Team:UNICAMP-Brazil/Yeastguard/Results

The Yeastguard: Results

We confirm the following Biobricks:

Mechanism of recognition

Parts:

• BBa_K284002 - JEN1 Promoter from Kluyveromyces lactis -
• BBa_K284003 - Partial DLD Promoter from Kluyveromyces lactis

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

Killing mechanism

Parts:

• BBa_K284001 - Lysozyme from Gallus gallus

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)

Yeast experiments: Lysozyme

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. After being plated on selective medium without uracil, the transformants were selected and transferred to liquid medium also selective. From the initial OD of ~1.71 the growth of non-induced and lactate-induced yeast was monitored for 5 hours (Figure 1).

grafico

The lysozyme expression were verified by SDS-PAGE (Figure x) and by lactobacilli killing induction (Figure y).

Growth curve

Lactococcus lactis

SDS-PAGE

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.

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.

20091021_SDSpage_extrato_levedura.png

20091021_sdspage_sobrenadantes.png