Difference between revisions of "Part:BBa K346005"

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As the main part of mercury bioabsorbant of our project, this device is designed to combine 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 T7promotor, they can be expressed when T7polymerase exists.
 
As the main part of mercury bioabsorbant of our project, this device is designed to combine 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 T7promotor, they can be expressed when T7polymerase exists.
  
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1 '''Metal binding pepside(MBP)'''  
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1 '''Metal Binding Pepside(MBP)'''  
  
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MBP was designed as a single polypeptide that could fold into an antiparallel coiled coil, just like MerR, the mercury-responsive metalloregulatory protein MerR dose. As a result, the engineered MBP has a similar mercury binding capacity as MerR. We got the the gene of mbp by PCR with the plasmid from Anna.
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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 will be an excellent candidate for the absorbent effector.The construction and structure of MBP are shown as followed.
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[[Image:MBP.jpg]]
  
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2 '''Dsba-mbp'''
 
  
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Dsba-mbp is a fusion protein aiming to transport the MBP protein to the periplasm. Dsba is a signal peptide, which can be recognized and transported to the periplasm.
 
  
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3 '''Lpp-ompa-mbp'''
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2 '''Dsba-MBP'''
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MBP was fused with DsbA, a periplasmic expression signal protein to construct periplasmic MBP.
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[[Image:Dsba-MBP.jpg]]
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3 '''Lpp-OmpA-MBP'''
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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.
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[[Image:Lpp-OmpA-MBP.jpg]]
  
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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.
 
  
  

Revision as of 15:57, 24 October 2010

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. Mercury figure1.jpg

Description:

As the main part of mercury bioabsorbant of our project, this device is designed to combine 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 T7promotor, 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 will be an excellent candidate for the absorbent effector.The construction and structure of MBP are shown as followed. MBP.jpg


2 Dsba-MBP

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

Dsba-MBP.jpg


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.

Lpp-OmpA-MBP.jpg