Sci-Phi 29

Home

The majority of substrates and environmental conditions are not utilized in synthetic biology. Due to the lack of characterized parts, this potential can not be harnessed at this moment. To solve this problem we have developed Sci-Phi 29: a platform enabling orthogonal replication and predictable expression to expand the repertoire of engineerable bacteria for synthetic biology.

Motivation

There are more than one trillion different bacterial species living on Earth (Locey & Lennon, 2016). Every single one of these bacteria occupies its own niche, providing unlimited potential for synthetic biology.

In an ideal world every single bacterium could be engineered for our own benefits, meaning that any type of substrate can be converted into any desired product. Exploring this potential would mean we can broaden the range of substrates and environmental conditions which is currently used in synthetic biology. However, nowadays, synthetic biology is limited to a very small subset of these organisms, where the most commonly used bacteria are Escherichia coli and Bacillus subtilis (Adams, 2016).

Without a doubt, E. coli is the most widely used chassis in synthetic biology. It has the largest toolkit of genetic parts and regulatory elements, such as promoters, regulatory binding sites and terminators, as well as DNA vectors (Adams, 2016). However, when exploiting the potential of a non-model bacterial organism, there is a lack of genetic tools (Calero & Nikel, 2019). When moving to an unconventional bacterial species, new genetic tools need to be developed. This includes characterized, species-specific promoters, replicative vectors and suicide vectors, to cover a wide range of expression levels and genome engineering tools, such as CRISPR devices (Calero & Nikel, 2019).

**Figure 1: Engineering organism-specific parts requires extensive characterization and is extremly laborious and expensive.**

The iGEM Registry of Standard Biological Parts by itself already contains over 20,000 documented parts. All these parts are characterized for expression in their specific host. We can, however, not express these parts in different bacterial species. Furthermore, the behaviour of these parts in different bacteria is unpredictable, because regulatory layers differ across species (Calero & Nikel, 2019). To solve these problems, we created Sci-Phi 29, a platform that allows anyone to express genetic parts across different bacterial species in a controllable and predictable manner.

Sci-Phi 29: Enabling Orthogonal Replication and Predictable Expression to Expand the Repertoire of Engineerable Bacteria

Sci-Phi 29 allows expression of your gene of interest in a controllable manner across different bacterial species and independently of the host due to the concept of orthogonal replication. To make Sci-Phi 29 a standardized and user-friendly platform we provide a Modular Cloning (MoClo) compatible collection. Sci-Phi 29 is a versatile platform to further explore the bacterial diversity, providing new opportunities for the advancement of synthetic biology.

Orthogonality

To express genetic tools across multiple bacterial species, we were inspired by the replication machinery of the phi29 bacteriophage, a unique protein-primed based DNA replication machinery. Protein primed replication, unlike the conventional DNA or RNA primed mechanism, greatly simplifies the design of replication systems. This machinery is able to replicate a linear piece of DNA by using only four proteins: DNA polymerase (DNAP, p2), Terminal Proteins (TP, p3) Single Stranded Binding Protein (SSB, p5) and Double Stranded Binding Protein (DSB, p6) (Van Nies et al., 2018).

For a more detailed visual of replication by the phi29 replication machinery watch the video below:

Video 1: The replication process begins with the binding of the phi29 DNAP and TP complex at both origins of replication (OriR and OriL), which flank the protein-primed linear plasmid. The DSB proteins aid in the process of replication and binds with a higher frequency at the origins of replication, destabilizing the region and facilitating strand displacement. The SSB proteins bind to the displaced DNA strand preventing strand switching of DNA polymerase and protecting the linear plasmid from host nucleases (Reference).

When trying to express the phi29 replication machinery in E. coli we came into contact with Chang Liu and Julian Willis, experts on orthogonal replication machinery (Link to iHP page), who informed us that the expression of these four proteins had to be tightly controlled in prokaryotes. If the expression is too high, these proteins can interfere with the host’s genome, while if the expression is too low replication might not occur at all. In light of this discovery we redesigned our experiments by expressing these proteins using different T7 promoter variants and different IPTG concentrations for induction of these proteins. (Link to results page: viability).

By using the PURE system we demonstrated replication in vitro of our own linear construct (Link to result page-constructed parts), which is flanked by the phi29 origins of replication. (Link to results page - replication).

Controllability

Using orthogonal replication allows us to transfer and replicate genetic parts between bacterial species. However, many variables play a role in the behaviour of synthetic circuits inside cells. This includes variation in plasmid copy number, transcription, and translation. Therefore, it is difficult to introduce reliable parts for genetic engineering in different bacteria (Segall-Shapiro et al., 2018), since the same parts may behave differently across organisms.

To tackle the issue of variation in expression across species, we took our platform to the next level by integrating the concept of controllability, which is based on a systems engineering approach (Link to Design).

To make expression host-independent, we included an incoherent feed-forward loop (iFFL) in our design. An iFFL can be used to make the output of a system independent of the input (Figure 2).

Figure 2: Left: Scheme of incoherent Feed Forward Loop. Right: The increasing ‘red’ line indicates how the output normally increases linearly with the input. The stable ‘green’ line depicts the addition of a repressor which results in independence of the output to the input.

According to our model (Link to Model) and experimental validation (Link to Results), regulation of these interbacterial variables ensures stable expression across different bacterial species.

Standardization of Sci-Phi 29

Through concepts of orthogonality and controllability, we have shown that existing parts can be expressed in a standardized manner across bacterial species. To achieve standardization of our platform, we made our part collection Modular Cloning(MoClo) compatible (Weber et al., 2011). Our part collection provides 35 MoClo compatible parts (link to parts collection page) that can be used for predictable expression of genes across different bacterial species. To overcome the need to identify parts for different bacterial hosts, our collection provides regulatory elements (promoters, RBSs, terminators) that work in a plethora of bacterial species (Link to Design Page).

Impact of Sci-Phi 29

Sci-Phi 29 enables orthogonal replication and predictable expression to expand the repertoire of genetically engineerable bacteria. To envision a future where Sci-Phi 29 can be used to tackle a real world problem, we created a hypothetical use-case scenario, where we used our platform to, theoretically, engineer P. putida using Sci-Phi 29 to be of use in removal of microplastics from waste water streams (Link to iHP-use case scenario).

This use case scenario showed us the potential our platform hold. With Sci-Phi 29 we are able to access the microbial diversity. And we as a team are fascinated by this microbial diversity and wanted to share our fascination with the rest of the world. That is why our goal this year was to introduce the general public, from children to your neighbor to your teacher, to the hidden world of microbes. We organized multiple events because we wanted to make sure that everyone has access to the invisible microbial world. During our ‘Foldscope’ event (Link to Education and Engagement-Foldscope), we taught participants how to fold their own origami microscope and how to use these microscopes. Besides showing people the potential of synthetic biology, we hope to inspire people so that they are to share our excitement for scientific exploration.