Part:BBa_K346005

Mercury (II) ions absorption device

Hg(II) Bioabsorption Device

DsbA-mbp(mercury metal binding peptide)+mbp(mercury metal binding peptide)+lpp-ompa-mbp(mercury metal binding peptide)

This part was designed to function as mercury(II) ions absorption device in our project. If T7 polymerase is inductively expressed, this device will be switched on and metal bind peptide will be dramatically expressed and translocated to cytosol, periplasm and outer membrane surface. Namely, bacteria bearing this device will function as whole-cell bioabsorbent in the presence of T7 polymerase.

Description:

As the main part of mercury bioabsorbant of our project, this device is designed to assemble three subparts----the T7promoter-rbs-DsbA-mbp-terminator, T7promoter-rbs-mbp-terminator and T7promoter-rbs-lpp-ompa-mbp-terminator (figure1), which can bind mercury separately in periplasm, cytosol and the membrane of E.coli, to make full use of the space and maximize the absorption of mercury. Since all these subparts are driven by their own T7 promotors, they can be expressed when T7polymerase exists.

1 Metal Binding Pepside(MBP)

MBP was designed as a single polypeptide that could fold into an antiparallel coiled coil. Previous work shows that artificial MBP chain still kept the in vivo metal-binding ability comparable to dimeric, full-length MerR, while it comprises less amino acids and will cost less for large-scale expression. Since our ultimate goal is to design a high-performance and less energy-consuming bioabsorbent, the MBP is an excellent candidate for the absorbent effector.The construction and structure of MBP are shown as followed.

2 DsbA-MBP

MBP was fused with DsbA, a periplasmic expression signal protein to construct periplasmic MBP.

3 Lpp-OmpA-MBP

Lpp-OmpA-mbp is designed as a fusion protein consisting of the signal sequence and first 9 amino acid of Lpp, residue 46~159 of OmpA and the metal binding peptide(MBP). The signal peptide of the N-termini of this fusion protein targets the protein on the membrane while the trans-membrane domain of Ompa serves as an anchor. MBP is on the externally exposed loops of OmpA, which can be anchored to the outer membrane.

4 assemble

To make full use of the spaces and reduce the energy consumption to the least while improve the efficiency of metal binding, we assemble the three genes together: mbp, Dsba-mbp, lpp-ompa-mbp, which are expressed separately in cytoplasm, periplasmic space and on the membrane.More information about this design will be given in the "Part design".

Result

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We first use SDS-PAGE and Western blotting to analyse the expression of this engineered MBP protein. We can see from the result of them that the MBP is truly expressed in a large quantity. We then analyze the different form of MPB fusion protein, the DsbA-MBP which is expressed in the periplasm, and the lpp-OmpA-MBP on the surface, also using Western blotting and SDS-PAGE. The result indicated that the right fusion protein have been expressed correctly to their location.

Figure 1: The result of SDS-PAGE and Western blotting, indicating the expression of MBP, DsbA-MBP, lpp-OmpA-MBP in different location.

We then use IPC-AES to detect the mercury binding capacity of the engineered MBP. From the result we can see that the surface displayed Lpp-OmpA-MBP has the highest capacity and efficiency.Though the mercury absorption device didn't work completely as predicted, it is still obvious that the efficiency of the device is higher than the MBP and Dsba-MBP,indicating that the design to combine these three subparts can indeed increase the efficiency. However, since the expression of exogenous proteins places harder burden on bacteria, the efficiency of expression will be affected. This can explain why the efficiency of the device is lower than that of Lpp-OmpA-MBP.

Figure 2: Overnight cultures were diluted 1:100 and grown to final OD600=0.6-1.0. We then resuspend the culture with 1% PBS and make samples for ICP-AES according to the protocol(See more about this, please visit our wiki.)

Mercury (II) ions absorption device

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal PstI site found at 529
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal PstI site found at 529
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 317
  Illegal BamHI site found at 1148
  Illegal BamHI site found at 2111
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal PstI site found at 529
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal PstI site found at 529
  Illegal AgeI site found at 149
  
• 1000
  COMPATIBLE WITH RFC[1000]