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).

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:

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).

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.