Difference between revisions of "Part:BBa K4134012"

 
 
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<partinfo>BBa_K4134012 short</partinfo>
 
<partinfo>BBa_K4134012 short</partinfo>
  
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AggC is a gene downstream to the chromosomal bpfA. The 1000bp-long fragment of BpfA and the 1000bp-long fragment of AggC are attached to our vector for homologous recombination so as to fuse the destination fragment (silver-binding protein) to the C-terminus of BpfA. This design enables the destination gene to be displayed on the surface of S. oneidensis. Considering the large size of BpfA, this fusion expression does nearly no harm to the normal physiological state of S. oneidensis and we have approved it through a biofilm growth test and bacterial density measurement.  
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==Description==
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After all the considerations above, we made the pUC57-BpfA-Atox&#8544;/AgBP2-Kana-loxP-aagC fusion vector. Its core DNA box consists of the coding sequence of a silver binding protein (either Atox1 or AgBP2) and the kanamycin resistance gene (Kana, for recombinant screening). In the following experiment, proliferated vectors were digested into two fragments before electroporation because the linearized expression vector exhibited a high site-specific recombination frequency after being inserted into the S. oneidensis genome. The digestion of the plasmid DNA (20 mg) was carried out with the restriction enzymes Kpn&#8544;(10 U) and EcoR&#8544;(10 U) in a volume of 200 ml.  
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<p>
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In short, we displayed a silver-binding protein Atox&#8544; onto the cell surface of S. oneidensis by fusing it to the C-terminus of BpfA, a large surface protein, and used the surface displayed Atox&#8544; as a receptor to capture the silver ions in the matrix of S. oneidensis biofilms.
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AggC is a gene downstream to the Shewanella membrane protein, BpfA. </p>
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<p>
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[[Image:Nanjing-China-recombination-device-AgBP2.jpeg|400px|thumb|right|Homologous recombination device design]]
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The C-terminal 1000bp fragment of BpfA and the N-terminal 1000bp-long fragment of AggC are attached to our vector for homologous recombination so as to fuse the destination fragment (silver-binding protein) to the C-terminus of BpfA. This design enables the destination gene to be displayed on the surface of S. oneidensis. Considering the large size of BpfA, this fusion expression does nearly no harm to the normal physiological state of S. oneidensis and we have approved it through a biofilm growth test and bacterial density measurement. </p>
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<p>
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After all the considerations above, we made the pUC57-BpfA-Atox&#8544;/AgBP2-Kana-loxP-aagC fusion vector. Its core DNA box consists of the coding sequence of a silver binding protein (either Atox1 or AgBP2) and the kanamycin resistance gene (Kana, for recombinant screening). In the following experiment, proliferated vectors were digested into two fragments before electroporation because the linearized expression vector exhibited a high site-specific recombination frequency after being inserted into the S. oneidensis genome. The digestion of the plasmid DNA (20 mg) was carried out with the restriction enzymes Kpn&#8544;(10 U) and EcoR&#8544;(10 U) in a volume of 200 ml. </p>
 +
<p>
 +
In short, we displayed a silver-binding protein Atox&#8544; onto the cell surface of S. oneidensis by fusing it to the C-terminus of BpfA, a large surface protein, and used the surface displayed Atox&#8544; as a receptor to capture the silver ions in the matrix of S. oneidensis biofilms.</p>
  
  

Latest revision as of 14:38, 13 October 2022


AggC (N-terminal 1000bp)

Description

AggC is a gene downstream to the Shewanella membrane protein, BpfA.

Homologous recombination device design
The C-terminal 1000bp fragment of BpfA and the N-terminal 1000bp-long fragment of AggC are attached to our vector for homologous recombination so as to fuse the destination fragment (silver-binding protein) to the C-terminus of BpfA. This design enables the destination gene to be displayed on the surface of S. oneidensis. Considering the large size of BpfA, this fusion expression does nearly no harm to the normal physiological state of S. oneidensis and we have approved it through a biofilm growth test and bacterial density measurement.

After all the considerations above, we made the pUC57-BpfA-AtoxⅠ/AgBP2-Kana-loxP-aagC fusion vector. Its core DNA box consists of the coding sequence of a silver binding protein (either Atox1 or AgBP2) and the kanamycin resistance gene (Kana, for recombinant screening). In the following experiment, proliferated vectors were digested into two fragments before electroporation because the linearized expression vector exhibited a high site-specific recombination frequency after being inserted into the S. oneidensis genome. The digestion of the plasmid DNA (20 mg) was carried out with the restriction enzymes KpnⅠ(10 U) and EcoRⅠ(10 U) in a volume of 200 ml.

In short, we displayed a silver-binding protein AtoxⅠ onto the cell surface of S. oneidensis by fusing it to the C-terminus of BpfA, a large surface protein, and used the surface displayed AtoxⅠ as a receptor to capture the silver ions in the matrix of S. oneidensis biofilms.


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]