Overview

Alternative Roots is a mulit-component project proposing that plant endophytes - microbes that live harmlessly within plant tissues - can be engineered to enhance beneficial plant : microbial interactions. This may be achieved, for example, by engineering plant endophytes to synthesise chemoattractants of free-living, nitrogen-fixing bacteria. We examined both the feasibility of the biology and considered how this technology might be viably deployed in our local community. Here we highlight some of the conclusions from our work.

Chassis Development

1. We have demonstrated root colonisation by our Pseudomonas species.
2. We have demonstrated that this Pseudomonas species is genetically transformable.
3. Importantly, we have demonstrated that our genetically transformed Pseudomonas can colonise root tissues.
4. We have identified an origin of replication and two selection methods (gentamicin and streptomycin) that can be used with this Pseudomonas chassis.

Microbial Community Engineering

1. We have characterised the responses of three free-living, nitrogen-fixing bacteria to the chemoattractant naringenin and demonstrated that two are attracted to this flavonoid.
2. In tandem with our experimental characterisation we have built an agent-based model that indicates that naringenin biosynthesis by a plant endophyte would result in the formation of a biofilm by N₂ -fixing bacteria.
3. We have built a kinetic model describing flux through the naringenin biosynthetic pathway demonstrating an improved design for balancing flux, creating two, two-gene operons instead of a single, four-gene operon.

Measurement, Automation and Reproducibility

1. Any chassis development requires good characterisation and measurement. We examined how this works within the InterLab study and created an Internal Standard for each test device. Our new devices worked, and demonstrated that even simple systems such as the InterLab test devices are highly context-dependent.
2. We demonstrated small-scale, parallel automation and optimisation of E. coli transformation protocols using the Opentrons OT-2 robot that can now be used to optimise transformation of our Pseudomonas chassis.
3. We have demonstrated the value of a chemically-defined media to chassis optimisation and parts characterisation.

Hardware and Software

1. We have demonstrated a functional, low-cost programmable plant growth chamber capable of housing >1300 seedlings that permits high-throughput plant experiments and physically simulates some of the growth conditions proposed for Newcastle's Victoria Tunnel by Alternative Roots.
2. Underpinning our development of the plant growth chamber NH-1 and use of the OT-2 robotics is the need for software. We have developed and successfully deployed code for both NH-1 and OT-2.

Integrated Human Practices

1. All of this work was guided by our dialogue with those working in and around the agricultural and food production sectors. We designed an underground production facility that is consistent with local legislation and iGEM safety considerations relating the use and release of genetically modified organisms and microbes.
2. These plans provided an opportunity to engage with the public on issues such as local food production and different aspects of synthetic biology.
3. Finally, regardless of the progress Alternative Roots makes as a synthetic biology project within the iGEM community, our project has started a conversation amongst local food producers and social movements about the potential to develop Newcastle’s largely disused Victoria Tunnel network to grow fresh, local produce for the city.