OVERVIEW
| Part | Link to The Registry Page | Type | Function |
|---|---|---|---|
| BBa_K3887008 | https://registry.igem.org/parts/bba-k3887008 | Inducible Promoter | Expresses the MS2 polycistronic transcript only in the presence of lactose or IPTG |
| BBa_K5044019 | https://registry.igem.org/parts/bba-k5044019 | Packaging signal | Binding site for the MS2 Coat Protein to encapsulate the RumiVec transcript |
| BBa_255Z0P1J (New) | https://registry.igem.org/parts/bba-255z0p1j | RBS | Ribosome binding site for the Maturation Protease CDS extracted from the wild-type sequence. |
| BBa_253BMF18 (New) | https://registry.igem.org/parts/bba-253bmf18 | CDS | Coding sequence for the MS2 Maturation Protease |
| BBa_255HNR31 (New) | https://registry.igem.org/parts/bba-255hnr31 | RBS | Custom ribosome binding site with initiation rates similar to the wild-type MS2 |
| BBa_25JYSJJN (new) | https://registry.igem.org/parts/bba-25jysjjn | CDS | Coding sequence for the MS2 Coat Protein |
| BBa_J23100 | https://registry.igem.org/parts/bba-j23100 | Constitutive Promoter | Responsible for constitutive expression of the LacZα reporter for blue-white screening |
| BBa_K329005 (RBS+LacZα) | https://registry.igem.org/parts/bba-k329005 | RBS + CDS | Ribosome binding site and coding sequence for the LacZα peptide, which serves as a reporter for screening clones. |
| BBa_B1006 | https://registry.igem.org/parts/bba-b1006 | Terminator | Stops transcription |
For the development of Rumino, the team needed a way to validate the biosensor using a viral sample. Ideally, we would test our device using avian influenza samples directly. However, due to the risks of using a pathogenic virus, this would not be feasible.
To have a way to safely validate nucleotide-based viral biosensors without the need of using the real target viruses, we developed a new phagemid system called RumiVec. Phagemids are a specific type of plasmid that can be transformed into cells to produce bacteriophages. In line with this, our new composite part can produce single-stranded RNA phages with a test sequence embedded into its genome. As far as we are aware, there is no other phagemid system in the iGEM registry that is able to produce phages like this for use as a model organism. With a whole village dedicated to diagnostics and another one for infectious diseases, many team projects revolve around developing biosensors for quick viral detection. With our contribution we hope that teams are able to test and validate their viral sensors without relying on high-risk organisms. We decided to make a system for production of the MS2 phage specifically due to its small genome size, simplicity, and higher yield in synthetic-production methods compared to other single-stranded RNA bacteriophages [1].
MS2 Virion Structure [3]
The MS2 phage structurally consists of a single-stranded RNA genome encapsulated by 90 dimers of the Coat protein (Cp), and a single Maturation protein (Mat) [2]. Assembly of the phage technically only requires the Cp, but the presence of the Maturation protein greatly enhances stability and resistance against endonuclease degradation [2]. The RNA genome is mainly encapsulated by the binding of the Cp to a specific sequence of the transcript known as the Packaging Signal (Pac), which forms a special stem-loop that's recognized by the protein [2].
There are three main production methods for MS2 phages using different numbers and types of plasmid [2]. For our purposes, we decided to follow the one-plasmid packaging system which allows for packaging of up to 2 kb of RNA [2]. This system uses a plasmid which codes for the Cp and Mat proteins and contains an insert sequence that is packaged along with the rest of the transcript produced [2]. Out of the three methods, we decided to use this system due to its simple construction and ease of use for production [2]. RumiVec consists of two parts: The phage factory, and a modular cassette for simple insertion of other sequences using Type IIS assembly with BsaI.
PHAGE FACTORY
The phage factory is responsible for production of all the parts necessary for MS2 phage assembly. It uses BBa_K3887008 as an IPTG-inducible promoter for expression of the RumiVec transcript to control production of the virus. Downstream of the promoter is an altered pac sequence [4], which binds to the Cp for encapsulation of the transcript into the progeny. Following this, there is a pair of Ribosome Binding Site-CDS units which produce the Mat protein followed by the Cp. Downstream of the Cp CDS there's another pac sequence.
The pac sequence used was obtained from a study published by Wei, et al., and has a much higher affinity and packaging efficiency for RNA molecules by the Cp compared to the wild-type sequence [4]. To ensure that the transcript produced from RumiVec is efficiently encapsulated into phage particles, the predicted secondary structures of the sequence were analyzed using Vienna's RNAFold [5] to ensure that the necessary stem loop forms on the respective pac sites. The resulting model predicts with high-confidence the formation of the secondary structure in the pac sites required for adequate interactions with the Coat protein [4], meaning that RumiVec will be packaged into the phage progeny at high efficiency.
Figure 1. RNA Fold of the two pac sites (red arrows) in the transcript expressed by RumiVec.
The CDS for the proteins were designed by inputting the corresponding amino acid sequences obtained from the phage entry in NCBI (NC_001417.2) [6] into the IDT codon-optimization tool.
The RBS were chosen based on the translation initiation rates (TIRs) predicted by the SalisLab Operon/RBS Calculator [7]. The wild-type (WT) untranslated region upstream of the Mat CDS was used as the RBS as it had similar TIRs as the WT transcript. However, the WT RBS for the Cp CDS in RumiVec had a TIR significantly lower than expected. Although the Operon Calculator has an option to design RBS, it always gives a sequence that maximizes TIR. Regardless, this option was chosen and then the sequence obtained was randomly mutated through different iterations to obtain an RBS with similar TIR as the WT transcript.
