Part:BBa_K597102:Design
VioA-AviTag [AviTagged violacein enzyme A]
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 1012
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Design Notes
The AviTag is capable of being on either the C- or N-terminus of the protein of interest. There is little difference in the binding efficiency or protein expression between the two configurations. The Cornell iGEM team chose to put the AviTag on the C-terminus for the convenience of using a primer dimer (annealed primers with 5' and 3' overhangs) to insert the iGEM suffix into the sample from the Parts Registry.
Violacein Pathway:
Figure from Balibar et. al. 2
With the modified enzymes, we performed experiments to show their expression and utilization of tryptophan using a UV/Vis spectrometer at the peak wavelengths at which tryptophan responds to (590,638, and 562 nm). The OD measurements of solutions containing equivolume of VioA, VioB, 40 mM Hydrogen Peroxide (for oxidation of tryptophan) and VioE lysate with varying concentration of tryptophan. The results are shown below.
The green points represent readings at 562 nm, blue at 590 nm, and red at 638 nm at 0.008 g/mL concentration of L-Tryptophan and 40 mM H202. As you can see, the absorbance of tryptophan noticably decreases over a time course of 1 hour. This experiment was recreated varying the ratio of enzymes and concentration of initial tryptophan. The results showed a similar decreasing trend.
Source
AviTag: Oligonucleotide (single-stranded DNA) synthesized by Integrated DNA Technologies and annealed to form a short double-stranded sequence. Sequence for the C-terminus AviTag reference can be found in Cull and Schatz.1
VioOperon:BBa_K274004 (Cambridge 2009, Distribution 2011) 2
References
1. Cull MG, Schatz PJ (2000). “Biotinylation of proteins in vivo and in vitro using small peptide tags.” Methods of Enzymology Vol 326, 2000, Pages 430-440.
2. Balibar CJ, Walsh CT (2006). "In Vitro Biosynthesis of Violacein from l-Tryptophan by the Enzymes VioA−E from Chromobacterium violaceum." Biochemistry Volume 45 Issue 51, Pages 15444-15457