Part:BBa_K194001:Experience
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Applications of BBa_K194001
For the purpose of characterization, we connected the Cln2 (BBa_K194000) degradation signal downstream of GFP (BBa_K194001) and thereby created BBa_K194002. BBa_K194001 inserted on the same plasmid (pRS416: single copy yeast plasmid with URAA marker) without the degradation signal was used as reference. Both genes were expressed from the strong constitutive TEF promoter.
Results for a fermentation of the biobricks in a BioLector machine
Description of the construction of the plasmids can be found on our iGEM 2009 wiki at http://2009.igem.org/Team:DTU_Denmark/results
Growth rates (h-1) obtained for the two strains containing the two biobricks ( GFP_Cln and GFP) with glucose as carbon source
Strain | Growth Rate |
| GFP_Cln | 0,080 ± 0,003 |
GFP |
0,129 ± 0,001 |
The growth profile of these two strains in Glucose and in a mixed carbon source of Glucose and Galactose is presented bellow.
Both strains show very similar behavior. With Glucose as the solely carbon source, the curve presents a distinct exponential phase corresponding to the consumption of this substrate. After Glucose is consumed, OD seems to drop. Although there is not a decrease in cells, this drop might be due to morphological changes in the cells typical of a shift in the metabolism. As for the curves representing the mixed carbon, there are two exponential phases, the first corresponding to the glucose consumption and the second to the Galactose.
The next two graphs represent the variation of the GFP signal corrected and normalized with OD to subtract any variability that could come from the differences of growth
As one can see, there is a significant difference between this two constructs in terms of GFP signal. As expected, the signal from the degradable GFP is much lower than the signal showed by the non-degradable GFP, as the GFP that is not being degraded is accumulating and giving a stronger signal. This results go in line with the results published by Avery and Mateus [1] that state that the degradable GFP has a 14 times shorter half-life than the non-degradable. Therefore, based on this results we demontrate that the biobrick BBa_K194001 submited, presents a good option as a functionel GFP for yeast. We show that the addition of the sequence from Cln2 results in a strain with lower levels of GFP when the genes are expressed from the same promoter in plasmids with same copy number in the same strain.
References
[1] Mateus, C, Avery, S V 2000. Destabilized green fuorescent protein for monitoring dynamic changes in yeast gene expression with flow cytometry . Yeast 16:1313-1323.
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