Difference between revisions of "Part:BBa K5302005"
 
Line 5: Line 5:
 
This year, the USTC iGEM team has utilized the competitive binding of vascular endothelial growth factor (VEGF) to develop a targeted bacterial therapy for solid tumors. Our quest for the optimal VEGF-binding protein(or peptide) led us to an in-depth exploration of proteins structurally akin to the vascular endothelial growth factor receptor (VEGFR), which we have named VEGFR-like. 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.
 
This year, the USTC iGEM team has utilized the competitive binding of vascular endothelial growth factor (VEGF) to develop a targeted bacterial therapy for solid tumors. Our quest for the optimal VEGF-binding protein(or peptide) led us to an in-depth exploration of proteins structurally akin to the vascular endothelial growth factor receptor (VEGFR), which we have named VEGFR-like. 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.
 
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 1. </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>
 +
            <b>Figure 2. </b> Cartoon representation of the crystal structure of the mini-Z highlighting randomized residues shown as sticks and coloring the helices
 +
        </caption>
 +
    </div>
 +
</div>
 +
</html>
 +
 +
<html>
 +
<div style="text-align:center;">
 +
    <img src="https://static.igem.wiki/teams/5302/images/part-registry-miniz-3.png"
 +
        width="60%" style="display:block; margin:auto;" alt="Jamboree Program" >
 +
    <div style="text-align:center;">
 +
        <caption>
 +
            <b>Figure 3. </b> Colony PCR results of pBBR-INP-miniZ
 +
        </caption>
 +
    </div>
 +
</div>
 +
</html>
 +
 +
<html>
 +
<div style="text-align:center;">
 +
    <img src="https://static.igem.wiki/teams/5302/images/part-registry-miniz-4.png"
 +
        width="60%" style="display:block; margin:auto;" alt="Jamboree Program" >
 +
    <div style="text-align:center;">
 +
        <caption>
 +
            <b>Figure 4. </b> SDS-PAGE analysis of pBBR1MCS-INP-miniZ expression in Escherichia coli Nissle 1917
 +
        </caption>
 +
    </div>
 +
</div>
 +
</html>
 +
 +
<html>
 +
<div style="text-align:center;">
 +
    <img src="https://static.igem.wiki/teams/5302/images/part-registry-miniz-5.png"
 +
        width="60%" style="display:block; margin:auto;" alt="Jamboree Program" >
 +
    <div style="text-align:center;">
 +
        <caption>
 +
            <b>Figure 5. </b> Colony PCR results of pBBR-OmpA-miniZ
 +
        </caption>
 +
    </div>
 +
</div>
 +
</html>
 +
 +
<html>
 +
<div style="text-align:center;">
 +
    <img src="https://static.igem.wiki/teams/5302/images/part-registry-miniz-6.png"
 +
        width="60%" style="display:block; margin:auto;" alt="Jamboree Program" >
 +
    <div style="text-align:center;">
 +
        <caption>
 +
            <b>Figure 6. </b> SDS-PAGE analysis of pBBR1MCS-OmpA-miniZ expression in Escherichia coli Nissle 1917 (1)
 +
        </caption>
 +
    </div>
 +
</div>
 +
</html>
 +
 +
<html>
 +
<div style="text-align:center;">
 +
    <img src="https://static.igem.wiki/teams/5302/images/part-registry-miniz-7.png"
 +
        width="60%" style="display:block; margin:auto;" alt="Jamboree Program" >
 +
    <div style="text-align:center;">
 +
        <caption>
 +
            <b>Figure 7. </b> SDS-PAGE analysis of pBBR1MCS-OmpA-miniZ expression in Escherichia coli Nissle 1917 (2)
 +
        </caption>
 +
    </div>
 +
</div>
 +
</html>
 +
  
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

Latest revision as of 08:45, 1 October 2024


miniZ

This year, the USTC iGEM team has utilized the competitive binding of vascular endothelial growth factor (VEGF) to develop a targeted bacterial therapy for solid tumors. Our quest for the optimal VEGF-binding protein(or peptide) led us to an in-depth exploration of proteins structurally akin to the vascular endothelial growth factor receptor (VEGFR), which we have named VEGFR-like. 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 1. sequence of miniZ and its KD with VEGF

Jamboree Program
Figure 2. Cartoon representation of the crystal structure of the mini-Z highlighting randomized residues shown as sticks and coloring the helices

Jamboree Program
Figure 3. Colony PCR results of pBBR-INP-miniZ

Jamboree Program
Figure 4. SDS-PAGE analysis of pBBR1MCS-INP-miniZ expression in Escherichia coli Nissle 1917

Jamboree Program
Figure 5. Colony PCR results of pBBR-OmpA-miniZ

Jamboree Program
Figure 6. SDS-PAGE analysis of pBBR1MCS-OmpA-miniZ expression in Escherichia coli Nissle 1917 (1)

Jamboree Program
Figure 7. SDS-PAGE analysis of pBBR1MCS-OmpA-miniZ expression in Escherichia coli Nissle 1917 (2)


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]