Difference between revisions of "Part:BBa K2165003:Design"

 
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===Design Notes===
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<p>This biobrick utilizes the VioD promoter. VioD was codon optimized in hopes of improving the expression. The biobrick ends with the ADH1 terminator [https://parts.igem.org/Part:BBa_K801012 BBa_K801012] for yeast that was submitted by the TU Munich team in 2012. For more information about each part, view their respective pages.
  
  
 
===Source===
 
===Source===
  
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VioD is derived from the violacein pathway in &#8203;Chromobacterium&#8203; &#8203;Violaceum&#8203;, a type of Proteobacteria. The Gal1 promoter is from the genomic DNA of S. cerevisiae in the GAL1 locus. The ADH1 terminator is also from the genomic DNA of S. cerevisiae.
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VioD is derived from the violacein pathway in &#8203;Chromobacterium&#8203; &#8203;Violaceum&#8203;, a type of Proteobacteria. The original VioD sequence was aquired from the Duber laboratory at the University of California Berkley and was codon optimized through IDT's codon optimizer web application. The Gal1 promoter is from the genomic DNA of S. cerevisiae in the GAL1 locus. The ADH1 terminator is also from the genomic DNA of S. cerevisiae.
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===Characterization===
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[[File:BBa_K2165000_Violcultures.png|thumb|left|200px|Figure 1: Constituatively active VioABCDE yeast in synthetic media, along with a positive control (regular yeast) and a negative control (no yeast)]]
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[[file:BBa_K2165002_Gel.jpeg|thumb|left|200px|Figure 2: A gel electrophoresis of four parts related to the University of Washington's project.]] Though typical biobrick characeterization involves in-vitro data, this is unavailabe due to the lack of an available chassis containing the three enzymes necessary to produce the substrate for VioD (VioA, VioB, and VioE). Because of the nature of this biobrick, it is reasonable to look at information from other sources to see the expected results. An image displaying the violacein pathway can be found [[https://static.igem.org/mediawiki/2016/7/72/T--Washington--Wetlab_ViolaceinPathway.png here]] (Lee 2013; Kim 2016). This composite part should produce VioD in the presence of galactose, assuming no glucose is present. Providing VioA, VioB, and VioE are in solution, this will produce a tealish color pigment. When VioC is also present, a violet will be produced (shown in figure 1 utilizing a constitutionally active Violacein plasmid). Figure 2 shows a diagnostic gel indicating that the biobrick is the proper size. Expected bands when cut with EcoRI and PstI are 2029 (backbone) and 2007 (composite part). This suggests the part was assembled successfully.
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===Works Cited===
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Kim, S. et al. PubChem substance and compound databases. Nucleic Acids Research 44, (2016).
  
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===References===
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Lee, M. E., Aswani, A., Han, A. S., Tomlin, C. J. & Dueber, J. E. Expression-level optimization of a multi-enzyme pathway in the absence of a high-throughput assay. Nucleic Acids Res. 41, 10668–10678 (2013).

Revision as of 18:47, 29 October 2016


Violacein D gene with Gal1 promoter + ADH1 terminator


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 150
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 1177
    Illegal SapI.rc site found at 1558


Design Notes

The VioD gene was codon optimized for use in yeast to optimize response time of the violacein system due to an introduction of galactose.


Design Notes

This biobrick utilizes the VioD promoter. VioD was codon optimized in hopes of improving the expression. The biobrick ends with the ADH1 terminator BBa_K801012 for yeast that was submitted by the TU Munich team in 2012. For more information about each part, view their respective pages.

Source

VioD is derived from the violacein pathway in ​Chromobacterium​ ​Violaceum​, a type of Proteobacteria. The original VioD sequence was aquired from the Duber laboratory at the University of California Berkley and was codon optimized through IDT's codon optimizer web application. The Gal1 promoter is from the genomic DNA of S. cerevisiae in the GAL1 locus. The ADH1 terminator is also from the genomic DNA of S. cerevisiae.


Characterization

Figure 1: Constituatively active VioABCDE yeast in synthetic media, along with a positive control (regular yeast) and a negative control (no yeast)
Figure 2: A gel electrophoresis of four parts related to the University of Washington's project.
Though typical biobrick characeterization involves in-vitro data, this is unavailabe due to the lack of an available chassis containing the three enzymes necessary to produce the substrate for VioD (VioA, VioB, and VioE). Because of the nature of this biobrick, it is reasonable to look at information from other sources to see the expected results. An image displaying the violacein pathway can be found [here] (Lee 2013; Kim 2016). This composite part should produce VioD in the presence of galactose, assuming no glucose is present. Providing VioA, VioB, and VioE are in solution, this will produce a tealish color pigment. When VioC is also present, a violet will be produced (shown in figure 1 utilizing a constitutionally active Violacein plasmid). Figure 2 shows a diagnostic gel indicating that the biobrick is the proper size. Expected bands when cut with EcoRI and PstI are 2029 (backbone) and 2007 (composite part). This suggests the part was assembled successfully.


Works Cited

Kim, S. et al. PubChem substance and compound databases. Nucleic Acids Research 44, (2016).

Lee, M. E., Aswani, A., Han, A. S., Tomlin, C. J. & Dueber, J. E. Expression-level optimization of a multi-enzyme pathway in the absence of a high-throughput assay. Nucleic Acids Res. 41, 10668–10678 (2013).