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Sci-Phi 29

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Sci-Phi 29: Enabling orthogonal replication and predictable expression to expand the repertoire of engineerable bacteria

Many of the world’s issues, like pollution and climate change, could be tackled with biotechnology, by using microorganisms that digest certain substrates, like plastic or CO₂. On earth, we have a huge biodiversity of microbes that could potentially utilize all these substrates and convert it to useful products. However, in reality, biotechnology sticks to a limited number of microbial chassis; since there is a lack of characterized parts for genetic engineering of unconventional microbes, we cannot harness the potential of all microbes on the planet.

Sci-Phi 29 is a versatile platform which allows expressing 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 of parts. Sci-Phi 29 is a versatile platform to further explore the bacterial diversity providing new opportunities for the advancement of 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 bacteria could be engineered to 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 with 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, whenever exploiting the potential of a non-model bacterial organism for the first time there is a lack of genetic tools (Calero & Nikel, 2019). Therefore, when moving to an unconventional bacterial species, there is the need of developing new genetic tools. This includes characterized species-specific promoters, replicative and suicide vectors, to cover a wide range of expression levels and genome engineering tools, such as CRISPR devices (Calero & Nikel, 2019).

**Figure 1: Genetic tools that are required to express genetic circuits in non-model bacteria.**

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

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).

The mechanism of the phi29 replication machinery is visualized in Video 1:

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 (Mencia et al., 2011).

When trying to express the phi29 replication machinery in E. coli we came into contact with Dr. Chang Liu and Dr. Julian Willis, experts on expression of orthogonal replication machinery, 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. See results here.

By using the PURE system we demonstrated replication in vitro of our own linear construct, which is flanked by the phi29 origins of 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 genetic circuits inside cells. This includes variation in plasmid copy number, transcription, and translation. Therefore, it is difficult to introduce reliable parts for the genetic engineering of 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. 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 an 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 and experimental validation, 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 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.

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, engineer P. putida using Sci-Phi 29 to be of use in removal of microplastics from waste water streams. This use case scenario showed the potential our platform holds.

We as a team are fascinated by the microbial diversity and wanted to share our fascination with the rest of the world. That is why our goal this year was to introduce as many people as possible 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, we taught participants how to fold their own origami microscope and how to use these microscopes.With the proceeds of this workshop, we were able to send 100 of these microscopes to urban areas in India, so the children there also have access to the fascinating world of microbiology. Besides showing people the potential of synthetic biology, we hope to inspire people so that they are to share our excitement for scientific exploration.

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