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

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Unknown
• 21
  INCOMPATIBLE WITH RFC[21]

  Unknown
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE 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.

Production of cellular envelopes with immobilised DyP (ghost shells)

The plasmids pET28a-GG: Lysis E and pSB1C3:DyP were transformed into BL21 (DE3) cells and incubated on the plates with both chloramphenicol and kanamycin. Only pSB1C3-RFP grown on the plate with chloramphenicol and pET28a-GG plasmids grown on the plate with chloramphenicol or kanamycin displayed pink colonies, while no colonies were found on other plates (data not shown), indicating successful transformation process and there might be leakage of the expression system of pET28a-GG. In conclusion, producing empty cellular envelopes (ghost shells) with immobilized peroxidase was not achieved on this project.

Future Improvement

1. Only crude proteins were adopted in this project, which is difficult to determine whether the extracellular peroxidase DyP was fully expressed. SDS-PAGE shall be done to analyze whether the protein is expressed in intact cells, cytoplasm and membrane separately. An N-terminal His-tag could assist in confirming whether the immobilized DyP or secreted DyP exists on the SDS-PAGE gel.

Troubleshooting can include new methods of measuring expression of Lysis E; For example, a recently application of fluorescent dye technology is able to accurately keep track of the E. coli’s membrane permeability, which can thus react to the Lysis protein induction. Additionally, another approach to consider is the use of thermo-controlled expression system to output bacterial empty shells. This system allows to inserte the Lysis E into varying vectors including the strong major leftward (pL) and/or rightward (pR) promoters [3].