Chemical Exchanges and Actuation in Liposome-Based Synthetic Cells: Interaction with Biological Cells

Abstract

The development of new synthetic biology frontiers has led to scenarios where the embodied information-processing capability of biological organisms are implanted, in minimalistic version, in liposome-based synthetic cells. These are cell-like systems of minimal complexity resembling biological cells. Although not yet alive, synthetic cells are useful for generating basic biological understanding, and can become interesting biotechnological tools. In 2012 we devised a research program aimed at the design and construction of synthetic cells capable of exchanging chemical signals with biological micro-organisms (in particular bacteria). Here we review the fundamental steps leading to this innovative research field and comment on the most relevant experimental results obtained by us and others.

Appendices

List of Abbreviations

$\alpha$-HL:

$\alpha$-haemolysin

AI-2:

autoinducer-2

AHL:

N-acyl homoserine lactone

AI:

artificial intelligence

C4-CoA:

butyryl coenzyme A

C4-HSL:

N-butyryl homoserine lactone

C.V.:

coefficient of variation (standard deviation/mean)

HSL:

homoserine lactone

ICTs:

information and communication technologies

IPTG:

isopropyl $\beta$-d-1-thiogalactopyranoside

SAM:

S-adenosylmethionine

SB:

synthetic biology

SC:

synthetic cell

TL:

translation (from RNA to protein)

TX:

transcription (from DNA to RNA)

TX-TL:

coupled transcription-translation reactions

 

Appendix A

With reference to Fig. 1, here we give explanatory comments on the role and relevance of three technologies and on their intersections.

1: Cell-free systems have been traditionally used in biochemical and molecular biology to study biological processes outside the cell. Known since decades, the TX-TL systems have been recently re-discovered, also thanks to the introduction of a purified kit whose composition is minimal and perfectly known, the so-called PURE system [44].

2: Liposome technology has been developed mainly for producing liposomes for drug-delivery applications and for biophysical studies. Several methods are available to prepare empty and solute-filled liposomes, depending on the lipid type and vesicle size and morphology. Of particular interest are the so-called ‘giant’ vesicles (GVs) because they mimic biological cells and can be directly visualized by optical microscopy, for a review on GVs, see [49].

3: Microfluidic devices have been recently introduced in order to manipulate solutions at the micrometer scale. The use of microfluidics for producing water-in-oil droplets is one of the most important application of this technique, but the relevant goal is the production of giant vesicles directly in the microfluidic apparatus (for a review, see [11]). Currently the strategy is based on the formation of water-in-oil-in-water double emulsion droplet, followed by solvent removal.

1–2: These systems are the most used (to date), and consist of liposomes formed by non-microfluidic methods with TX-TL systems (or other biochemical machineries) encapsulated in their aqueous lumen or embedded in their membrane. For a review, see [45]. Note that the populations of spontaneously formed liposomes are generally quite heterogeneous with respect to several parameters (C.V. often >50%).

1–3: Cell-free systems included in microfluidic devices are also a possible combination useful for synthetic biology. For example, work has been carried out to study the dynamics of transcription-translation processes [17].

2–3: To this section belong all work aiming at constructing vesicles, and in particular GVs, by microfluidic devices. It is important to remark that the formation of vesicles in microfluidic devices occurs by repetitive reconstitution of microscopic conditions, and thus all vesicles have very similar structure and a homogeneous population is obtained (C.V. <5–10%). Pioneering work can be found here [27, 36].

1–2–3: This triple overlapping region is probably the ‘Holy Grail’ of bottom-up SB, namely, the construction by microfluidic devices of solute-filled vesicles, so to have a homogeneous population of bioreactors that can be designed and build according to the general requirements of SB, namely modularity, programmability, reproducibility, etc. An example can be found in [26].