Difference between revisions of "Part:BBa K1985013"
 
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[[File:PSB1A3+promoter+61.jpeg||400px|thumb|centre|Figure 1.1% agarose gel of the restriction digest of BBa_K1985013 in pSB1A3 plasmid backbone with EcoRI and BamHI]]
 
[[File:PSB1A3+promoter+61.jpeg||400px|thumb|centre|Figure 1.1% agarose gel of the restriction digest of BBa_K1985013 in pSB1A3 plasmid backbone with EcoRI and BamHI]]
  
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===Congo Red Assay===
 
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We then validated our part using a Congo Red agar plate assay with the antibiotics chloramphenicol and ampicillin present in order to select the VS45 strains expression our fusion protein. As a negative control, we used VS45 with pVS105, as this plasmid contains CsgAss and Sup35M, it does not have the ability to self-assemble into amyloid nano-wires. These strains were plated in 2 quarter segments of the plate and left to incubate for 24 hours at 37°C. This resulted red colonies of VS45 with CsgAss-Sup35-1-61 due to the binding of Congo Red to the amyloid fibres produced by the colonies. White colonies of VS45 with the negative control plasmid, as expected, showed no export of amyloid-forming protein, whilst red colonies of VS45 with the positive control plasmid can be seen. The Sup35-1-61 cells are a similar colour to the positive control PVS72. (shown in fig.2).
 
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[[File:Congoredcytochromerita.jpeg||400px|thumb|centre|Figure 2 shows our Congo Red plate. PVS105 indicates the negative control and PVS72 indicates the positive control. The section labelled 61 shows this BioBrick, indicating that amyloid is being formed as red colonies can be seen]]
 
 
===References===
 
===References===
 
[1] Men, D., Zhou, J., Li, W., Leng, Y., Chen, X., Tao, S., and Zhang, X. "Fluorescent protein nanowire-mediated protein microarrays for multiplexed and highly sensitive pathogen detection". ACS Appl. Mater. Interfaces (June 2016) DOI: 10.102.
 
[1] Men, D., Zhou, J., Li, W., Leng, Y., Chen, X., Tao, S., and Zhang, X. "Fluorescent protein nanowire-mediated protein microarrays for multiplexed and highly sensitive pathogen detection". ACS Appl. Mater. Interfaces (June 2016) DOI: 10.102.

Latest revision as of 23:17, 27 October 2016

Sequence coding for Sup35 with residues 1-61 with an arabinose inducible promoter

This part is an improved version of a previously designed BioBrick (Part:BBa_K1739002) from the Kent 2015 iGEM team. This part contains three segments, an arabinose inducible promoter designed by the Imperial 2014 iGEM team (Part:BBa_K1321333), the CsgA signal sequence and only the first 61 aminoacids residues of the prion domain Sup35. Our improved BioBrick aims to optimize the self-assembly process of amyloid fibrils with the addition of these residues as they have been considered to be a suitable building block for the assembly of functional nanostructures[1]. The addition of the arabinose inducible promoter allows for tighter control for the expression of amyloid fibres rather than a constitutive promoter as was previously used. This part was inserted into part (Part:BBa_K1985016), which is a pSB1A3 backbone with (Part:BBa_K1321333).

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 1205
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 1144
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 979
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI site found at 961


Validation

Restriction Digest

The plasmid was analysed through a diagnostic double restriction cut, using the enzymes EcoRI and BamHI. This was followed by agarose gel electrophoresis. The enzymes cleave the pBAD Ara-C promoter at 1165 bp, with the remainder plasmid and the insert being 2526 bp. The size of the two fragments were compared with size of the Invitrogen 1kB DNA marker and was found that our fragments were the correct.

Figure 1.1% agarose gel of the restriction digest of BBa_K1985013 in pSB1A3 plasmid backbone with EcoRI and BamHI

References

[1] Men, D., Zhou, J., Li, W., Leng, Y., Chen, X., Tao, S., and Zhang, X. "Fluorescent protein nanowire-mediated protein microarrays for multiplexed and highly sensitive pathogen detection". ACS Appl. Mater. Interfaces (June 2016) DOI: 10.102.