Difference between revisions of "Part:BBa K2165002:Design"

Latest revision as of 18:48, 29 October 2016

Violacein C gene with CUP1 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
COMPATIBLE WITH RFC[25]
1000
INCOMPATIBLE WITH RFC[1000]
Illegal BsaI.rc site found at 370

Design Notes

This biobrick utilizes the University of Washtion's Cup1 promoter. It has had one base pair mutated to remove an illegal XbaI cut site. VioC 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

VioC is derived from the violacein pathway in Chromobacterium Violaceum, a type of Proteobacteria. The original VioC 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 CUP1 promoter was identified in the 5' untranslated region of the metallothionein gene in S. cerevisiae. 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 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. 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 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 1963 (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).

@@ Line 16: / Line 16: @@
 ===Characterization===
 [[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)]]
-[[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 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. 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). Figure 2 shows a diagnostic gel indicating that the promotor is the proper size. Expected bands when cut with EcoRI and PstI are 2029 (backbone) and 1963 (composite part). This suggests the part was assembled successfully.
+[[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 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. 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 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 1963 (composite part). This suggests the part was assembled successfully.