Team:Bielefeld-CeBiTec/Proof Of Concept
Abstract
We developed a detection system for degradation products of chemical weapons that is highly specific and allows cost-efficient screening of large areas while being easy to use. Our plant indicates the presence of toxic chemicals by changing its color to red. We were able to successfully detect the uptake and storage of the chemical weapon degradation products methylphosphonic acid (MPA), thiodiglycol (TDG) and benzenetricarboxylic acid (BTCA) using GC/MS. Furthermore, by transfecting the signaling cascade into E. coli we showed that our computationally designed receptor is activated by BTCA and binds BTCA with a higher specificity than the receptor it is based on. For the visual report in plants we successfully transmutated a reporter system called RUBY into Nicotiana benthamiana, enabling the synthesis of the plant pigment betalain, which turned the leaf bright red over an extended period of time. We further proved that RUBY can be induced by estrogen when coded under control of the estrogen inducible lexA promoter. Therefore, we showed full functionality of every part of our detection system.
Uptake test hydroculture
As detectable chemicals, we chose MPA, diisopropyl methylphonate (DIMP), diethyl methylphosphonate (DEMP), TDG and BTCA). After a pre-test on toxicity which showed no negative effect on N. benthamiana after 7 days of cultivation, we tested the actual uptake for a period of nine days. To compare the uptake we also cultivated a hydroculture with estradiol as positive control and a negative control. BTCA, MPA and TDG could be detected via a combination of gas chromatography and mass spectrometry (GC/MS) over the entire period of time. While BTCA and TDG are degraded by N. benthamiana and reach their highest peak after one day, MPA akkumulates.
Computational design Model
Since there exists no receptor for any of our tested chemicals, we modeled binding proteins using computational protein design.We used the protein structure prediction tool Rosetta in combination with EvoDock, an algorithm for simulated evolution. We also proved the activation of the bacterial signaling cascade and induced expression of a reporter upon BTCA binding by the modeled receptor. We thereby could show the successful computational engineering of a receptor for BTCA.
Bacterial Chemotaxis
We reached our goals in establishing a signaling cascade in E. coli for the specific chemical detection. We have determined, that the E. coli can tolerate significant concentrations of our chemical weapon related chemicals. Additionally, we showed that our computationally designed receptors can bind BTCA with a higher specificity than the receptor it is based on. This is a proof of concept for our project and shows that it is possible to design de novo receptors by computational means and thus engineer organisms to detect certain chemicals and act as a biosensor with our approach. We constructed the bacterial signaling cascade encoded by a one plasmid system and the single parts were ordered as gene synthesis products and BTCA activated the signaling cascade, thus GFP expression.
Nicotiana benthamiana as a test system
We used the model organism N. benthamiana since it is suitable for screening of synthetically built constructs by applying agroinfiltration. We were able to successfully prove the transfection of the RUBY reporter in N. benthamiana, its time stability and that its expression can be induced.
Co-transformation
The distribution of the signaling cascade on two plasmids required to transform two plasmids into N. benthamiana. Hence, one of our first steps was to test whether co-transformation can be applied with agroinfiltration. We transformed tobacco plants with the plasmid pK7FWG2 carrying an EGFP and the RUBY reporter plasmid. Thereby, we were able to prove that RUBY as well as EGFP were expressed in the infiltrated spots by confocal laser scanning microscope (CLSM). Thereby, a co-transformation in N. benthamiana was successfully proven.
