Team:NUS Singapore/Proof Of Concept

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iGEM Wiki

Proof Of Concept

Testing our Blue Light Flocculation System

pGH3CF(BY474D) contained a plasmid encoding GAL1 controlled HBD2 production as well as our synthetic 3C120-CYC-LacO promoter controlling flocculation, transformed into a strain with our improved EL222 expression driver to promote stronger blue light flocculation.

pGH3CF(BY474D) was cultured in a dark incubator at 30 degrees, 220rpm shaking with YNB-URA and glucose as a carbon source overnight to achieve high cell mass. The next day, galactose was added to induce production and secretion of HBD2 into the media.

After HBD2 induction over 3 days, flask was switched to our DIY bioreactor. Blue light was coded to automatically switch on to induce flocculation (Figure 1).

Figure 1: Culture in bioreactor before and after flocculation induction facilitated by optogenetic reactor

Testing the Functionality of Human Beta Defensin (HBD)

Supernatant was drained from the bioreactor automatically using the peristaltic pump, and concentrated using centrifugation and a size filter to 100x concentration. It was then directly used in an MIC assay against E.coli without purification (Figure 2). To ensure the yeast itself did not directly produce any compouds with anti-microbial activity, the media of BY474D strain without HBD producing plasmid, as well as the flowthrough from the concentration containing remaining media were included as negative controls.

Supernatant from flocculated culture showed anti-microbial activity compared to negative controls, demonstrating a successful proof that our system can be used to produce environmentally friendly, human safe pesticides in a cost effective manner using an automatic 3D printed bioreactor, optogenetics, and flocculation based purification.

Figure 2: pGH3CF (BY474D) supernatant concentrated (Top left), 1mg/ml Ampicillin positive control (Top right), BY474D media negative control (Bottom left), flowthrough from media concentration (Bottom right).