Team:Wageningen UR/overview/results
From 2014.igem.org
Line 8: | Line 8: | ||
<li class="menu-item"> | <li class="menu-item"> | ||
- | <a href="#overview"> | + | <a href="#overview">Fungal sensing</a> |
</li> | </li> |
Revision as of 17:26, 15 October 2014
Results
With all the results, obtained from this project. It's hard to go through all of them in one go. Here you can find all of our key results highlighted together.
Fungal sensing
In literature it was never sure if there is really a fusaric acid dependent promoter. In this project we proved that there is such a promoter. A fusaric acid dependent promoter (isolated from Pseudomonas putida KT2440) was cloned in front of GFP(BBa_E0040), transformed in Pseudomonas putida KT2440 (P.putida). And flouresence were measured in different fusaric acid concentration. We were able validated and characterized new fusaric acid promoter (Bba_K1493000).
For more information, read fungal sensing.
Fungal inhibition
Upon sensing fusaric acid, three genes and a gene cluster will be activated that will lead to production production of certain antifungal. Those genes and their function being:- phlABCDE gene cluster, able to produce 2,4-Diacetylphloroglucinol(2,4-DAPG)
- Methionine-γ-lyase, Dimethyldisulfide (DMDS) and dimethyltrisulfide (DMTS)
- Pfri, produce pyoverdine in pressence of iron
- Chitinase, overexpresses chitinase activity
Methionine-γ-lyase and Pfri were bopth 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 pressence of iron in the medium.
All transformants were co-inoculated with Fusarium oxysporum cubense TR4 on agar plates in order to test its inhibition ability. Controls used were wild type P.putida KT2440 with F.oxysporum and just F.oxysporum.
With transformants giving (slightly) smaller F.oxysporum growth (figure 3). It can be said that each transformants had a slight increase of inhibition towards F.oxysporum making our chassis P.putida better as being a biocontrol. For more information, read fungal inhibition.
Killswitch
Promoter design model
With the killswitch being designed, it was never quiet sure it would work as it is a pretty complicated circuit. So we looked at a statistical mechanics model has led to the experimentalists decision to opt for a new set of designed promoters and build two kill-switches in parallel. The model has predicted the newly designed promoters to have a higher stability. For more information, read kill-swtich promoter design.
Performance model
System cost
Having all the whole system in P.putida is great but can P.putida the metolic stress, so a model was developed that would predict the cost of the whole system. In the model it is indicated that metabolic stress is not a bottleneck for the production of anti-fungals in our activated system. For more information read system cost