Summary of Parts
Satellite phage represent an underexplored frontier of synthetic biology, brimming with potential for useful new parts. Currently, helper phage, the evolutionary counterparts that satellites depend on for reproduction, form the basis of many core BioBrick parts. This includes some of the most popular promoters, terminators and coding domain sequences on the parts registry, as well as many important circuits for SynBio including the original repressilator. With further exploration of the treasure trove of parts that satellites offer, these mobile genetic elements have the potential to match or even exceed the utility of phage-derived parts, both by augmenting existing systems and allowing for entirely new applications.
Our parts collection shows off various utilities of satellite phage and enables bioengineers to access some of their massive potential, particularly for gene delivery at a large scale to a flexible and engineerable range of hosts. For this, we champion two systems.
The first is the P4 cosmid, a flexible vector for gene delivery enhanced with parts of the well-characterized P4 satellite phage. We believe the P4 cosmid system performs excellently in its role as a model phage satellite system for its ease of use and its versatility, showing promise in multiple environments and possessing a tunable host range including commonly used bacteria in SynBio. Our iterations on the P4 cosmid expand its utility to perform an even wider variety of roles, including targeted gene silencing and upregulation.
The second are the mycobacteria phage satellites, or “phagelets”, a novel class of mobile genetic elements completely new to the world of synthetic biology.
We tested both of these systems in environments that simulate two popular areas of SynBio applications: the mammalian gut, which is the intended area of deployment for a variety of therapeutic and diagnostic applications of SynBio, and the soil, which plays a similar role for bioremediation and agricultural applications. We hope that our parts will be useful to future iGEM teams working in these areas. More broadly, we hope that by following our approach and designs, future iGEM teams will be able to more easily characterize the behavior and performance of genetic circuits in simulated environments, approximating the conditions they are intended to work in and contributing to a new standard of rigor for testing biologically engineered constructs.
While the addition of these parts to the iGEM registry more than doubles the number of current parts derived from phage satellites, our collection is only the tip of the iceberg when it comes to satellite SynBio. We hope that our contributions in this area inspire further research exploring the breadth and depth of phage satellite families. Further, we hope that our methods, protocols, and bioinformatics tools empower future researchers, including future iGEM teams, in their efforts to discover and engineer systems with this unique array of genetic elements.
Cosmid Collection
The P4 cosmid, originally designed by Dr. Fa-Arun, is a versatile system for gene delivery based on the P4 cos site and sid operon. When used with a specially engineered helper-phage lysogen, the system can produce a pure and concentrated titer of transducing units. This enables researchers to transduce genetic circuits into a desired host without contamination with replicative phage, which can interfere with experiments and in some cases kill stock bacteria, potentially leading to loss of engineered circuits.
Additionally the transducing units of this system have a host range which is far easier to engineer than alternative systems, which, for the cosmid, is achieved via simply swapping out an accessory tail fiber plasmid. Though many lab strains of bacteria are phage-resistant, not all are. These properties of flexible host range and pure titer synergize to produce a system which is applicable to a range of interesting chassis, which could be expanded by future host range engineering projects.
The original cosmid contains a Cas9 cassette with BsaI cut and fusion sites for easy insertion of guide RNA sequences. The Cas9 cassette can be used to induce site specific killing of transductants, provided the target bacteria lacks a non-homologous end joining repair pathway, as is the case with most bacteria. It can also be used for gene insertion with the use of a repair template sharing homology with the target site.
Our iterations on the P4 cosmid system expand the functionally of the P4 cosmid system to include a wider variety of genetic manipulations, including gene silencing, targeted upregulation, and reporting with a fluorescent protein.
The BioBrick Compatible P4 cosmid is a generalized version of the original P4 cosmid designed for use as a backbone in standard assembly or in the last step of a 3A assembly process. By using a different set of primers the BioBrick Compatible Cosmid can also be made to work with iGEM type IIS assembly and/or MoClo as well. This expands the compatibility of the P4 cosmid system to include nearly all BioBrick parts, and works as a straightforward and versatile method of transducing genetic circuits.
| Name | BioBrick Identifier | Type | Description |
|---|---|---|---|
| KanR Targeting P4 Cosmid | https://parts.igem.org/Part:BBa_K5179101 | Transducing Vector | P4 cosmid with neomycin phosphotransferase targeting crRNA, used to characterize efficacy of P4 cosmid in simulated natural environments |
| dCas9 P4 Cosmid | https://parts.igem.org/Part:BBa_K5179102 | Transducing Vector | P4 cosmid with inhibited Cas9 DNA cleavage activity for CRISPR Interference |
| dCas9-Omega P4 Cosmid | https://parts.igem.org/Part:BBa_K5179104 | Transducing Vector | P4 cosmid with inhibited DNA cleavage activity and fusion of RNAP Omega subunit to C-terminus for targeted gene upregulation |
| RFP P4 Cosmid | https://parts.igem.org/Part:BBa_K5179105 | Transducing Vector/Reporter | P4 Cosmid with CRISPR cassette replaced with RFP reporter device |
| BioBrick Compatible P4 Cosmid | https://parts.igem.org/Part:BBa_K5179110 | Transducing Vector Backbone | P4 Cosmid with Cas9 cassette and RFC10 and 1000 illegal cut sites removed, BioBrick prefix and suffix added; designed to be used in final step of 3A assembly, most suitable for transduction of genetic circuits between 5-6kb |
“Phagelet” Collection
“Phagelets” are a class of satellite phage that were discovered by High School students as part of William and Mary’s 2018 iGEM outreach. They are known to not only parasitize but also trigger the excision of the mycobacteriophage HerbertWM and are the only known phage satellite family in mycobacteria.
