Introduction

As detailed before, we aim to develop a versitale ROS-sensing platform for agritech and health applications. Therefore, we decided to focus our platform on the two main engineered micro-organisms: yeast and bacteria. Our model disease for agritech is fireblight and IBD for medecine.

Design considerations

Species and strain choice

The most studied yeast strain is Saccharomyces cerevisiae, which can live epiphytically on plants, and the most engineered gut-friendly lab strain is the bacterial strain Escherichia coli Nissle 1917. We had considered other organisms such as the yeast in Blossom Protect biocontrol spray Aureobasidium pullulans, but as a sporulating mycete it would have been to complex to engineer in a few months. We have also considered bacteria for the agritech application, such as the C9-1 white variant of Pantoea vagans. Indeed, it has a short life-expectancy but long enough to last the blossoming period (1-2 weeks) w which would fit in safety measures for GMOs, and Pantoea species are widely used for plant biocontrol and epiphytic applications, but we were already using a bacteria for the medecine application and thought it would be pertinent for our platform to encompass

Transcription factor choice

To detect fireblight, we wish to engineer our yeast to produce a GFP fluorescent signal upon oxydative stress detection. Indeed, during fireblight, reactive oxygen species (ROS) soar up to 10-fold. A ROS-sensitive transcription factor ("TF" from now on) tuned to react to the relevant H2O2 concentration could allow the yeast to express GFP only during infection. For this TF, we chose to test in parallel the yeast's native YAP1, and plant TF involved in H2O2-directed immune responses TGA2. Both have specificities to take into account for design which we will go over in the next section. To locally and timely produce anti-oxidant enzymes (such as Superoxide Dismutase SOD) to help with inflammation in IBD, we engineered our bacteria to first produce GFP (as a reporter) in a ROS-sensitive manner. The TF chosen here, is bacteria's most characterized and widespread ROS-sensitive TF: OxyR.

Transcription factor choices' implication in design

YAP1 and OxyR are already expressed by the yeast/bacteria, which means that we cannot engineer the TF itself but rather adjust the sensitivity of the promoter. We chose to achieve random-mutagenesis of the promoter region through error-prone PCR.

Final construct designs

epPCR design

Building the plasmids

Testing?