Team:Valencia Biocampus/Results2

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=== '''Are you fermenting?''' ===
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=== '''How long have you been fermenting?''' ===
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The experiments with our glucose-sensitive construction were carried out by three different ways: (1) containing only glucose as a carbon source, (2) containing galactose as an extra carbon source and (3) containing sodium acetate as an extra carbon source, since we checked in previous tests that low concentrations of glucose compromised cell growth. All tubes had, in addition to the glucose gradation, also IPTG (see information on the <html><a href="https://2012.igem.org/Team:Valencia_Biocampus/Molecular#LACTOSE-INDUCED_PROMOTER"><b>molecular mechanism</b></a></html>). For fluorescence intensity (FI) measures cell growth, i.e. OD600, was taken into account. We worked with a 0D600 close to 0.1 in order to be able to use high sensitivity in the fluorimeter.
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In order to check if both constructions are functional, we induced expression using several media as depicted in this <a href=”yeast induction”> protocol </a>. The supplemented SD media allows the selective growth of transgenic yeasts to obtain a big inoculant which will grow better in a glucose-rich media (YPD8%), leaving us with a culture in exponential phase and high biomass. The expression of ZsGreen1 takes place when our yeasts are deprived of glucose and there is presence of ethanol. Further information about molecular mechanisms is <a href=”molecular mechanism”> here </a>.
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In order to quantify cell growth and normalize fluorescence, the DO at 600 nm of each sample was measured. Fluorescence intensity was measured at an excitation wavelength of 493 nm and an emission wavelength of 505 nm.
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Figure 1 shows expression of ZsGreen1 in our transformed yeast –and not in non-transformed yeast– in <a href=”ypre broth”>YPRE broth</a>, that is, in presence of ethanol when glucose is absent. Figure 2 shows intracellular expression of our protein.
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Further experiments are required for a better characterization of our constructions, in which we are already working, but cannot be shown
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As you can see in the graphs below, <b>the less glucose you have, the more fluorescence you get</b>. And this results replicate well when galactose (Figure 2) or sodium acetate (Figure 3) were added to the medium as supplementary carbon sources. The threshold for a boost in the fluorescence intensity seems to be 0.1 g/L of glucose, since from this concentration the values get really high. Normal values are 10 g/L, and we got the best results when we added 10<sup>-4</sup> glucose concentration of that of the canonical values.
 

Revision as of 15:07, 25 September 2012




    Talking to yeast


    How long have you been fermenting?


    In order to check if both constructions are functional, we induced expression using several media as depicted in this <a href=”yeast induction”> protocol </a>. The supplemented SD media allows the selective growth of transgenic yeasts to obtain a big inoculant which will grow better in a glucose-rich media (YPD8%), leaving us with a culture in exponential phase and high biomass. The expression of ZsGreen1 takes place when our yeasts are deprived of glucose and there is presence of ethanol. Further information about molecular mechanisms is <a href=”molecular mechanism”> here </a>.
    In order to quantify cell growth and normalize fluorescence, the DO at 600 nm of each sample was measured. Fluorescence intensity was measured at an excitation wavelength of 493 nm and an emission wavelength of 505 nm.
    Figure 1 shows expression of ZsGreen1 in our transformed yeast –and not in non-transformed yeast– in <a href=”ypre broth”>YPRE broth</a>, that is, in presence of ethanol when glucose is absent. Figure 2 shows intracellular expression of our protein.
    Further experiments are required for a better characterization of our constructions, in which we are already working, but cannot be shown


    Figure 1. Fluorescence intensity (FI) normalized by the optical density of the
    culture (OD) for differents amounts of glucose present in the medium.

    Figure 2. Fluorescence intensity (FI) normalized by the optical density of the
    culture (OD) for differents amounts of glucose in a medium supplemented with
    3 g/L galactose.

    Figure 3. Fluorescence intensity (FI) normalized by the optical density of the
    culture (OD) for differents amounts of glucose in a medium supplemented with
    3 g/L sodium acetate.



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