temperature (53h ± 11h at 40 °C) [2], suggesting its high stability in extreme working environment. However, as the physicochemical environment in the wastewater is significantly different from the original environment within the cells. The low adaptability of the enzymes may result in the failure of biodegradation. So one-step expression and immobilisation of DyPs is feasible for pursuing an accelerated processing.

Cytb5 is the oligonucleotides for the cytochrome b5 membrane anchor from rabbit liver. The hydrophobic domain (HP) of cytochrome b5 incorporates the information, which is needed to target polypeptides including this domain to the membrane [3].

To enable contact between the immobilised enzymes and their substrates in the extracellular environment, the lysis protein E (BBa_K2500009), originating from phage PhiX174, was used to open up the cytosolic membrane of the bacterial cell via a conformational modification that leads to the fusion of the inner membrane with the outer membrane and forms a lysis pore (40–200 nm) [4]. The fusion of the membrane, subsequently, resulted in the release of cytosolic plasma under osmotic pressure and the formation of the empty enzyme-anchored cell membrane.

Sequence and Features