Difference between revisions of "Part:BBa K1739000"
 
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<partinfo>BBa_K1739000 short</partinfo>
 
<partinfo>BBa_K1739000 short</partinfo>
  
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We have improved a previously designed BioBrick (Part:[https://parts.igem.org/Part:BBa_K401001 BBa_K401001]) from the [http://2010.igem.org/Team:Valencia Valencia 2010 iGEM team] that encoded the Sup35 protein from Saccharomyces cerevisiae. The previously designed BioBrick contained two illegal cut sites for Pstl and one for Bsal within the coding region that reduce compatibility for digestion and modification of the part. Our improved BioBrick has used genome optimisation in order to remove these cut sites, producing a part compatible with the iGEM part submission standards. Validation of this part used a diagnostic Congo Red plate that demonstrated the presence of amyloid by formation of red colonies.  
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Sup35 is derived from the yeast prion protein Sup35p and excludes the C-terminal domain. The N-terminal domain allows self-assembly of functional amyloid [1][2].
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This BioBrick is an improved version of a previously designed BioBrick (Part:[https://parts.igem.org/Part:BBa_K401001 BBa_K401001]) from the [http://2010.igem.org/Team:Valencia Valencia 2010 iGEM team] that encoded the Sup35 protein from Saccharomyces cerevisiae. The previously designed BioBrick contained illegal BioBrick restriction sites. Our improved BioBrick has optimized in order to remove these cut sites and we have produced a part compatible with the iGEM part submission standards.  
  
  
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===Validation===
 
===Validation===
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To validate the nature of our plasmid, we analysed it through agarose gel electrophoresis. By comparing the sizes of the fragments to a marker we discovered the size of our fragment (as seen in figure 1) to be slightly less than 850 base pairs, matching the actual size of 800 base pairs of the Sup35NM sequence. Also, our gel shows a fragment to be about 2000 base pairs, confirming it is PSB1c3 which is 2030 base pairs in size.
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To validate our plasmid, we analyzed it through a diagnostic double restriction cut, followed by agarose gel electrophoresis. A restriction digest of our plasmid was carried out using EcoRI and PstI. These enzymes cleave pSBIC3 at 2029bp, with our insert being 780bp. By comparing the sizes of our inserts to the Invitrogen 1kB DNA Marker, we found that our fragments were the correct sizes.  
  
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[[File:Team KentSUP35gel2.jpg|thumb|center|400px|Figure 1. Shows the agarose gel produced, following the restriction digest of our part.]]  
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[[File:Team KentSUP35gel2.jpg|thumb|center|400px|Figure 1. Agarose gel of the restriction digest of BBa_K1739000 in pSCB13 plasmid backbone with EcoRI and PstI.]]
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Function Validation of the Sup35NM sequence was performed on [https://parts.igem.org/Part:BBa_K1739000 (BBa_K1739002)] by diagnostic Congo Red plate assay and AFM imaging that demonstrated the presence of amyloid by formation of red colonies.
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===Part improvement===
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Team USTC 2016 improve this part to make it shorter and more effective to be utilized now. See the [[Part:BBa_K1739000:Experience|Experience page]] for details.
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Team Kent 2016 improved this part by removing the constitutive promoter, allowing the user choice as to which promoter they use. See (Part:[https://parts.igem.org/Part:BBa_K1985012 BBa_K1985012]) for more detail.
  
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<!---[[File:Team Kent SUP35gel.jpg|thumb|center|400px|Figure 1. Shows .]]--->
 
 
===References===
 
===References===
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[1] Frederick, K., Debelouchina, G., Kayatekin, C., Dorminy, T., Jacavone, A., Griffin, R. and Lindquist, S. (2014). Distinct Prion Strains Are Defined by Amyloid Core Structure and Chaperone Binding Site Dynamics. Chemistry & Biology, 21(2), pp.295-305.
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[2] Glover, J., Kowal, A., Schirmer, E., Patino, M., Liu, J. and Lindquist, S. (1997). Self-Seeded Fibers Formed by Sup35, the Protein Determinant of [PSI+], a Heritable Prion-like Factor of S. cerevisiae. Cell, 89(5), pp.811-819.

Latest revision as of 22:43, 29 October 2016

Sequence coding for amyloid Sup35NM


Sup35 is derived from the yeast prion protein Sup35p and excludes the C-terminal domain. The N-terminal domain allows self-assembly of functional amyloid [1][2]. This BioBrick is an improved version of a previously designed BioBrick (Part:BBa_K401001) from the [http://2010.igem.org/Team:Valencia Valencia 2010 iGEM team] that encoded the Sup35 protein from Saccharomyces cerevisiae. The previously designed BioBrick contained illegal BioBrick restriction sites. Our improved BioBrick has optimized in order to remove these cut sites and we have produced a part compatible with the iGEM part submission standards.


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
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 522


Validation

To validate our plasmid, we analyzed it through a diagnostic double restriction cut, followed by agarose gel electrophoresis. A restriction digest of our plasmid was carried out using EcoRI and PstI. These enzymes cleave pSBIC3 at 2029bp, with our insert being 780bp. By comparing the sizes of our inserts to the Invitrogen 1kB DNA Marker, we found that our fragments were the correct sizes.

Figure 1. Agarose gel of the restriction digest of BBa_K1739000 in pSCB13 plasmid backbone with EcoRI and PstI.

Function Validation of the Sup35NM sequence was performed on (BBa_K1739002) by diagnostic Congo Red plate assay and AFM imaging that demonstrated the presence of amyloid by formation of red colonies.

Part improvement

Team USTC 2016 improve this part to make it shorter and more effective to be utilized now. See the Experience page for details. Team Kent 2016 improved this part by removing the constitutive promoter, allowing the user choice as to which promoter they use. See (Part:BBa_K1985012) for more detail.

References

[1] Frederick, K., Debelouchina, G., Kayatekin, C., Dorminy, T., Jacavone, A., Griffin, R. and Lindquist, S. (2014). Distinct Prion Strains Are Defined by Amyloid Core Structure and Chaperone Binding Site Dynamics. Chemistry & Biology, 21(2), pp.295-305.

[2] Glover, J., Kowal, A., Schirmer, E., Patino, M., Liu, J. and Lindquist, S. (1997). Self-Seeded Fibers Formed by Sup35, the Protein Determinant of [PSI+], a Heritable Prion-like Factor of S. cerevisiae. Cell, 89(5), pp.811-819.