An early meeting with Team MSP-Maastricht inspired several design aspects and had a significant impact on the direction of our project. At this initial meeting, we learned about their project - Aestuarium, the goal of which is to engineer an organism capable of performing biodesalination of sea water into fresh water for agricultural and industrial uses. We learned that their plan was to use the Cyanobacterium Synechococcus PCC11901 as their chassis organism, as this is a quick-growing lab strain of marine Cyanobacteria.
We quickly saw how we could partner together to improve both our projects. During our discussions, we learned that Team MSP-Maastricht was researching methods by which their engineered cyanobacteria could be removed from a water source when desalination is complete or immobilized to prevent their engineered organism from being freed in the water source in the first place. One of the original applications we envisioned for our ghost phage was its use in tagging a Cyanobacteria host. While we initially envisioned this tagging for use in detection, we realized that our phage might be useful for immobilization of a target cell and could therefore be used by Team MSP-Maastricht to achieve their goal. We realized that our project would benefit from testing by an outside team. Not only would Team MSP-Maastricht be able to evaluate the host specificity of our ghost phage against a common Cyanobacteria chassis, but their testing of our system would also be a useful proof of concept.
To make this partnership work, we had to develop a system that would allow us to immobilize the ghost phage on a solid surface, while allowing it to adhere to its target cell freely. T7-like phages utilize tail-fiber proteins to bind to a host. So, to prevent hindrance of this interaction we elected to add an immobilization tag to the capsid protein, which makes up the icosahedral head region of the phage. Previous work has demonstrated that biotin-tagged viruses can be immobilized by streptavidin coated surfaces and can capture target bacteria at concentrations as low as 10 cells/mL of sample (1). We therefore decided to include a biotin tagged capsid in our toolkit.
We envisioned adding a biotin tag to the capsid of the virus, and then immobilizing the virus on a solid streptavidin-coated substrate, such as a chip, or beads. A sample of desalinated water could be passed over the chip, or the bead-phage complexes could be added to the water, where the phage would bind and immobilize the engineered bacteria. The water could then be collected as it runs off the chip, or separated from beads via filtration, centrifugation, or use of a magnet (if magnetic beads were used).
Schematic diagram of Cyanobacteria immobilization using a biotin tagged capsid
To further this partnership, we designed a version the S-TIP37 capsid protein with a biotin tag on the C-terminus. This part has been entered into the parts repository under part number BBa_K4268003. The sequence of this tag, along with a 6 amino acid linker was taken from the literature (1, 2). We also used this sequence to design a complete version of the phagemid that contains the biotin tag, which can be found in the repository as the composite part BBa_K4268020. We were successfully able to clone the basic part and are still working on constructing the higher-level assemblies needed to create the final phagemid.
Unfortunately, we ran out of time to complete assembly of the full phagemid. We planned to send the completed DNA construct to Team MSP-Maastricht for testing, along with protocols and materials for isolation and testing of the ghost phage. In turn, we hoped to obtain their data regarding the ability of the phage to attach to Synechococcus PCC11901.
References:
- Edgar, R., McKinstry, M., Hwang, J., Oppenheim, A. B., Fekete, R. A., Giulian, G., Merril, C., Nagashima, K., & Adhya, S. (2006). High-sensitivity bacterial detection using biotin-tagged phage and quantum-dot nanocomplexes. Proceedings of the National Academy of Sciences of the United States of America, 103(13), 4841–4845. https://doi.org/10.1073/pnas.0601211103
- Stolz, J., Ludwig, A., & Sauer, N. (1998). Bacteriophage lambda surface display of a bacterial biotin acceptor domain reveals the minimal peptide size required for biotinylation. FEBS letters, 440(1-2), 213–217. https://doi.org/10.1016/s0014-5793(98)01454-9