Key Results

In this section we will discuss the key results the BananaGuard project (figure 1), which is a combination of both experimental and modelling. What we have achieved is a system that can sense fusaric acid, produce fungus growth inhibitors coupled together with a Kill-switch and have models that support our system. We will shortly discuss sensing, inhbition, Kill-switch, promoter design, system model cost and performance.

Sensing

While fusaric acid dependent protection systems have been observed in microorganisms and mechanisms for this protection have been predicted in silico after genome sequencing, no evidence of a validated fusaric acid dependent promoter could be found in the literature.In this project we characterised and validated such a promoter. A putative fusaric acid dependent promoter along with its hypothesized regulator (isolated from Pseudomonas putida KT2440) were cloned in front of GFP(BBa_E0040), transformed in Pseudomonas putida KT2440 (P.putida). And fluoresence were measured in different fusaric acid concentration. We were able to validate and characterize a novel fusaric acid dependent promoter (Bba_K1493000).

For more information, read fungal sensing.

Inhibition

Upon sensing fusaric acid, three genes and a gene cluster will be activated that will lead to production of certain fungal growth inhibitors. They were cloned behind a IPTG inducible promoter. Those genes and their function being:

1. phlABCDE gene cluster, able to produce 2,4-Diacetylphloroglucinol(2,4-DAPG)
2. Methionine-γ-lyase, Dimethyldisulfide (DMDS) and dimethyltrisulfide (DMTS)
3. PfrI, produce pyoverdine in presence of iron
4. Chitinase, overexpresses chitinase activity

Methionine-γ-lyase and PfrI were both made into biobricks, Bba_K1493300 and Bba_K1493200 respectively. With both biobricks validated, and for PfrI characterized. PfrI has shown to give a four fold increase of pyoverdine production in the presence of iron (31μM) in the medium. Pyoverdine is a compound that chelates iron and is naturally only produced when there is no iron available.

All transformants were co-inoculated with Fusarium oxysporum cubense TR4 on agar plates in order to test their inhibition ability. Controls used were wild type P.putida KT2440 with F.oxysporum and just F.oxysporum.

In general, it was hard to distinguish the increased inhibition effect of the fungal growth inhibitors producing P. putida against F. oxysporum. This is because the P. putida chassis we have chosen is already very good at inhibiting F. oxysporum naturally, which probably makes it hard to observe increased growth inhibition by our synthetic, growth inhibitor producing P. putida strains. However, with the Methionine-γ-lyase(MgL) strain, we have a clear indication that there is an enhanced growth inhibition of F. oxysporum (figure 3) and with others, producing 2,4-DAPG, chitinase or pyoverdine, we can say that there is an indication of a slight increase of growth inhibition (figure 8, 12 and 13) on top of the natural inhibition. For more information, read fungal inhibiton.

Kill-Switch

Once fungal growth inhibitors are produced and F. oxysporum is no longer in the soil BananaGuard has done it's job and is no longer needed in the soil. Therefore we have implemented a Kill-switch into our system, which works like a toggle switch that senses when fusaric acid is around, and when it has dissipated toxins will be produced that eliminate BananaGuard. Toxins will be produced that eliminate BananaGuard itself, with the kill switch regulatory system in figure 4.

CIλ (induced by rhamnose) has shown to suppress the pcIλ/Tet promoter inhibiting GFP production when induced with rhamnose (figure 5). In addition, CIλ and lacI has also shown to suppress GFP production of the pcI/lac promoter (figure 6). A toggle-switch was constructed ( Bba_K1493702, Bba_K1493703) containing pCI/lac promoter + TetR together with ptet + LacI + GFP. After establishing that induction by IPTG leads to adequately low GFP expression (the off-state), whereas induction by aTc results in high GFP expression (the on-state), we concluded that the toggle switch mechanism suits our intended application purpose (figure 7). For more information, read Kill-switch and rhamnose characterization.

Promoter design model

The kill-switch design is relatively intricate and therefore requires in silico analysis in order to test and improve its architecture. In order to accommodate this aim, we exploited statistical mechanics to derive a model of the system. Not unexpectedly, the new insight obtained strongly favoured some adaptations to the current design, which included reallocation of promoters as well as parallel placement of an additional kill-switch, which according to the predictions would yield a more stable system. For more information, read model kill-switch promoter design.

Back to the lab

With new output from the promoter model, new promoters were made with different inhibitor binding sites with the BioBrick standard in mind. These promoters were then coupled in front of GFP and all 5 out of the 6 promoters we have designed have shown to work (Bba_K1493801, Bba_K1493802, Bba_K1493804, Bba_K1493805 and Bba_K1493806), by showing GFP expression (see figure 10). For more information, read promoter design results.

System model

Cost

Having the whole system in P. putida is great however there is always metabolic stress in everything that we want P. putida to produce. Therefore another model was developed to predict the cost of the whole system, using a genome-scale constraint based metabolic model. The model indicates that the metabolic stress introduced by fungal growth inhibitors production should not pose a bottleneck. For more information read system cost.

Performance

Knowing that P. putida is able to cope with the whole system, the next objective is to assess the performance of the system; will the kill-switch function according to our expectations? Will the kill-switch kill P. putida in advance of performing its intended role as a fungicide due to imbalance of the toxin anti-toxin system?In order to answer these questions we created a stochastic whole system model, incorporating metabolic stress, leakiness of each individual promoter and the toxin anti-toxin syswtem. The results of this analysis are depicted in figure 4.

The stochastic model (figure 11) has shown that different basal production levels of CIλ can have different effects on population dynamics, cell growth and the stability of the kill-switch, a point of attention for final construct of the system. Finally, the kill-switch will perform with 98% efficiency given the slow growth rate in the soil predicted by the metabolic model. For more information read model performance.

Green house

We were able to establish a collaboration with the plant research international group of Wageningen, which gave us the unique opportunity to test the system, not only against F. oxysrporum, but in a setting that mimics the situation outside the lab as closely as possible with banana plants. At present we have banana plants in the green house (figure 12) that have been inoculate with our engineered P. putida and which were also infected with F. oxysporum. However, plants grow at a much slower pace than bacteria. So results were not possible to obtain before the wiki-freeze (see green house ).

In short

We as an iGEM team have achieved quite a lot during these couple of months. Here is a short list of what we have achieved:

• Validated and characterized a novel working fusaric acid dependent promoter
• Proved that pyoverdine can be produce in an iron rich environment
• Improve inhibition of P. putida towards F. oxysporum
• Show the proof of concept of the Kill-Switch using input output plasmid
• Did an extensive characterization of the rhamnose promoter
• Have a stable toggle switch that can be activated
• Feedback loop with model and wetlab by promoter design
• Have new promoters made and validated for future iGem team to use!
• Have a model that predicts the metabolic cost of our whole system BananaGuard
• Have a model that shows the performance of our whole system BananaGuard

Apart from all things in the lab, we have also been waiting patiently to see the results of the greenhouse. How will our engineered P.putida behave in the its final application against F. oxysporum! Is it good enough in the soil? is it enough to help the banana plants? Sadly, the results were not here before the wiki-freeze however we will present them at the jamboree! So stay tuned and come to our presentation!