Team:Amsterdam/Project/testing rom
Testing Romance
Lasting bonds or one-night stand?
Overview
Background
The ultimate test for any romance is lovers living together. Will they cooperate in mutually beneficial ways? Can true microbial romance be sustained? Or will our microbial relationships last no longer than a one night stand? And, in the case of (photo)synthetic consortia designed to benefit mankind by delivering products: can it be used to convert CO2 into a valuable compounds?
Aim
We developed extensive theory about consortia by an interplay of in vivo and in silico approaches shaping each others design. However, all the experimental information we've gathered to help quantitatively understand the consortia was done by cultivating the partners in isolation, trying to mimic the environment they would meet in the co-culture as much as possible. Here, we aim to validate our assumptions and efforts thus far: we want to put the members of our rationally-designed consortium - the carbon-sharing Synechocystis strain and a product-producing E. coli - together in the same compartment to test growth and product formation.
Approach
To assess the development of our consortia and determine final organism ratios, we innoculated 48-well plates with acetate and lactate-producing Synechocystis strains and E. coli. To test product formation, we constructed two consortia: one in which we transformed an E. coli with Bielefeld’s iso-butanol biobrick for the of CO2 to acetate to isobutanol; and another consortium based on an acetoin producing E. coli and Synechocystis engineered to produce glycerol and meso-butanediol from acetoin, showcasing the benefits of compartmentalization in consortia. Both product-producing consortia were tested in batch and turbidostat experiments.
Results
We show growth and organism ratio convergence. And, on the very last day of our project, we finally showed meso-butanodiol production in our consortium! This demonstrates how consortia can be used to implement complex redox production pathways that would be infeasible in a single organism. .
Connections
- Stable Romance: Measure stability.
- Engineering Romance: Using software tools to select targets
- Measuring Romance: Turbidostat experiments.
The first step: showing growth
The first thing we wanted to demonstrate was co-culture growth in which an E. coli culture was able to grow on the carbon shared by Synechocystis. We innoculated 48-well plates with different starting ratios of Synechocystis and E. coli. We used both the lactate-producing Synechocystis strains whose genetic instability we showed earlier, as well as the acetate-producing strain based on the acs knock-out. Cells were counted using a coulter-counter before innoculation and re-counted every 24 hours for seven consecutive days. Results are shown in figure 1 for the acetate producer & E. coli consortium, while results for the lactate-producer & E. coli consortium are shown in figure 2. Overall, all consortia seem to converge to a common final ratio, showing robust population dynamics governed by the carbon-sharing dependency of E. coli on Synechocystis. This result is perfectly aligned with our in silico simulations {link} that had predicted the existence of attractor states, quite robust to fluctuations in model parameters such as the initial ratio of Synechocystis:E. coli.
The next step: demonstrating production
Approach 1: Acetate-production and isobutanol
Our first attempt to demonstate product-production in a synthetic consortium involved our stable acetate-producer and an isobutanol-producing E. coli strain. The latter was obtained via the isobutanol pathway biobrick that was part of this year’s distribution kit and submitted by Bielefeld’s iGEM team last year. Their isobutanol pathway consists of four enzymes under the control of an inducible promoter. Unfortunately, despite succesfully transforming an E. coli strain, we could not demonstrate subsequent isobutanol production in neither stand-alone E. coli growing on various carbon sources nor E. coli driven by Synechocystis stable supply of acetate.
Approach 2: Three-step meso-butanodiol production
In an ambitious last attempt to demonstrate product formation using a consortium, we combined an acetoin-producing E. coli strain available in our lab with a previously-engineered Synechocystis strain that produces glycerol via insertions of a gene coding for phospho-glycerol phosphatase (ggp), which catalyzes the dephosphorylation of glycerol-3-phosphate to glycerol (Savakis, 2015). We reasoned that with a modified glycerol-producing strain also created in our lab by Philipp Savakis, the separate compartments of Synechocystis and E. coli could be used to more favourably drive a reaction towards the production of meso-butanediol, an important chemical precursor for a variety of industrial chemicals and biofuels. Previous attempts involved producing meso-butanediol from acetoin within the same compartment in a single cell (figure 2). The flux to meso-butanediol, however, is hampered by the series of redox-reactions with overall unfavourable thermodynamics due to presence in the same compartment. To overcome this, we designed the following consortium:
After several days of turbidostat innoculation, we managed to show meso-butanediol production using HPLC. Although we obtained these results mere hours before the wiki-freeze and are as such far from being fully exploited, the result demonstrates the potential of using a consortium for successfully driving product-flux using compartmentalisation in what would otherwise be an unfavourable thermodynamic environment.
