Team:Bielefeld-CeBiTec/Plant

Plant Synthetic Biology



The aim of our project 'P.L.A.N.T. Plant-based Ligand Activated Noxious agent Tracker - make the invisible visible' is to develop a biosensor for the detection of chemical weapon degradation products.

As a first step, we identified several chemicals of interest to be detected. They are degradation products of chemical weapons, which require low safety measurements. A crucial step in the development of a biosensor is to find out whether the molecules of interest can come in contact with the detector unit of the biosensor. In our project, the detection takes place in the apoplast, the extracellular space between the plant cells. Therefore, it is important for us to investigate the uptake of our chemicals of interest by our chassis N. benthamiana. In addition, we wanted to know whether the chemicals, which are taken up in the roots, are transported into the leaves.

To accomplish this, we cultivated N. benthamiana in a hydroculture and added the chemicals of interest to the culture medium. We were able to prove the successful uptake of several chemicals of interest and their transport into the leaves already after one day of cultivation in hydroculture (see figure 1).

In order to transduce the signal triggered by the detection of the chemical of interest through the cell, it is necessary to establish a signaling cascade. In our case, the signal is percepted by an modified extracellular Ribose Binding Protein. Upon ligand binding, the receptor protein activates the intracellular histidine kinase of a transmembrane fusion protein, resulting in the phosphorylation of the transcription factor PhoB-VP64. The phosphorylation leads to the nuclear translocation of PhoB-VP64 and thereby to the activation of target gene expression (see figure 2). The components of this signaling cascade are combined in the composite parts BBa_K3900050 and BBa_K3900051.

Besides the described signaling cascade for the use in N. benthamiana, we also introduced an analogous signaling cascade for E. coli (see figure 2). We used this bacterial version to analyze the properties of our engineered receptor protein. This way, we were able to prove that our computationally engineered receptor protein for benzenetricarboxylic acid (BTCA) specifically binds its ligand and activates the signaling cascade (BBa_K3900054). It can be assumed, that with some modifications like codon optimization and the insertion of introns, this receptor protein can successfully be integrated into our plant signaling cascade for the specific detection of BTCA.

Taken together, all these results imply that our plant-based detection system with the components of the signaling cascade can be successfully realized.
Figure 1: GC/MS analysis of N. benthamiana leaves after 1 day and 9 days of cultivation in hydroculture with different chemicals added. We used BTCA (benzenetricarboxylic acid) to substitute TNT, MPA (methylphosphonic acid) and TDG (thiodiglycol) are chemical weapon degradation products, β-Estradiol is used as positive control.
Figure 2: Schematic overview of the chemical detection signaling cascade in the variants adapted for bacteria (left) and plants (right)