Transformation Preparation Cycle 1

Design

Initially, we believed that we had to grow the leucine-deficient yeast strain (BY4742) in the same conditions necessary for successful transformation (plasmid uptake) — meaning we thought the yeast could stabilize even under leucine-deficient conditions and grow to reach required density.

Build & Test
  1. Plated 10μL of the yeast in YND CSM-Leu agar plates (prepared with 100 mL dH₂O, 0.67 g YNB, 2 g glucose, 0.83 g CSM-Leu, and 1.5 g agar powder).
  2. Incubated plates overnight at 37°C.
  3. Checked plate for growth the next day and found insufficient density to complete transformation.
Cycle 1 build step 1
Cycle 1 build step 2
Learn

We learned that the yeast strain requires nutrient-rich medium (including leucine) and cannot reproduce on its own without leucine.

Transformation Preparation Cycle 2

Design
  1. Took into account results from Cycle 1 and searched for nutrient-rich medium.
  2. Decided to opt for YPD to facilitate growth.
  3. Increased the yeast volume plated to improve chances of higher yield.
Cycle 2 design step
Build & Test
  1. Prepared 1% YPD powder mix solution as recommended by the manufacturer.
  2. Added the YPD mix to agar plates instead of the CSM-Leu.
  3. Observed that the desired growth was still not achieved.
Learn

We recalled that our yeast strain had been spilled when we opened the packaging. This suggested it was thawed during delivery and refrozen, which could have significantly compromised yeast viability and affected density yield during fermentation.

Cycle 2 learn step