Part:BBa_K2940010
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
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 30 - 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 995
Illegal SapI site found at 425
Illegal SapI site found at 783
Illegal SapI.rc site found at 939
Characterization
We successfully assembled pSB1C3: DyP-Cytb5 and pET28a-GG: Lysis E plasmid and transformed it into BL21 E. coli cells.
Expression of proxidases
The SDS-PAGE analysis of the crude extract cells of peroxidases shows that the supplement of hemin in the culture medium facilitated the formation of a stronger band at around 45 kDa for both DyP and DyP-Cytb5 (Figure 1). However, similar bands were also shown in cell crude extracts without hemin induction and some bands were also disappearing upon induction. Unfortunately, the SDS-PAGE analysis was not repeated to confirm the protein expression due to the time limit.
Figure 1. Protein expression of DyP and DyP-Cytb5 examined via SDS-PAGE. Visible protein bands at 45 kDa are shown in the image.
Expression of Lysis E
Lysis E was expressed with IPTG added while the OD at 600 nm was up to 0.4. After 3 hours, SDS-PAGE gel was used to analyze the crude extracts induced with IPTG and non-induced as control. Furthermore, the expression time of Lysis E caused by IPTG was determined. When OD600nm was up to 0.4, by adding IPTG to the liquid culture medium, the induction was conducted and then OD600 was measured each hour to monitor the cell growth.
SDS-PAGE gel failed to reveal the expected band at 10 kDa. OD600 value was measured each hour, but the cell growth of BL21 (DE3) cells harbouring pET-LE did not decrease (Figure. 2), possibly cell did not die.
Figure 2. The growth kinetics of the E. coli empty shell harboring the pET-LE plasmid. Till the mid-log phase indicated by an arrow and OD600 reaches to 0.4, cells growing at 37°C was split in two cultures. The first was under the induction of 1 mM IPTG, while the second was the non-induced control. Cell growth was monitored through the measurement of the OD600 of the culture every hour.
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