Team:Wageningen UR/overview/results

From 2014.igem.org

Revision as of 17:34, 15 October 2014 by Wenchies (Talk | contribs)

Wageningen UR iGEM 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).

Figure 1. The measurement is based on GFP fluorescence in P. putida at increased concentrations of fusaric acid to prove and characterize the activity of the fusaric acid induced promoter, BBa_K1493000. For comparison, the well characterized pLac promoter (BBa_K741002, uninduced by IPTG, see part registry) was used to quantify the activity of this promoter at different concentrations of fusaric acid. The promoter does not respond to low concentrations up to 170nmol/ml. From 255nmol and up, the activity increases. The maximum measured activity of the promoter is 0.21 RPU at 425nmol/ml.

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:
  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 pressence of iron
  4. 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.

Figure 2.Pyoverdine absorbance at 400nm with error bars,OD corrected.


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.

Figure 3.In vivo assay, P.putida co-inoculated with F.oxysporum. Red circle indicates area occupated by F.oxysporumgrowth. Foc control=Fusarium oxysporum cubense TR4, WT=wild type P.putida KT2440, DAPG=P.putida containing phlABCDE gene cluster, MgL=P.putida containing methionine-γ-lyase, chitinase=P.putida overexpressing chitinase, pfri= P.putida overexpressing pfri and mix(all 4)=all 4 tranformants mixed.

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.


Kill-switch

Promoter design model

With the kill-switch 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.


Color maps indicating functioning and non-functioning systems. Each letter represents different repressor binding site configurations. Each small square within the colour maps represents a score for a simulation of the system with a unique set of parameters. The colours correspond to the previously given description

2: The system performs to design, after a rhamnose input the toggle switch changes state and GFP is produced when CIλ leaves the system

1: The system performs less efficiently, though the toggle switch changes state, the GFP promoter is leaky


0: The system does not work, the toggle switch is out of balance and does not function, the system favors either LacI or TetR

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

Figure 2: The relative growth rate compared to the wild type P. putida for different carbon uptake rates. The optimal solution is with glucose as carbon source, the realistic solution is with the banana exudates as carbon source. The expected carbon uptake rate of P. putida in the rhizosphere is indicated with transparent red.

Green house

Retrieved from "http://2014.igem.org/Team:Wageningen_UR/overview/results"