After surveying publications on invertases, we came across a group of proteins called anti-invertases/invertase inhibitor which regulate invertase and control sugar hydrolysis.

We also discovered that anti-invertase has quite a small half-life. This meant that the invertase would have to be secreted at the site of action (i.e., inside the sugarcane).

To decrease the nutrient load on our bacterial chassis and have a controlled release of our protein, we decided on a biosensor mechanism.

Since the concentration of fructose was a more or less direct result of invertase activity, we wanted to calibrate a self-regulatory mechanism that modulated the secretion of the anti-invertase in tandem with the concentration of fructose in the medium. We were able to achieve that by using a pFruB-Cra system.

First, we ascertained the trigger to be the swift and abrupt change in atmospheric conditions (anaerobic inside the sugarcane stem and aerobic outside)

Second, we found a toxin-antitoxin pair that was quite fatal to bacteria but did not affect human cells.

After an extensive search of the registry, we found the FNR promoter that was only activated in anaerobic conditions.

Using these parts, we designed our killswitch genetic circuit.

We started researching polymer-based inoculants that had longevity and were organic in nature.

We determined the optimal inoculant composition of a Carboxymethylcellulose-based polymer with bentonite suspending agent and sorbic acid preservative.

We optimized the viscosity to minimize the inoculant's toxicity, and ensured that it is close to that of water for efficient transport.

This inoculant would be supplied to the farmer and can be used through an injector mechanism. In this manner the sugarcane's inherent transport mechanism due to transpirational pull is leveraged for the uniform distribution of the bacteria through the plant.