Difference between revisions of "Part:BBa K2384001"

 
 
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<partinfo>BBa_K2384001 short</partinfo>
 
<partinfo>BBa_K2384001 short</partinfo>
  
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It is well known that the structure of proteins determines its function.The two disulfide bond plays an important role in the folding and assembly of a structure with biological activity, and stability of this structure(Especially in containing more than one subunit protein or polypeptide).Disulfide bond formation protein A(DsbA) is folded enzyme catalyzed two sulfur bond formation in Escherichia coli periplasm cavity in a nascent protein folding process.It can greatly improve the folding rate of the nascent peptide chain, thus enabling Escherichia coli to ensure that the nascent polypeptide chains can be folded in a timely and correct fashion.
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It is highly expected that when fused with N-terminal fragment of Outer membrane protein A (OmpA), the lead metal binding peptide(MBP)will be translocated and displayed on the surface of bacteria, which will contribute a lot to increase the mental binding capacity when combined will other collection parts. In the Lpp-Omp display system with potential for displaying macromolecular proteins,it contains signal peptide and the first nine amino acids of Lpp and forty-sixth to 159th amino acids of OmpA.The former contains all the information that carries the protein to the outer membrane and anchors it to the inside of the outer membrane,and the latter can anchor the target fusion protein to the outside of the membrane.The MBP that follows it can be expose.
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DsbA is the first protein found in the Dsb family.It consists of 208 amino acids, and has a C terminal signal peptide of 19 amino acids.It has an active central structure.The process of the formation and reduction of two sulfur bonds is achieved by exchanging sulfhydryl and two sulfur bonds between the active sites and the sulfhydryl or two sulfur bonds of the target proteins.(Thompson JD&#65292;Gibson TJ&#65292;Piewniak F&#65292;et a1&#65294;The ClustalX windows interface&#65306;flexible strategies for multiple sequence alignment aided by quality analysis tools&#65294;Nucleic Acids Research,1997,24:4876 -4882.)
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PbrR is a member of the MerR family of metal-sensing regulatory protein, acts as an effective Pb(II) capturer. According to earlier research, the DNA binding domain and metal binding domain can function individually and the constructed peptide can form a stable dimer with its mercury and lead binding affinity remaining. In order to reduce side effects of over-expression, Peking University tandemed two copies of metal binding domain of PbrR.
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DsbA has a stronger oxidation than Thioredoxin.Experiments have shown that its strong oxidation comes from Cys30 residues with abnormally low pKa and unstable oxidation structure.(Zapun A,Cooper L,Creighton TE.Replacement of the active-site cysteine residues of DsbA,a protein required for disulfide bond formation in vivo.Biochemistry,1994,33:1907-1914.)Also,experimental results show that DsbA can improve the efficiency of protein folding in intracellular and extracellular.
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Our sequence optimization is based on the sequence provided by Peking University's IGEM Wiki in 2010.It is more suitable for expression in <i>Bacillus megaterium</i>.
  
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===Usage and Biology===
 
===Usage and Biology===
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<b>Optimization Report</b>
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Gene Name: Lpp-OmpA+MBP(Pb)
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Expression System:Bacillus megaterium
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Gene Length:939 (bp)
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1.Codon Used Adjustment
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The best value is 1 for sequence optimization.
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<table><tr>
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<th>[[Image:T--FAFU-CHINA--Lpp-OmpA-MBP(Pb)1.png|thumb|400px|'''Figure 1:Before Codon Adjustment''']]</th>
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</tr></table>
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<table><tr>
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<th>[[Image:T--FAFU-CHINA--Lpp-OmpA-MBP(Pb)2.png|thumb|400px|'''Figure 2:Codon Adjustment''']]</th>
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</tr></table>
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2.Codon Used Distribution
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Show the relative codon used distribution
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<table><tr>
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<th>[[Image:T--FAFU-CHINA--Lpp-OmpA-MBP(Pb)3.png|thumb|400px|'''Figure 3:Before Optimization''']]</th>
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</tr></table>
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<table><tr>
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<th>[[Image:T--FAFU-CHINA--Lpp-OmpA-MBP(Pb)4.png|thumb|400px|'''Figure 4:After Optimization''']]</th>
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</tr></table>
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3.GC Content:
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The comparison of  GC content between original sequence and optimized sequence
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<table><tr>
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<th>[[Image:T--FAFU-CHINA--Lpp-OmpA-MBP(Pb)5.png|thumb|400px|'''Figure 5:Before Optimization''']]</th>
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</tr></table>
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<table><tr>
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<th>[[Image:T--FAFU-CHINA--Lpp-OmpA-MBP(Pb)6.png|thumb|400px|'''Figure 6:After Optimization''']]</th>
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</tr></table>
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<table><tr>
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<th>[[Image:T--FAFU-CHINA--Lpp-OmpA-MBP SDS PAGE.png|thumb|400px|'''Figure 7:The expression of Lpp-OmpA-MBP detected using SDS-PAGE ''']]</th>
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</tr></table>
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<table><tr>
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<th>[[Image:T--FAFU-CHINA--M-Lpp-OmpA+MBP(Pb).png|thumb|400px|'''Figure 8:Plasmid digested by PvuII''']]</th>
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</tr></table>
  
 
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Latest revision as of 18:53, 1 November 2017


Lpp-OmpA-MBP(Pb)

It is highly expected that when fused with N-terminal fragment of Outer membrane protein A (OmpA), the lead metal binding peptide(MBP)will be translocated and displayed on the surface of bacteria, which will contribute a lot to increase the mental binding capacity when combined will other collection parts. In the Lpp-Omp display system with potential for displaying macromolecular proteins,it contains signal peptide and the first nine amino acids of Lpp and forty-sixth to 159th amino acids of OmpA.The former contains all the information that carries the protein to the outer membrane and anchors it to the inside of the outer membrane,and the latter can anchor the target fusion protein to the outside of the membrane.The MBP that follows it can be expose. PbrR is a member of the MerR family of metal-sensing regulatory protein, acts as an effective Pb(II) capturer. According to earlier research, the DNA binding domain and metal binding domain can function individually and the constructed peptide can form a stable dimer with its mercury and lead binding affinity remaining. In order to reduce side effects of over-expression, Peking University tandemed two copies of metal binding domain of PbrR. Our sequence optimization is based on the sequence provided by Peking University's IGEM Wiki in 2010.It is more suitable for expression in Bacillus megaterium.

Usage and Biology

Optimization Report

Gene Name: Lpp-OmpA+MBP(Pb)

Expression System:Bacillus megaterium

Gene Length:939 (bp)


1.Codon Used Adjustment The best value is 1 for sequence optimization.


Figure 1:Before Codon Adjustment
Figure 2:Codon Adjustment


2.Codon Used Distribution Show the relative codon used distribution

Figure 3:Before Optimization
Figure 4:After Optimization

3.GC Content: The comparison of GC content between original sequence and optimized sequence

Figure 5:Before Optimization
Figure 6:After Optimization
Figure 7:The expression of Lpp-OmpA-MBP detected using SDS-PAGE
Figure 8:Plasmid digested by PvuII

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]