Proposed Implementation

CyanoSpectre centers around the reverse engineering of a phagemid that, when expressed in an E. coli chassis, would result in the synthesis of ghost phages – phage capsids devoid of any genetic material. While an empty viral capsid might not seem particularly useful by itself, our intent is that the ghost phage will be a vehicle that can be customized by end users (synthetic biologists and future iGEMers) for their own needs. Making the design customizable and expressible During the design of the ghost phagemid, we elected to use a MoClo strategy when building the phagemid to allow the user to customize expression of each of phagemid component to suit their needs. If the user desires to fine-tune any component, they can swap the selected promoter, RBS, or terminator sequence. In preparation for this customization, we elected to use a set of T7 promoters with varying strengths to allow for tunable expression under the control of a single regulator, T7 RNA polymerase. In addition, the final phagemid assembly contains dummy sequences, which can be replaced with additional coding sequences as needed by the user based on their needs (Figure 1).


Figure 1: Modular assembly of the ghost phagemid. Each transcriptional unit contains a T7 promoter and RBS from a collection within the iGEM parts repository, permitting easy comparison of expression efficiency and modular assembly of transcriptional units (TU). The units rely on an inducible T7-based expression system in E. coli and contain space for the addition of up to 9 additional TU.

Additional information regarding the design and assembly of the phagemid can be found in the Project Design, Phagemid Construction, and Engineering sections of our wiki.

Customization Examples

Examples of how the phage might be adapted for a specific user are given below:

Example 1: A synthetic biologist uses CyanoSpectre to deliver a DNA construct to a susceptible Cyanobacterial chassis for engineering purposes.
In this case, one would need to add a DNA delivery function to CyanoSpectre. To do this, the phage would be modified by adding genes required for DNA packaging and delivery. The S-TIP37 genome does contain a putative gene coding for a DNA packaging protein gp55 (NCBI Gene ID 54998430) (1). This protein could potentially be added to the phagemid to introduce DNA packaging capabilities. Because of lack of research into the virus, it is unclear how this protein may recognize the S-TIP37 genome, making it difficult to ascertain what genomic sequences might be necessary to ensure DNA packaging. In T7, introduction of the T7 oriC and the end of the concatemer junction site are needed for efficient packaging of plasmid DNA into the viral capsid (2), so it is feasible such sequences might serve similar functions in S-TIP37.

Another feature that a scientist may want to exploit, is the possibility that S-TIP37 may integrate into the host genome as a lysogen. S-TIP37 contains a putative Integrase gene and attP recombination site but does not become permanently integrated into the host genome (3). It should be noted that the authors of this study were using replication-competent viruses, and therefore S-TIP37 was free to utilize its normal lytic lifecycle. A ghost phage missing lytic lifecycle genes may stably integrate, allowing for permanent engineering of the host. In this case, a user may wish to design a plasmid containing the putative DNA packaging protein and Integrase (NCBI gene ID 54998389) gene into the phagemid in place of dummy sequences. Introduction of the phagemid into E. coli alongside the payload DNA modified with the S-TIP37 oriC, end of the concatemer junction site, and the attP site may allow stable integration.

Example 2: Modification of CyanoSpectre to enable its use as a nanodelivery system of compounds to Cyanobacteria.

There is great interest in the use of viruses and virus-like particles as delivery mechanisms for the delivery of drugs, vaccine platforms, and their use in imaging (4,5). Generally, this involves tagging capsid components with compounds and relying on the natural specificity of viruses to deliver that compound directly to a host cell. A variety of alternative strategies that can be used to get viruses to carry compounds are shown in Figure 2.



Figure 2: Strategies used to introduce cargo into viruses. a) Introduce cargo into host and rely on viral self-assembly around cargo by altering pH and buffer conditions b) Infuse cargo into assembled viruses by changing pH or salt concentration, c) genetically engineer the viruses to produce conjugated capsid scaffolding proteins, d) use bioconjugation techniques to add compounds to the capsid surface. Image from (4).
We envision that scientists may want to modify CyanoSpectre to target and deliver compounds to Cyanobacteria – particularly those that cause Harmful Algal Blooms (HABs). Currently, management of HABs involves prevention (e.g., through pollution reduction), mitigation of risk (e.g., closing of lakes and beaches, blocking fishing activities), and physical and chemical means of control (e.g., spraying of clay to absorb excess nutrients, crop dusting with antimicrobial chemicals such as Copper sulfate). (6) None of these methods are targeted to a specific strain of Cyanobacteria. CyanoSpectre could be adapted to deliver antimicrobial compounds directly to Synechococcus spp. that cause HABs, while not affecting other species of Cyanobacteria and phytoplankton. Such work would involve learning more about the host specificity of S-TIP37, and engineering changes into the tail-fiber like protein (part number K4268007). It would also require identifying substances that would specifically inhibit the growth of the offending HAB species. One way a scientist might achieve this is by identifying and packaging into CyanoSpectre an antimicrobial peptide or bactericidin that inhibits the growth of the target strain. Bactericidins may represent a particularly promising option, as a recent study has indicated that these are widely produced in Cyanobacterial communities (7).

Example 3: Modification of CyanoSpectre for use in the capture and immobilization of Cyanobacteria.


