Integrated Human Practices

Prior to even creating our system we sought advice on organizing our team and how to come up with ideas effectively. From there, we had initially planned to deliver antimicrobial peptides to cyanobacteria. However, after hearing concerns about the lack of target specificity of such a system, we switched to a CRISPR-Cas13a system. This system allows us to target a gene that is unique to Microcystis aeruginosa cyanobacteria. The tests our wet lab team performed are prerequisites to testing in mesocosm and experimental lakes facilities: a goal of ours that developed after extensive interviews with researchers. This will provide an imperative expansion to regular laboratory bench experiments, which are limited in their ability to simulate aquatic environmental conditions.The input of experts has permitted us to optimize details and take the required steps to bring the project beyond the wet lab.

Modeling

We used modeling to better understand the interaction between the mlrA protein and the MC-LR toxin. By using autodocking, we could see the most likely conformations that MC-LR would take upon its interaction with mlrA. Using molecular dynamics, we created trajectories that mimicked how the mlrA proteins would function in our projected environment for our project.

We implemented previous year’s homology models to run through minimization, heating and production in order to simulate environmental conditions. These models were then ran though grid inhomogeneous solvent theory analysis in order to determine whether locations of water molecules matched the same location that was hypothesized by Xu et al. (2019). We also used the two replicates of our simulated molecules, and a negative control non-simulated homology model, also underwent autodocking simulations with the mclr toxin using open webservers in order to determine favored conformations of the toxin in and around the mlrA receptor protein.

Safety and Security

Our project this year aims to address the widespread harm caused by blue-green algae, or cyanobacteria, through the delivery of a CRISPR-Cas13a system to cyanobacteria cells. This will be achieved by using a phage–like particle (PLP) encapsulating our system. Upon being delivered into the cells, our system offers the advantage of being target-specific: CRISPR-Cas13 uses a crRNA, which is designed to be complementary with a cyanobacteria cell-specific messenger RNA. Once the mRNA–crRNA interaction occurs, the Cas13 enzyme will activate and cleave all surrounding target RNA within the cell, resulting in cell death. We also plan to deliver the enzyme mlrA to cyanobacteria cells. MlrA inactivates harmful microsystem toxins produced by cyanobacteria, mitigating their toxic effects in bodies of water. Our system will provide a safer alternative to current methods used to eliminate blue-green algae by employing a CRISPR-Cas13a system. We plan to carry out experiments to test and ensure the specificity and safety of our CRISPR system in a biological environment through the use of mesocosm facilities.

For safety, there is the need to control both the levels of SuperNova and the vectors that are introduced into a body of water. Therefore, we have planned to use phage-like particles (PLPs), since they cannot self-replicate when they contain the SuperNova plasmid. Additionally, PLPs target only the species of bacteria that they have been engineered to bind to, killing harmful cyanobacteria without depleting other bacterial populations within an aquatic ecosystem (Team:Aix-Marseille, 2017). SuperNova is also a relatively safe protein to release into a body of water, since it kills through the buildup of ROSs in a cell and is not itself a toxic molecule (Takemoto et al., 2013). For this reason, it should not be toxic to other organisms, unless ROS levels in the body of water were to eventually become too high, which reiterates the importance of being able to control SuperNova levels and convert ROSs into oxygen and water using the enzymes superoxide dismutase and catalase (Biotek 2014).

Creating models developed with dry-lab software resources will also allow the team to study and determine specific times that the system can be introduced while ensuring its efficiency and maintaining safety. The in vitro controlled testing, followed by modeling serves a crucial role to explicate potential impacts to the environment before our genetically engineered biosystem is introduced into the ecosystem. The experimentation carried out using the resources will inform our genetic design and also direct the optimal application of the system, in tandem, letting the team identify and devise counteracting measures to any impacts on the overall safety of the system.

Takemoto, K., Matsuda, T., Sakai, N. et al. SuperNova, a monomeric photosensitizing fluorescent protein for chromophore-assisted light inactivation. Sci Rep 3, 2629 (2013). https://doi.org/10.1038/srep02629

Wiki

Our teams wiki exemplifies the work of all the different subgroups that make up our teams. Although each subgroup worked on their own separate portions of the project, our wiki is where you see all of our work come together and of the impacts that each subgroup has on one another. Each week we would have meetings with the entire team and would discuss important advances and ideas. Towards the end of the season, the whole team came together to work on our wiki. It was interesting to see the influence that different subgroups had on one another. The wiki demonstrates the roles each subgroup has and how they all came together to form our project.