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. For this, we successfully transfected 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. If the targeted chemical is present, it specifically binds to a receptor, which then activates a signaling cascade, resulting in the activation of RUBY. To enable the detection of chemical weapon degradation products, 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. After the activation of the signaling cascade within Eschericia coli with the original ribose receptor, the BTCA receptor has been shown to successfully bind and activate the signaling cascade in E. coli as well.

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 showed its functionality by activation through a ribose binding protein. We further determined, that the E. coli can tolerate significant concentrations of our chemical weapon related chemicals We could also show that our computationally designed receptors can bind BTC with a higher specificity than the receptor it is based on. We thereby showed a successfull computational protein design to adapt our detection system for the detection of further chemicals.

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.