Design

Distribution

Inspired by network theory and by the knowledge that distributed networks are more stable than centralised ones, we have designed our project to be distributed. This means that, despite having the same overarching goal, we have divided the project in several independent subprojects. The failure in a subset of these subprojects would not prevent us from reaching a certain success in the project.

Figure. 1: Dividing our project in into several subgroups to make it more stable in case of part-failures, similar to a house standing on multiple instead of one pillar.

Occam’s razor

The Occam’s razor states that the simplest explanation is generally true. In engineering, one applies this principle to create the simplest system able to perform a given task. In synthetic biology, it corresponds to creating the needed biological device using the smallest sets of building blocks. We followed this principle when selecting the types of compartments to work with: we chose those requiring the smallest number of proteins.

Modularity

Modularity is arguably the most known –and applied– design principle in synthetic biology. We followed this principle selecting to work with a universal localisation system, whereby proteins of interest (POIs) are sequestered into the compartments via fusion to a special protein (SpyCatcher [1] or SnoopCatcher [2]) rather than via direct fusion to the compartment-forming protein. Like this, users of our compartmentalisation toolbox only need to clone their POI into the appropiate plasmid, leaving all the rest unchanged. While this still requires a cloning step, the plasmid encoding the proteins forming the compartments are typically more complex and difficult to clone.

Multiplexing

When designing a new synthetic biology tool, multiplexing is often considered. This means the possibility of achieving multiple objectives with a single component. A well-known example is the CRISPR/Cas system, whereby nuclease dead Cas9 (dCas9) is combined with a modified guide RNA into which RNA stem loops have been inserted [3]. In this case, regulatory domains (transactivation, repression, methylation domains, etc.) are brought to the DNA of interest by RNA-binding proteins –such as the MS2 coat protein (MCP)– instead than by dCas9 itself (due to genetic fusion). This ingenious design allows using a single dCas9 protein to perform simultaneously repression, activation and other regulatory functions at different genomic loci, achieving multiplexing.
Inspired by the work done by the Kerfeld lab [4], in which the SpyTag and the SnoopTag have been fused to the T1 protein to bring into the nanocompartment two different POI, we also fused these tags to the SPD-5 protein, that forms liquid droplets in E. coli. Like this, we wanted to achieve multiplexing by recruiting two POIs into the droplets using a single engineered SPD-5.

Orthogonality

This is perhaps the most important principle in synthetic biology. Because we work with cells, it is crucial to employ building blocks (e.g. proteins) that do not impact endogenous cellular processes. We followed this principle manyfold, for instance by selecting compartment-forming proteins that are not endogenous to E. coli, our selected chassis.
To incorporate non-canonical amino acids (ncAAs) into proteins, we also relied on orthogonal pairs of tRNA synthetase-tRNA, which originally do not come from E. coli. In this case, the orthogonality ensures that only this pair can “read” the amber stop codon as if it were a normal triplet instead of a stop codon.

Standardisation

The iGEM registry was one of the first attempts to establish the concept of standardisation in synthetic biology. This design principle wants parts to be constructed following a standard assembly process, so that they can be easily re-used and expanded by future users. We followed this principle making all our constructs compatible with the registry (see our parts-page).

Optimisation

After initial experiments to test a prototype, typically one proceeds with its optimisation. We decided to optimise our compartments by incorporating ncAAs at desired positions in some of the compartment-forming proteins.
We also thought of optimising the system using genome-reduced E. coli strains, which are supposed to be better suited for the production of useful compounds due to the reduced burden linked to expression of unnecessary proteins [5].
We also performed codon-optimisation to enhance the expression in E. coli of our compartment-forming proteins (all originally found in other organisms) as well as the XiaI enzyme (naturally a gene of Streptomyces sp. SCSIO 02999) involved in the metabolic pathway for indigo/indirubin production.