RESULTS

Protein modeling experiments

The protein modeling experiments gave two main results:

• a model using Rosetta scripts to evaluate the theoretical activity of a mutated dehalogenase with different substrates
• mutated sequences for dehalogenase that score better on the aforementioned evaluation model

These models and mutated sequences have not yet been validated in a wet lab environment, which is the next step. For more information about these modeling experiments, please visit the modeling page.

See the files below to view the data tables summarizing the experiments.

Result #1

Results for experiments editing dock and relaxation protocol parameters to develop an evaluation model.

Summary: most favorable energy (dH) scores found using 6ZXX structure, relaxed with two distance constraints with SCALARWEIGHTEDFUNC = 50, and the relaxation ramp parameter = TRUE, and no relax:coord_constrain_sidechains flag.

Result #2

Mutations generated for three ligands (total of four orientations) using ligand MPNN protocols. Includes mutation reversion experiments.

Result #3

Mutations generated for three ligands (total of four orientations) using resfiles and a Rosetta Scripts protocol.

Laboratory results

Figure 1. Gas chromatography mass spectroscopy (GC-MS) data for PCB-11 in cyclohexane.

Due to the difficulty of procuring cyanobacteria and plasmids in time for our project, we were unable to finish transformations and test the effectiveness of our modified dehalogenase. If we were able to successfully transform the E. Coli, we would measure the amount of PCB-11 in the water before and after exposure to the transformed E. Coli with the modified dehaolgenase protein and identify if there was a significant change. Using gas chromatography mass spectroscopy (GC-MS) we were able to verify that PCB-11 could be detected and identified by GC-MS making it an ideal method to measure the amount of PCB-11 was dechlorinated after being exposed to E Coli. transformed with the modified dehalogenase.

Next Steps

Once we have confirmed that our protein design is efficient at breaking down our selected PCB congeners, our next steps on the pathway towards implementation are transforming cyanobacteria to express our enzyme, engineering the cyanobacteria to form a stable biofilm, and testing our biofilm in wastewater conditions. We plan to use Synechocystis sp. PCC 6803 as our cyanobacteria due to its status as a prominent model organism, as well as its well documented ability to form biofilms [1]. We are excited to move forward with our project post iGEM in order to turn our project into a reality.

References

1. Axenic Biofilm Formation and Aggregation by Synechocystis sp. Strain PCC 6803 Are Induced by Changes in Nutrient Concentration and Require Cell Surface Structures - PMC