Team:DTU Denmark/results



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As expected, both strains showed different profiles of growth accordingly to the substrate. For Glucose and Galactose, the growth curve showed two exponential phases, the first one corresponding to the growth on Glucose and the second corresponding to the growth on Galactose. Although the initial OD was similar in both cases, the growth on Galactose showed an even longer lag phase than the glucose. Finally, as expected, the substrate reporting less growth was ethanol. For the Redoxilator, the growth solely on Glucose showed a longer lag phase compared to the mixed substrate, probably due to a lower initial OD.



Regarding the GFP signal, all substrates showed different profiles. Nevertheless, the fermentative substrates, Glucose and Galactose, showed higher values of GFP than the respirative substrate, Ethanol.



Although these results are accordingly to what was expected, it is important to mention that these cultures didn’t have comparable growths, so, in order to subtract this variability of the growth, one should normalize the GFP expression with OD. Additionally, to the GFP values, the residual values for each case were subtracted.



One other aspect that is worth instigating is the variation of the GFP signal with growth. In other words, if growth is correlated with GFP signal.

Results of the fermentations and demonstration of biobrick BBa_K194001 and BBa_K194002

Growth rates (h-1) obtained for the two strains containing the two biobricks BBa_K194002 (GFP_Cln) and BBa_K194001 (GFP) with glucose as Carbon source

The growth profile of these two strains in Glucose and in a mixed carbon source of Glucose and Galactose is presented below.



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 [3] 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_K194002 submited, presents a good option as a fast degradable GFP for yeast. We show that both biobricks codes for Green Fluorescent Protein and 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] Lund, A. H., M. Duch, and F. S. Pedersen. 1996. Increased cloning efficiency by temperature-cycle ligation. Nucleic Acids Res. 24:800-801. doi:l50400 [2] Mark A. Sheff and Kurt S. Thorn. 2004. Optimized cassettes for fluorescent protein tagging in Saccharomyces cerevisiae. Yeast 24:800-801. doi:10.1002/yea.1130 [3] 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.

  Achievements

Redox sensing device Two novel genes have been designed and synthesized each comprised of 5 genetic elements. Together they function as a device termed the Redoxilator that can sense the internal redox state of a yeast cell, and output a reporter signal. Biobricks DNA of several new biobricks have been designed and submitted including a yeast optimized GFP reporter protein, a protein degradation sequence and a fast degradable yeast GFP. (Bronze medal) We have demonstrated that our USER fusion biobrick works as expected and documented it (silver medal) USER fusion Assembly standard A new biobrick assembly standard that allows the rapid construction of multi-part devices have been developed and documented. The assembly standard offers many benefits: All restriction sites are allowed, multiple biobricks can be joined in one step, the result is scar-free making it ideal for protein fusions and more. (Gold medal) USER-fusion primer design software A novel and very useful software tool have been developed that can automatically design the optimal primers for USER fusion assembly of 2-9 biobricks, taking several parameters into account.