Though much research is still needed to completely characterize the inner workings of this novel family, they have already shown great promise in evading prophage immunity in mycobacteria lysogens. In order to utilize this unique ability, we are using the “phagelet” satellite system in Mycobacterium aichiense to increase the host range of the helper phage, HerbertWM. Although HerbertWM belongs to a family of phages known to infect M. smegmatis, the host range of HerbertWM does not extend beyond M. aichiense. We designed chimeric tail fibers for the prophage that will be assembled as the “phagelets” trigger the packaging of HerbertWM.
Our collection of “phagelets” enables future work with phage satellite systems in mycobacteria, and our collection of engineered tail fibers provides a foundation for future host range engineering projects.
| Name | BioBrick Identifier | Type | Description |
| Mycobacteria Phage Satellite Anya | https://parts.igem.org/Part:BBa_K5179200 | Whole Satellite Phage | Satellite phage of HerbertWM |
| Mycobacteria Phage Satellite ChrisB1 | https://parts.igem.org/Part:BBa_K5179201 | Whole Satellite Phage | Satellite phage of HerbertWM |
| HerbertWM Gp3 Mutant | https://parts.igem.org/Part:BBa_K5179212 | CDS + RBS | Chimeric tail protein containing the RBS and initial homologous region associated with Gp3 of Mycobacteriophage HerbertWM, followed by the remainder of the corresponding gene from Mycobacteriophage L5 Gp6 |
| WT L5 Gp6 | https://parts.igem.org/Part:BBa_K5179213 | CDS + RBS | Wild-type Gp6 Gene from Mycobacteriophage L5 |
| L5 Δ homolog | https://parts.igem.org/Part:BBa_K5179214 | CDS + RBS | Chimeric tail protein containing L5 Gp6 gene from Mycobacteriophage L5, with a homologous region of the sequence substituted with that of Mycobacteriophage HerbertWM’s Gp3 gene |
Basic Parts
Our team also designed a number of basic parts based on the immunity factor of satellite phage P4. An immunity factor works to keep a phage in its lysogenic phase by preventing the transcription of its excisionase. In phage these immunity factors are commonly peptides, but satellite bacteriophage P4’s immunity factor has been shown to be a small RNA species known as the CI RNA (Sabbattini et al., 1995).
The CI RNA works by binding with a number of complementary regions in nascent transcripts from P4’s chromosome, causing them to form a termination structure and prematurely stop transcription before RNA polymerase can transcribe P4 late genes.
The CI RNA and its related terminators caught our attention when our literature search indicated that point mutations in specific regions of the CI RNA prevented its functioning, but that its functionality could be restored by introducing complementary point mutations in a specific region of CI dependent terminators (Forti et al., 2002). With this in mind we re-designed the CI RNA and P4 terminators into a family of translation independent genetic switches. We hypothesize that these will function with similar parameters, since they are all derived from the P4 CI RNA, but also function orthogonal to each other as CI double mutants do in mutational analysis experiments (Forti et al., 2002). Since P4 immunity machinery functions independently of translation we also hypothesize these parts will impose little burden as they do not exhaust host tRNA reserves, and that they will affect translation with less latency than protein-based regulators.
We haven’t finished characterizing this family of parts just yet, but we wanted to make sure that future iGEM teams had access to our designs for this potentially very useful group of basic parts.
| Name | BioBrick Identifier | Type | Description |
| P4 CI RNA | https://parts.igem.org/Part:BBa_K5179300 | RNA | Minimal P4 CI RNA with immunity-determining region replaced with flanking BsaI cut sites, can be made into any CI RNA ortholog by cloning an 8bp insert in a golden gate reaction with BsaI |
| P4 T1 | https://parts.igem.org/Part:BBa_K5179301 | Terminator | Minimal P4 CI RNA dependent terminator with immunity-determining region replaced with flanking BsaI cut sites, can be made into any P4 T1 ortholog by cloning an 8bp insert in a golden gate reaction with BsaI |
| CI Expression Device (Constitutive) | https://parts.igem.org/Part:BBa_K5179302 | Device | Expression device for P4 CI RNA with a constitutive promoter |
| CI Expression Device (Heat-Shock) | https://parts.igem.org/Part:BBa_K5179305 | Device | Expression device for P4 CI RNA with a heat shock promoter |
| T1 Terminator Test Device | https://parts.igem.org/Part:BBa_K5179304 | Device | Dual fluorescent protein device for characterizing CI RNA orthologs |