Team:UNICAMP-Brazil/Notebooks/October 20

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