Difference between revisions of "Part:BBa K5302021"
 
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<partinfo>BBa_K5302021 short</partinfo>
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This work is derived from pBBR1MCS-OmpA-mCherry and pUC19-miniZ-Z3C, and it has undergone codon optimization. This composite part combines OmpA(21.4kda) and miniZ(3.8kda), we succeeded in transferring this plasmid into Escherichia coli Nissle 1917 and let it express miniZ. The plasmid uses lac promotor and has kanamycin resistence.
 
This work is derived from pBBR1MCS-OmpA-mCherry and pUC19-miniZ-Z3C, and it has undergone codon optimization. This composite part combines OmpA(21.4kda) and miniZ(3.8kda), we succeeded in transferring this plasmid into Escherichia coli Nissle 1917 and let it express miniZ. The plasmid uses lac promotor and has kanamycin resistence.
  
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This part is derived from three helix 58-residue Z-domain of staphylococcal protein A. And through stabilizing mutations and the addition of a disulfide constraint the Z-domain is reengineered into a two-helix 34-residue “mini-Z” version that retains the parent's affinity. This is supposed to be more potent binders against VEGF.
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We used pBBR1MCS-2 plasmid as a backbone and transfered miniZ into Escherichia coli Nissle 1917, and finally succeeded in expressing miniZ.
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<div style="text-align:center;">
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    <img src="https://static.igem.wiki/teams/5302/images/part-registry-miniz-1.png"
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        width="60%" style="display:block; margin:auto;" alt="Jamboree Program" >
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    <div style="text-align:center;">
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        <caption>
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            <b>Figure 4. </b> sequence of miniZ and its KD with VEGF
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    <img src="https://static.igem.wiki/teams/5302/images/part-registry-miniz-2.png"
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        width="60%" style="display:block; margin:auto;" alt="Jamboree Program" >
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    <div style="text-align:center;">
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        <caption>
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            <b>Figure 5. </b> Cartoon representation of the crystal structure of the mini-Z highlighting randomized residues shown as sticks and coloring the helices
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        </caption>
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<!-- Add more about the biology of this part here
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===Usage and Biology===
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<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K5302021 SequenceAndFeatures</partinfo>
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<!-- Uncomment this to enable Functional Parameter display
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===Functional Parameters===
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<partinfo>BBa_K5302021 parameters</partinfo>
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Latest revision as of 01:45, 2 October 2024


pBBR-OmpA-miniZ

This work is derived from pBBR1MCS-OmpA-mCherry and pUC19-miniZ-Z3C, and it has undergone codon optimization. This composite part combines OmpA(21.4kda) and miniZ(3.8kda), we succeeded in transferring this plasmid into Escherichia coli Nissle 1917 and let it express miniZ. The plasmid uses lac promotor and has kanamycin resistence.

Jamboree Program
Figure 1. Colony PCR results of pBBR-OmpA-miniZ

Jamboree Program
Figure 2. SDS-PAGE analysis of pBBR1MCS-OmpA-miniZ expression in Escherichia coli Nissle 1917 （1）

Jamboree Program
Figure 3. SDS-PAGE analysis of pBBR1MCS-OmpA-miniZ expression in Escherichia coli Nissle 1917 （2）

This part is derived from three helix 58-residue Z-domain of staphylococcal protein A. And through stabilizing mutations and the addition of a disulfide constraint the Z-domain is reengineered into a two-helix 34-residue “mini-Z” version that retains the parent's affinity. This is supposed to be more potent binders against VEGF. We used pBBR1MCS-2 plasmid as a backbone and transfered miniZ into Escherichia coli Nissle 1917, and finally succeeded in expressing miniZ.

Jamboree Program
Figure 4. sequence of miniZ and its KD with VEGF

Jamboree Program
Figure 5. Cartoon representation of the crystal structure of the mini-Z highlighting randomized residues shown as sticks and coloring the helices

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 4025
    Illegal XbaI site found at 3140
    Illegal PstI site found at 1967
    Illegal PstI site found at 3128
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 4025
    Illegal PstI site found at 1967
    Illegal PstI site found at 3128
    Illegal NotI site found at 1008
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 4025
    Illegal BglII site found at 1754
    Illegal BamHI site found at 3146
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 4025
    Illegal XbaI site found at 3140
    Illegal PstI site found at 1967
    Illegal PstI site found at 3128
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 4025
    Illegal XbaI site found at 3140
    Illegal PstI site found at 1967
    Illegal PstI site found at 3128
    Illegal NgoMIV site found at 2418
    Illegal NgoMIV site found at 2701
    Illegal NgoMIV site found at 4683
    Illegal AgeI site found at 4523
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 1330
    Illegal SapI.rc site found at 2267
    Illegal SapI.rc site found at 2477