Test tube condensates

Several condensation modules have been used for the generation of test tube designer condensates. Pick the one that best matches your needs.





Building up complexity

The study of individual proteins phase separating under test tube conditions has proven extraordinarily useful for understanding the biophysical interactions underlying this process. Yet, while this approach allows for the exploration of the molecular details governing the behavior of a single protein, such condensates are but a glimmer of their cellular counterparts. The latter are often multi-layered or take up complex topologies, consisting of subcompartments with different composition and material properties. On the other hand, high-throughput studies have shown that several membraneless organelles can contain up to hundreds of different proteins and thousands of RNA species. Can engineered test tube condensates mirror this in vivo complexity?

Several studies have approached this question via different angles:

1. Engineering morphological complexity using protein mixtures: Condensating proteins with a differential molecular grammar can drive the spontaneous formation of multi-layered and demixed assemblies in vitro (Feric et al., 2016; Putnam et al., 2019

2. Engineering morphological complexity using RNA mixtures: RNA sequence features relating to its base pairing capability or differential self/non-self interactions can drive the formation of different complex morphologies (Boeynaems et al., 2019). By altering the ratio of RNA to protein one can generate vacuolated condensates (Banerjee et al., 2017).

3. Reconstituting minimal-component membraneless organelle mimics: Membraneless organelle mimics can be reconstituted with a minimal set of client and scaffold proteins (Woodruff et al., 2017; Mitrea et al., 2016; Schütz et al., 2017; Sheu-Gruttadauria et al., 2018).

4. Biophysical enrichment strategies for high throughput identification of client molecules: By inducing phase separation in a complex mixture of biomolecules once can enrich endogenous scaffolds and clients via centrifugation and identify them via mass spec (Boeynaems et al., 2017) or RNAseq (Van Treeck et al., 2018).

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Functionality in a droplet

As illustrated in the above sections, we now have a firm grasp on some of the common sticker-spacer modules and are slowly understanding how these can be combined to drive recruitment of biopolymers into tunable condensates. Thus, we have the basic tools in hand to start engineering designer condensates with the aim to create smart and functional biomaterials.

Numerous papers over the past few years have described the generation of in vitro condensates that can encode and regulate enzymatic reactions, show active behavior, or have technological applications.

You can find through the link below a curated list of such examples of in vitro condensates with different functionalities.