Difference between revisions of "Part:BBa K5302029"

 
 
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This work is derived from pBBR1MCS-INP-mCherry and pUC19-miniZ-Z3C, and it has undergone codon optimization. This composite part combines INP(about 30kda) 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-INP-mCherry and pUC19-miniZ-Z3C, and it has undergone codon optimization. This composite part combines INP(about 30kda) 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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    <img src="https://static.igem.wiki/teams/5302/images/part-registry-p-inp-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 1. </b> Colony PCR results of pBBR-INP-miniZ
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    <img src="https://static.igem.wiki/teams/5302/images/part-registry-p-inp-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 2. </b> SDS-PAGE analysis of pBBR1MCS-INP-miniZ expression in Escherichia coli Nissle 1917
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        </caption>
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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.
 +
 +
<html>
 +
<div style="text-align:center;">
 +
    <img src="https://static.igem.wiki/teams/5302/images/part-registry-miniz-1.png"
 +
        width="60%" style="display:block; margin:auto;" alt="Jamboree Program" >
 +
    <div style="text-align:center;">
 +
        <caption>
 +
            <b>Figure 3. </b> sequence of miniZ and its KD with VEGF
 +
        </caption>
 +
    </div>
 +
</div>
 +
</html>
 +
 +
<html>
 +
<div style="text-align:center;">
 +
    <img src="https://static.igem.wiki/teams/5302/images/part-registry-miniz-2.png"
 +
        width="60%" style="display:block; margin:auto;" alt="Jamboree Program" >
 +
    <div style="text-align:center;">
 +
        <caption>
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            <b>Figure 4. </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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    </div>
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<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

Latest revision as of 02:04, 2 October 2024


pBBR-INP-miniZ

This work is derived from pBBR1MCS-INP-mCherry and pUC19-miniZ-Z3C, and it has undergone codon optimization. This composite part combines INP(about 30kda) 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-INP-miniZ

Jamboree Program
Figure 2. SDS-PAGE analysis of pBBR1MCS-INP-miniZ expression in Escherichia coli Nissle 1917

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 3. sequence of miniZ and its KD with VEGF

Jamboree Program
Figure 4. 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 4383
    Illegal XbaI site found at 3189
    Illegal PstI site found at 2016
    Illegal PstI site found at 3177
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 4383
    Illegal PstI site found at 2016
    Illegal PstI site found at 3177
    Illegal NotI site found at 1057
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 4383
    Illegal BglII site found at 1803
    Illegal BamHI site found at 3195
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 4383
    Illegal XbaI site found at 3189
    Illegal PstI site found at 2016
    Illegal PstI site found at 3177
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 4383
    Illegal XbaI site found at 3189
    Illegal PstI site found at 2016
    Illegal PstI site found at 3177
    Illegal NgoMIV site found at 2467
    Illegal NgoMIV site found at 2750
    Illegal NgoMIV site found at 3616
    Illegal NgoMIV site found at 5048
    Illegal AgeI site found at 4888
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 1379
    Illegal SapI.rc site found at 2316
    Illegal SapI.rc site found at 2526