Team:Newcastle/Demonstrate

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Alternative Roots

Alternative Roots

Demonstrate

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Alternative Roots

What worked - and didn’t work - in 2018 for Alternative Roots

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 colonisation of plant tissues by our Pseudomonas sp.
  2. We have demonstrated that this species is genetically transformable.
  3. We have demonstrated that transformed our Pseudomonas sp. can colonise root tissues.
  4. We have identified an origin or replication and two selectable antibiotic markers (gentamicin and streptomycin) that can be used with our Pseudomonas sp.

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.

Metabolic Engineering

  1. We have assembled a four gene operon coding for the naringenin biosynthetic pathway, but were unable to test this experimentally…
  2. …however, we have built a kinetic model describing pathway flux that demonstrates that a better design for balancing flux can be achieved by creating two, two-gene operons instead of a single, four-gene operon.

Measurements and standards

  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 endophyte.
  3. We have demonstrated the value of a chemically-defined media to chassis optimisation and parts characterisation.