Figure 2. A.) Predicted Translation Initiation Rates of: the wild-type (WT) RBS of either the Maturation Protease (Mat) or Coat Protein (CP) with the wild-type sequence (WT RBS); the WT RBS of the two proteins within our construct (WT RBS in RumiVec); and the Custom RBS generated by SalisLab Operon Calculator inside our construct (Custom RBS). B.) TIRs of the different RBS sequences tested for the Cp CDS. Iteration 6 was chosen as the sequence for the construct.
MODULAR CASSETTE
The modular cassette of RumiVec allows for insertion of sequences into the RNA molecule encapsulated by the MS2 phages. This region contains a reporter unit flanked by two BsaI cut sites. The reporter is made up of a simple translational unit which combines BBa_J23100, a constitutive promoter, with BBa_K329005, a part containing a strong RBS for expression of the LacZα peptide. Outside of the cassette, the BBa_B1006 terminator is used which stops transcription of both the reporter and the RumiVec transcript. The flanking BsaI sites allows anyone to easily swap the reporter with an insert sequence using Type IIS assembly.
When the reporter is swapped out, the plasmid can no longer produce the LacZα peptide. If the Type IIS assembly reaction mixture is used to transform E. coli strains apt for blue-white screening (e.g. NEB5α) and then plated on X-Gal plates, the cells transformed with the successfully cloned insert will be white while those with the reporter will be blue.
Alternatively, our team has also developed a pair of primers for RumiVec that will amplify the sequence inside the cassette. As long as this sequence is of different length from the reporter, then the products can be visualized in an agarose gel to confirm successful cloning.
Using the most recent iteration of RumiVec (pMS2_500), our team successfully swapped the reporter for an insert sequence using the NEBridge® Golden Gate Assembly (BsaI-HF® v2) kit. Although cloning could not be confirmed with blue-white screening, the products from a Colony PCR indicate a successful Type IIS assembly as observed by the expected positions of the bands for each group.
Figure 3. Agarose Gel of different samples: Golden-Gate assembly reaction mixture (GG Assembly); Colony PCR from Colony 1 and 2 from the transformation of NEB5α cells using the Golden Gate reaction (GG Colony 1/2); Colony PCR from a colony of a transformation using the reporter-construct plasmid (pMS2_500 Transformant); and PCR on the reporter-construct plasmid (pMS2_500).
With this new composite part, RumiVec can engineer bacteria to assemble MS2 Phages containing a genome with a desired sequence. These phages can subsequently be used to validate nucleic-acid sensors for the purpose of viral detection.
FUTURE WORK
RumiVec provides the groundwork for a safer alternative for validating viral sensors without the need of high risk strains. This new composite part also provides an opportunity for many other teams to build upon it for the development of a more flexible system. For example, although blue-white screening is a simple and well-studied screening method, it requires special plates and strains limiting its flexibility. Future teams could work on implementing a different reporter cassette such as a fluorescent protein for easier screening of clones. Purification of the MS2 phage particles is also a technique that not many are familiar with and requires reagents that are not commonly found in synthetic biology labs [2]. By inserting an affinity peptide tag (such as a His-tag) into the Cp or Mat proteins, purification can be greatly simplified and the yield of the produced phages can be greatly increased [2]. Furthermore, RumiVec provides a system for production of single-stranded RNA phages with a modular cassette for insertion of control sequences. Development of other similar systems with different genome structures (e.g. single stranded DNA viruses) for the iGEM registry will broaden the scope of these types of systems which can greatly strengthen the development of viral biosensors.
REFERENCES
[1] Bauman, V. R., Markushevich, V. N., Kish, A. Z., & Gren, E. I.a (1976). Sravnitel'naia fiziologiia replikatsii RNK-soderzhashchikh bakteriofagov MS2 i Q beta [Comparative physiology of the replication of RNA-containing bacteriophages MS2 and Q beta]. Mikrobiologiia, 45(4), 685–689. https://pubmed.ncbi.nlm.nih.gov/979687/
[2] Mikel, P., Vasickova, P., & Kralik, P. (2015). Methods for Preparation of MS2 Phage-Like Particles and Their Utilization as Process Control Viruses in RT-PCR and qRT-PCR Detection of RNA Viruses From Food Matrices and Clinical Specimens. Food and Environmental virology, 7(2), 96–111. https://doi.org/10.1007/s12560-015-9188-2
[3] ViralZone. [date unknown]. Fiersviridae. Swiss-Prot Group; [accessed 2025 Sept 28]. https://viralzone.expasy.org/163
[4] Wei, B., Wei, Y., Zhang, K., Yang, C., Wang, H., Xu, R., Zhan, S., Lin, G., Wang, W., Liu, M., et al. (2008). Construction of Armored RNA Containing Long-Size Chimeric RNA by Increasing the Number and Affinity of the Pac Site in Exogenous RNA and Sequence Coding Coat Protein of the MS2 Bacteriophage. Intervirology. 51 (2), 144–150. https://doi-org.ezproxy.lib.ucalgary.ca/10.1159/000141707
[5] Lorenz, R., Bernhart, S. H., Höner Zu Siederdissen, C., Tafer, H., Flamm, C., Stadler, P. F., & Hofacker, I. L. (2011). ViennaRNA Package 2.0. Algorithms for molecular biology : AMB, 6, 26. https://doi.org/10.1186/1748-7188-6-26
[6] GenBank. National Center for Biotechnology Information. [cited 2025 Sep 28]. Available from: https://www.ncbi.nlm.nih.gov/nuccore/NC001417
[7] Salis, H. M., Mirsky, E. A., & Voigt, C. A. (2009). Automated design of synthetic ribosome binding sites to control protein expression. Nature biotechnology, 27(10), 946–950. https://doi.org/10.1038/nbt.1568