The Yeastguard: Results

Confirmed 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 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.

Figure 1: Yeast growth curves and the respectives Lactococcus lactis death experiment.

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).

Figure 1:

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.