While designing SugarGain, we had to keep various considerations in mind. The initial idea was broken down into a set of problem statements and these formed the major pillars of our project. The following diagrams illustrate the flow of our thought as we tried to solve these problems to the best of our ability. We also illustrate how we modelled our project and its interconnectedness with the experiments we designed to complement the modelled data. Furthermore, we have taken into consideration the different outcomes that these experiments might lead to and planned accordingly. Due to unavailability of lab access this year, we plan to finish these experiments in phase 2.
For more details about these experiments, please see our Experiments page.
pFruB-Cra System
Problem Statement
Our project aims to reduce the activity of invertase, an enzyme that converts useful sucrose into profitless glucose and fructose. Since this process was important to the life cycle of the plant, it wasn’t practical to genetically re-engineer sugarcane cells itself. The most harm this enzyme causes is post-harvest and hence, that seemed like the most logical time period for us to target.
Designing Process
1
After surveying publications on invertases, we came across a group of proteins called anti-invertases/invertase inhibitor which regulate invertase and control sugar hydrolysis.
3
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).
2
To decrease the nutrient load on our bacterial chassis and have a controlled release of our protein, we decided on a biosensor mechanism.
4
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.
5
Using these parts, we constructed our fructose regulated anti invertase.
Modelling
Biosensor will simulate output of invertase inhibitor for particular levels of fructose in the environment and will also link the promoter activity to a measurable output.
Experimental Design
Due to the global pandemic, we were unable to complement our modelling with experimental results. However, we prepared and planned for these experiments to strengthen our project design and help the next team carry out our project to completion.
We tried to get an insight into the protein-protein interaction through modelling of anti-invertase with invertase.
We tried to get an insight into the protein-DNA interaction through homology modelling of FruR.
Since FruR and its interactions are not extensively characterised, we had to conduct molecular docking studies in order to elucidate the interactions between FruR and fructose.
Identify the optimum Kd value for the system to work
In phase 2, prediction of overall efficiency of the pFruB-Cra system and the amount of inoculant required per sugarcane using information from experiments.
Anti-Invertase Assay to characterise the functioning of anti-invertase.
FruR Specificity Experiment
pFruB-FruR Characterisation
Gel shift assay to experimentally determine Kd value of pFruB.
Site-directed mutagenesis of the pFruB to bring the Kd value to the one established through the model
if inadequate
Atmosphere Regulated Killswitch
Problem Statement
Given the consumable nature of our product, we had to come up with a robust killswitch that was capable of effectively terminating the bacteria taking into account the toxicity of killswitch with respect to the humans.
Designing Process
1
First, we ascertained the trigger to be the swift and abrupt change in atmospheric conditions (anaerobic inside the sugarcane stem and aerobic outside)
2
Second, we found a toxin-antitoxin pair that was quite fatal to bacteria but did not affect human cells.
3
After an extensive search of the registry, we found the FNR promoter that was only activated in anaerobic conditions.
4
Using these parts, we designed our killswitch genetic circuit.
Modelling
We focussed on modelling the behaviour of the circuit to the change in concentration of oxygen in the atmosphere. We further predicted the response time for the same.
Experimental Design
Due to the global pandemic, we were unable to complement our modelling with experimental results. However, we prepared and planned for these experiments to strengthen our project design and help the next team carry out our project to completion.
Create a persister cell model in phase 2
Differential expression of the antitoxin in the presence and absence of oxygen
Time required for the buildup of toxin concentration sufficient to restrict bacterial growth
Identify the optimum Kd value for the system to work
Assay to check if the killswitch functions as intended
Killswitch response time assay to ascertain the time required after exposure for the cell to succumb
Gel shift assay to experimentally determine Kd value of the FNR promoter
Site-directed mutagenesis of the FNR promoter to bring the Kd value to the one established through the model
pH dependency of the FNR promoter (due to the fluctuation in pH in the sugar extraction process)
if high cell viability
if not quick enough
if inadequate Kd value
Polymer Inoculant
Problem Statement
Since our product includes live bacteria, we wanted to ensure that the product had a long shelf life. After consulting farmers in various belts across India, we also came across the need for the product to be organic, non-toxic and cheap.
Designing Process
1
We started researching polymer-based inoculants that had longevity and were organic in nature.
2
We determined the optimal inoculant composition of a Carboxymethylcellulose-based polymer with bentonite suspending agent and sorbic acid preservative.
3
We optimized the viscosity to minimize the inoculant's toxicity, and ensured that it is close to that of water for efficient transport.
4
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.
Modelling
Keeping in mind the concerns of the sugarcane farmers across major Indian belts,we devised ways to optimise the features of the polymer inoculant.
Experimental Design
Due to the global pandemic, we were unable to complement our modelling with experimental results. However, we prepared and planned for these experiments to strengthen our project design and help the next team carry out our project to completion.
Kinematic Viscosity model and Toxicity model
Proposed implementation and economic scalability model
Removal of chemical residues of polymer inoculant during processing of sugarcane juice
Validating toxicity model
Capillary action experiment will help with delivery mechanism model in proposed implementation