Team:UNICAMP-Brazil/Notebooks/October 20

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====Yeast experiments: Lysozyme====
====Yeast experiments: Lysozyme====
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*<p style=”text-align:justify;”>Today we began the tests with the yeasts transformed with the following constructions: YEP+pDLD-Lysozyme and YEP+Adh1-Lysozyme.</p>
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*<p style=”text-align:justify;”>Today we began the tests with the yeasts transformed with the following constructions: YEP+pDLD-Lysozyme and YEP+''ADH1''-Lysozyme.</p>
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*<p style=”text-align:justify;”>We inoculated the yeast pre inocula in 3 types of media: YNB Glucose Ura-, YNB Ethanol Ura- and YNB Glucose Ura+. The inocula grew for 4 hours in 30ºC and 150rpm before the induction with lactic acid. Four concentrations of lactic acid were used in the induction.</p>
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* The pre inocula made yesterday grew.
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* We will have 10 samples at the end of the tests:  
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a) Wild type: inoculated in Ura+ media. No plasmid, no induction, no lysozyme expression.
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b) Glucose ''ADH1''+Lys: plasmid with lysozyme under control of ''ADH1'', a constitutive promoter. No induction.
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c) Glucose pDLD+Lys: No induction. Lysozyme under control of pDLD, a lactate responsive promoter.
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d) Glucose pDLD+Lys 1: lysozyme under control of pDLD, a lactate responsive promoter. Induction with 5mM lactate.
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e) Glucose pDLD+Lys 2: lysozyme under control of pDLD, a lactate responsive promoter. Induction with 25mM lactate.
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f) Glucose pDLD+Lys 3: lysozyme under control of pDLD, a lactate responsive promoter. Induction with 50mM lactate
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g) Ethanol pDLD1+Lys: No induction. Lysozyme under control of Adh1, a constitutive promoter.
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h) Ethanol pDLD1+Lys 1: lysozyme under control of pDLD, a lactate responsive promoter. Induction with 5mM lactate.
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i) Ethanol pDLD1+Lys 2: lysozyme under control of pDLD, a lactate responsive promoter. Induction with 25mM lactate.
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j) Ethanol pDLD1+Lys 3: lysozyme under control of pDLD, a lactate responsive promoter. Induction with 50mM lactate
 +
 
 +
* We chose ethanol as a carbone source since it doesn't represses the lactate perception unlike glucose (Catabolic Repression). 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 1,3 before the induction with lactic acid.  
 +
 
 +
* We performed the induction 5 hours long. We mesured the OD every one hour to construct a growth curve. At the end of the 5 hours we pelleted the culture, colected the supernatant and freezed the pellet to analyze in SDS-PAGE tomorrow.
 +
 
 +
* We also dripped drops of the supernatant in filter paper discs and placed them in a ''Lactococcus lactis'' plate. We hope to see inhibition zones surrounding some of the filters.
 +
 
 +
* The Yeasts inoculated in YNB Ethanol media didn't grow enough to begin the induction until the end of the day.  

Latest revision as of 03:33, 22 October 2009

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YeastGuard

Yeast experiments: Lysozyme

  • Today we began the tests with the yeasts transformed with the following constructions: YEP+pDLD-Lysozyme and YEP+ADH1-Lysozyme.

  • The pre inocula made yesterday grew.
  • We will have 10 samples at the end of the tests:

a) Wild type: inoculated in Ura+ media. No plasmid, no induction, no lysozyme expression.

b) Glucose ADH1+Lys: plasmid with lysozyme under control of ADH1, a constitutive promoter. No induction.

c) Glucose pDLD+Lys: No induction. Lysozyme under control of pDLD, a lactate responsive promoter.

d) Glucose pDLD+Lys 1: lysozyme under control of pDLD, a lactate responsive promoter. Induction with 5mM lactate.

e) Glucose pDLD+Lys 2: lysozyme under control of pDLD, a lactate responsive promoter. Induction with 25mM lactate.

f) Glucose pDLD+Lys 3: lysozyme under control of pDLD, a lactate responsive promoter. Induction with 50mM lactate

g) Ethanol pDLD1+Lys: No induction. Lysozyme under control of Adh1, a constitutive promoter.

h) Ethanol pDLD1+Lys 1: lysozyme under control of pDLD, a lactate responsive promoter. Induction with 5mM lactate.

i) Ethanol pDLD1+Lys 2: lysozyme under control of pDLD, a lactate responsive promoter. Induction with 25mM lactate.

j) Ethanol pDLD1+Lys 3: lysozyme under control of pDLD, a lactate responsive promoter. Induction with 50mM lactate

  • We chose ethanol as a carbone source since it doesn't represses the lactate perception unlike glucose (Catabolic Repression). 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 1,3 before the induction with lactic acid.
  • We performed the induction 5 hours long. We mesured the OD every one hour to construct a growth curve. At the end of the 5 hours we pelleted the culture, colected the supernatant and freezed the pellet to analyze in SDS-PAGE tomorrow.
  • We also dripped drops of the supernatant in filter paper discs and placed them in a Lactococcus lactis plate. We hope to see inhibition zones surrounding some of the filters.
  • The Yeasts inoculated in YNB Ethanol media didn't grow enough to begin the induction until the end of the day.


Raíssa and Taís




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