We have already begun to adapt CyanoSpectre for use in the capture of Synechococcus spp. This application of our project stems from a partnership with Team MSP-Maastricht. Briefly, Team MSP-Maastricht is engineering a strain of Synechococcus sp. PCC11901 to desalinate water. As part of their project, they were looking for a way to efficiently remove their engineered bacteria from the water before its use in agricultural or industrial applications. We designed a system for immobilizing the bacteria using a modified version of CyanoSpectre, one in which the capsid protein is conjugated to biotin. The phage could then be bound to streptavidin coated chips or beads and would be free to capture Synechococcus cells from the water source. More information on this partnership can be found on the Partnership page.

Such a system would be straightforward to implement. We would first need to isolate phages from the E. coli chassis. This would involve lysing the cells through freeze thaw, sonication, or lysis in a hypotonic buffer. Cell debris could be removed, and the sample concentrated through gradient centrifugation or filtration (8-10). In a small-scale test, samples of CyanoSpectre could be mixed with a water-Synechococcus mixture and incubated to allow adherence of the phage to the cells. Streptavidin-coated magnetic beads could then be added, allowed to bind the biotin coated capsule, and then captured with a magnet. Such beads are currently available commercially. Alternatively, streptavidin-coated biosensor chips could also be employed, along with a device, for not only small-scale capture, but quantitation. Once we had completed a proof-of-concept experiment using the natural host species for S-TIP37, we would then provide Team MSP-Maastricht with materials so they could test CyanoSpectre phage immobilization on Synechococcus sp. PCC11901.

Barriers to Implementation

In addition to potential uses of our ghost phage, we have also considered possible barriers to the implementation of our toolkit. First, the lack of information regarding Synechococcus species and the S-TIP37 cyanophage would make customization for new functionality difficult without additional basic research. Safety issues would be a concern if CyanoSpectre were to be used outside of a controlled laboratory environment. For example, the propensity for horizontal gene transfer (HGT) in Cyanobacteria would need to be considered, especially since cyanophage are a known vehicle for HGT. This concern is mitigated by the fact that the CyanoSpectre toolkit is currently replication-incompetent. However, we would caution users to do extensive research and testing prior to releasing our ghost phage into a natural environment.

References

1. HOT80_gp55 DNA packaging protein [Synechococcus T7-like phage S-TIP37] - Gene - NCBI. (n.d.). Www.ncbi.nlm.nih.gov. Retrieved October 11, 2022, from https://www.ncbi.nlm.nih.gov/gene/54998430
2. Chung, Y. B., & Hinkle, D. C. (1990). Bacteriophage T7 DNA packaging. I. Plasmids containing a T7 replication origin and the T7 concatemer junction are packaged into transducing particles during phage infection. Journal of molecular biology, 216(4), 911–926. https://doi.org/10.1016/S0022-2836(99)80010-2.
3. Shitrit, D., Hackl, T., Laurenceau, R., Raho, N., Carlson, M., Sabehi, G., Schwartz, D. A., Chisholm, S. W., & Lindell, D. (2022). Genetic engineering of marine cyanophages reveals integration but not lysogeny in T7-like cyanophages. The ISME journal, 16(2), 488–499. https://doi.org/10.1038/s41396-021-01085-8.Chung, Y. H., Cai, H., & Steinmetz, N. F. (2020). Viral nanoparticles for drug delivery, imaging, immunotherapy, and theranostic applications.
4. Advanced drug delivery reviews, 156, 214–235. https://doi.org/10.1016/j.addr.2020.06.024.
5. Aljabali, A. A., Hassan, S. S., Pabari, R. M., Shahcheraghi, S. H., Mishra, V., Charbe, N. B., Chellappan, D. K., Dureja, H., Gupta, G., Almutary, A. G., Alnuqaydan, A. M., Verma, S. K., Panda, P. K., Mishra, Y. K., Serrano-Aroca, Á., Dua, K., Uversky, V. N., Redwan, E. M., Bahar, B., Bhatia, A., … Tambuwala, M. M. (2021). The viral capsid as novel nanomaterials for drug delivery. Future science OA, 7(9), FSO744. https://doi.org/10.2144/fsoa-2021-0031
6. Anderson D. M. (2009). Approaches to monitoring, control and management of harmful algal blooms (HABs). Ocean & coastal management, 52(7), 342. https://doi.org/10.1016/j.ocecoaman.2009.04.006.
7. Wang, H., Fewer, D. P., & Sivonen, K. (2011). Genome mining demonstrates the widespread occurrence of gene clusters encoding bacteriocins in cyanobacteria. PloS one, 6(7), e22384. https://doi.org/10.1371/journal.pone.0022384
8. Carvalho, C., Susano, M., Fernandes, E., Santos, S., Gannon, B., Nicolau, A., Gibbs, P., Teixeira, P., & Azeredo, J. (2010). Method for bacteriophage isolation against target Campylobacter strains. Letters in applied microbiology, 50(2), 192–197. https://doi.org/10.1111/j.1472-765X.2009.02774.x
9. Van Twest, R., & Kropinski, A. M. (2009). Bacteriophage enrichment from water and soil. Methods in molecular biology (Clifton, N.J.), 501, 15–21. https://doi.org/10.1007/978-1-60327-164-6_2
10. Millard A. D. (2009). Isolation of cyanophages from aquatic environments. Methods in molecular biology (Clifton, N.J.), 501, 33–42. https://doi.org/10.1007/978-1-60327-164-6_4