Difference between revisions of "Part:BBa K2165000:Design"

 
 
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===Design Notes===
 
===Design Notes===
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The online codon-optimization tool offered by Integrated DNA Technologies (https://www.idtdna.com/CodonOpt) was used to codon-optimize this sequence for use in yeast.  Combinations containing RFC 10 illegal restriction sites were actively avoided.
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The VioC developed by the University of Washington's iGEM team have been codon optimized for yeast in hopes to improve the efficiency of its translation.
  
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===Source===
  
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The CDS contained in this BioBrick was designed by running the VioC gene sent to the University of Washington by the [http://dueberlab.berkeley.edu/ Dueber Laboratory] at University of California-Berkley through IDT's Codon Optimization Tool for S. cerevisiae. A biobrick standard assembly prefix and suffix was added before the it was ordered as a geneblock through IDT.
  
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===Source===
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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.]]
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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 VioC (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. A protein BLAST shows that the sequence in this biobrick codes for "VioC" found in "cloning vector pET15b-vioC" with 100% quarry cover. 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 VioC in the presence of copper. Providing VioA, VioB, and VioE are in solution, this will produce a pinkish color pigment. When VioD is also present, a violet will be produced (shown in figure 1).
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While the diagnostic gel for VioC had interesting results (the gel only showed an empty plasmid backbone), we produced a biobrick with the proper size banding utilizing our BBa_K2165000, BBa_K2165002. We conclude that there must have been experimental error, but didn't have enough time to do another cut and gel before the jamboree. Figure 2 shows this diagnostic gel. VioC (lane 2) should have had an additional band at 1335 base pairs when cut with EcoRI and PstI; however, the [[https://parts.igem.org/Part:BBa_K2165002:Design VioC composite part]] has the expected band at 1963 base pairs.
  
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​VioC​ ​is​ ​originally​ ​from​ ​the​ violacein​ ​metabolic pathway​ ​in​ ​Chromobacterium​ ​Violaceum​, a type of Proteobacteria. The original vioC sequence for the bacteria chassis was submitted by the Alberta team and has been codon-optimized for use in yeast in this part.
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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).

Latest revision as of 19:00, 29 October 2016


Violacein C gene codon-optimized for S. Cerevisiae


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 BsaI.rc site found at 101


Design Notes

The VioC developed by the University of Washington's iGEM team have been codon optimized for yeast in hopes to improve the efficiency of its translation.

Source

The CDS contained in this BioBrick was designed by running the VioC gene sent to the University of Washington by the [http://dueberlab.berkeley.edu/ Dueber Laboratory] at University of California-Berkley through IDT's Codon Optimization Tool for S. cerevisiae. A biobrick standard assembly prefix and suffix was added before the it was ordered as a geneblock through IDT.

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 VioC (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. A protein BLAST shows that the sequence in this biobrick codes for "VioC" found in "cloning vector pET15b-vioC" with 100% quarry cover. An image displaying the violacein pathway can be found [here] (Lee 2013; Kim 2016). This composite part should produce VioC in the presence of copper. Providing VioA, VioB, and VioE are in solution, this will produce a pinkish color pigment. When VioD is also present, a violet will be produced (shown in figure 1).

While the diagnostic gel for VioC had interesting results (the gel only showed an empty plasmid backbone), we produced a biobrick with the proper size banding utilizing our BBa_K2165000, BBa_K2165002. We conclude that there must have been experimental error, but didn't have enough time to do another cut and gel before the jamboree. Figure 2 shows this diagnostic gel. VioC (lane 2) should have had an additional band at 1335 base pairs when cut with EcoRI and PstI; however, the [VioC composite part] has the expected band at 1963 base pairs.

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).