Harmful contaminants can accumulate in soil and groundwater and may accrue in plants that are exposed to the contaminated media. In many cases, testing plants or media for specific contaminants may require specialized lab equipment. Further, significant experience and knowledge are likely required to operate lab equipment and carry out the needed tests. These tests can be time consuming and are only immediately useful to a small, specialized group of people. Contaminants may instead be detected by observable changes that occur in organisms when a specific substance is present. Biosensing techniques can be developed into viable methods of detection that are more expedient than running a battery of tests on samples, and may be quite applicable across many disciplines.
This project consists of biosensing in two very different organisms that utilize very similar detection mechanisms. One portion of the project focuses on biosensing in E. coli to detect contaminants in tree sap. Core samples, which leave holes, are commonly taken from trees to test for groundwater contaminants. Our modified E. coli, when inserted into these holes in a containment device will consume the tree’s sap, and will be exposed to any contaminants that have accumulated therein. Exposure to the substance of interest will initiate a signal transduction pathway that produces a chromoprotein or fluorescent protein. This pathway is conserved in plants, hence the other portion of the project focused on biosensing in Arabidopsis thaliana. In the presence of the common groundwater pollutant trichloroethylene, the Arabidopsis produces ‘degreening’ proteins which degrade and prevent production of chlorophyll. The result is an obvious color change.
This biosensing technique is expedient for a number of reasons. It can be applied in many circumstances, and can be easily adapted to fit a variety of needs. Any site that monitors water, soil, and/or effluent waste may make use of such methods Additionally, it does not require an extensive background in life sciences to understand the meaning of color changes, and does not rely on specialized personnel to run time-consuming tests.
Histidine Kinases (HK) are proteins that function in signal transduction across membranes, and are highly conserved between bacteria, fungi, and plants (Atunes et al, 2009). In response to a signal, the HK dimer undergoes a conformational change which in turn phosphorylates a response regulator protein that initiates transcription. Because of the highly conserved nature of the protein domains in these signal transduction pathways, bacterial response regulators have been found to interact effectively with plant HKs, and have been observed translocating to plant nuclei (Atunes et al, 2009).
Similarly, our synthetic signal pathway targets a periplasmic binding protein from E. coli to the apoplasm of Arabidopsis Thaliana where it binds the ligand. After binding, the complex interacts with the E. coli chemotaxis receptor Trg and the HK PhoR. The resulting conformational change in PhoR phosphorylates the E. coli response regulator PhoB. The phosphorylated PhoB, which carries four copies of the transcriptional activator domain VP16, translocates into the nucleus and binds our PhoB-induced CaMV 35S promoter to initiate transcription of our degreening circuit. The gene products of this circuit degrade chlorophyll, leaving the white.
We attempted to redesign Ribose Binding Protein using short molecular dynamics trajectories of MODELLER-derived structures. We sequentially mutated residues at 11 active site positions, then simulated the apo and TCE-bound protein, with TCE treated by Density Functional Theory. At each round, the lowest average Molecular Mechanics Poisson-Boltzmann Surface Area energy structures which remained close to the native structure as indicated by RMSD were chosen for the next round of mutations. Unfortunately, we ran into trouble with our university’s computing cluster and AmberTools, then did not have time to fix the issues or try other methods (namely Rosetta).
After finding the Universal Acceptor Plasmid (BBa_P10500) for PhytoBricks in the parts registry has an extra ten bases before the suffix by sequencing, we removed the bases and submitted the new part. We tested the cloning efficiency for Golden Gate of a single part into our updated UAP.