Team:UNICAMP-Brazil/Yeastguard/Results

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===Yeast experiments: Lysozyme===
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====Yeast experiments: Lysozyme====
 
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Lysozyme constitutive expression and Lysozyme expression in response to lactate (DLD promoter) devices were transferred to an yeast expression vector (YEp358 ura+) and then were transformed into the Saccharomyces cerevisiae YF23 ura- strain.
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Lysozyme constitutive expression and Lysozyme expression in response to lactate (DLD promoter) devices were cloned into a yeast expression vector (YEp358 ura+) and then they were transformed into the Saccharomyces cerevisiae FY23 ura- strain. After being plated in selective medium without uracil, the transformants were selected and transferred to liquid medium (also selective) containing glucose or ethanol as carbon source.
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After being plated on selective medium without uracil, the transformants were selected and transferred to liquid medium also selective.
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From the initial OD of ~1.71 the growth of non-induced and lactate-induced yeast was monitored for 5 hours (Figure 1).  
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[[Image:Graficos.jpg|800px|center]]
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With these tests we expect to see if there is expression of lysozyme under control of the constitutive promoter(ADH1) and the lactate responsive (pDLD) promoters, the last one under lactate induction. The idea of using ethanol as carbon source is to see if there is catabolic repression of the pDLD promoter by glucose, it wouldn't work in glucose samples if this hypothesis were true.  
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We inoculated the pre inocula in 50mL of each medium (YNB Ethanol Ura- and YNB Glucose Ura-). The wild type yeast is Ura- so we grew it on YNB Glucose Ura+. The inocula grew for 4 hours in 30ºC and 150rpm until the OD 1,0 before the induction with lactic acid. When the cultures reached the OD600 of 1.0, we started the  lactate induction.
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The lysozyme expression were verified by SDS-PAGE (Figure x) and by lactobacilli killing induction (Figure y).
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We performed the induction 5-7 hours long. We measured the OD every one hour to construct a growth curve (Figure 1 - a,c,e) . At the end of the 5-7 hours we pelleted the culture, collected the supernatant and freezed the pellet to analyze in SDS-PAGE and in ''Lactococcus lactis'' death experiment.
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'''Growth curve'''
 
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[[Image:Graficos.jpg|630px|center]]
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Figure 1: Yeast growth curves and the respectives ''Lactococcus lactis'' death experiment ''(1 - control: non-induced)''.
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'''The samples were analyzed by:'''
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'''''Lactococcus lactis'''''
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I) Application of the yeasts culture supernatant in liquid cultures of ''Lactococcus lactis''(Figure 1 - b,d,f).
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II) SDS-PAGE of the supernatant and the total extract of the yeats cells (in case the protein has not been exported) to see if the protein was expressed (Figure below).
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[[Image:geis sds page final result.png|400px|center]]
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'''SDS-PAGE'''
 
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We made two SDS-PAGE gels, one for the supernatant and the other for the total yeast extract.
 
We couldn't load the pellet ethanol samples maybe because of residual ethanol at the pellets; the alcohol hinder the entry of the sample in the well.
We couldn't load the pellet ethanol samples maybe because of residual ethanol at the pellets; the alcohol hinder the entry of the sample in the well.
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Unfortunately we couldn't see any protein band in the gel neither in the supernatant one nor in the total extract one. Maybe we loaded insufficient amounts of sample.  
Unfortunately we couldn't see any protein band in the gel neither in the supernatant one nor in the total extract one. Maybe we loaded insufficient amounts of sample.  
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[[Image:geis sds page final result.png|400px|center]]
 
{{:Team:UNICAMP-Brazil/inc_rodape}}
{{:Team:UNICAMP-Brazil/inc_rodape}}

Latest revision as of 03:56, 22 October 2009

Topo l2.gif topo_r_igem.gif
topo_r_b.gif

The Yeastguard: Results

Confirmed Biobricks


Mechanism of recognition


Parts:

  • BBa_K284002 - JEN1 Promoter from Kluyveromyces lactis -
  • BBa_K284003 - Partial DLD Promoter from Kluyveromyces lactis
Biofusion-digestion.jpg


Devices to JEN1 and DLD promoters characterization:

  • BBa_K284020 - EYFP regulated by lactate responsive promoter (JEN1)
  • BBa_K284021 - EYFP regulated by lactate responsive promoter (DLD)
  • BBa_K284023 - EYFP regulated by constitutive promoter Adh1
20091021 promotores com YFP.jpg


Killing mechanism


Parts:

  • BBa_K284001 - Lysozyme from Gallus gallus
Lysozymedigestionresult.png


Devices to Lysozyme characterization:

  • BBa_K284016 - Lysozyme constitutive expression
  • BBa_K284017 - Lysozyme expression in response to lactate (DLD promoter)
  • BBa_K284015 - Lysozyme expression in response to lactate (JEN1 promoter)
Final adh1lys rwsult.png
20091021 promotores com lis final results.jpg


Yeast experiments: Lysozyme


Lysozyme constitutive expression and Lysozyme expression in response to lactate (DLD promoter) devices were cloned into a yeast expression vector (YEp358 ura+) and then they were transformed into the Saccharomyces cerevisiae FY23 ura- strain. After being plated in selective medium without uracil, the transformants were selected and transferred to liquid medium (also selective) containing glucose or ethanol as carbon source.

With these tests we expect to see if there is expression of lysozyme under control of the constitutive promoter(ADH1) and the lactate responsive (pDLD) promoters, the last one under lactate induction. The idea of using ethanol as carbon source is to see if there is catabolic repression of the pDLD promoter by glucose, it wouldn't work in glucose samples if this hypothesis were true.

We inoculated the pre inocula in 50mL of each medium (YNB Ethanol Ura- and YNB Glucose Ura-). The wild type yeast is Ura- so we grew it on YNB Glucose Ura+. The inocula grew for 4 hours in 30ºC and 150rpm until the OD 1,0 before the induction with lactic acid. When the cultures reached the OD600 of 1.0, we started the lactate induction.

We performed the induction 5-7 hours long. We measured the OD every one hour to construct a growth curve (Figure 1 - a,c,e) . At the end of the 5-7 hours we pelleted the culture, collected the supernatant and freezed the pellet to analyze in SDS-PAGE and in Lactococcus lactis death experiment.


Graficos.jpg

Figure 1: Yeast growth curves and the respectives Lactococcus lactis death experiment (1 - control: non-induced).

The samples were analyzed by:


I) Application of the yeasts culture supernatant in liquid cultures of Lactococcus lactis(Figure 1 - b,d,f).


II) SDS-PAGE of the supernatant and the total extract of the yeats cells (in case the protein has not been exported) to see if the protein was expressed (Figure below).


Geis sds page final result.png



We couldn't load the pellet ethanol samples maybe because of residual ethanol at the pellets; the alcohol hinder the entry of the sample in the well.

Unfortunately we couldn't see any protein band in the gel neither in the supernatant one nor in the total extract one. Maybe we loaded insufficient amounts of sample.





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