What remains from living cells in bacterial lysate-based cell-free systems

Abstract

Because they mimic cells while offering an accessible and controllable environment, lysate-based cell-free systems (CFS) have emerged as valuable biotechnology tools for synthetic biology. Historically used to uncover fundamental mechanisms of life, CFS are nowadays used for a multitude of purposes, including protein production and prototyping of synthetic circuits. Despite the conservation of fundamental functions in CFS like transcription and translation, RNAs and certain membrane-embedded or membrane-bound proteins of the host cell are lost when preparing the lysate. As a result, CFS largely lack some essential properties of living cells, such as the ability to adapt to changing conditions, to maintain homeostasis and spatial organization. Regardless of the application, shedding light on the black-box of the bacterial lysate is necessary to fully exploit the potential of CFS. Most measurements of the activity of synthetic circuits in CFS and in vivo show significant correlations because these only require processes that are preserved in CFS, like transcription and translation. However, prototyping circuits of higher complexity that require functions that are lost in CFS (cell adaptation, homeostasis, spatial organization) will not show such a good correlation with in vivo conditions. Both for prototyping circuits of higher complexity and for building artificial cells, the cell-free community has developed devices to reconstruct cellular functions. This mini-review compares bacterial CFS to living cells, focusing on functional and cellular process differences and the latest developments in restoring lost functions through complementation of the lysate or device engineering.

Keywords: Adaptation; Cell-free; E. coli; Homeostasis; Microfluidics; Prototyping; Spatial organization.

Fig. 1

Characterization of the in vivo features remaining in Bacterial lysate-based cell-free systems (CFS) across the main steps of its preparation. The 5-step protocol on the left is pictured with the equipment needed to produce CFS. (i) Regarding protein production and metabolism, CFS is a well-recognised platform for gene expression in which some metabolic pathways like those regenerating ATP are still functional. (ii) All the dynamics systems originally present in a living cell (response to an external stimulus, sensor and transporter ensuring exchanges with the external medium) does not exist anymore in CFS. (iii) While the living cells maintain homeostasis in a non-equilibrium steady state, CFS relaxes to biochemical equilibrium. Only some parameters such as pH and NTPs concentrations are stabilised for a few hours and minutes respectively. (iv) As it is a dilute and well-mixed reaction environment, CFS no longer presents any spatial organisation. The machine icons come from BioRender.com. TX-TL: Transcription and translation.

Fig. 2

Devices developed to restore structures and functions that are defective in CFS. (A) Almost all lost functions related to the central dogma can be restored by adding missing purified molecules. (B) Crowding agents coupled with encapsulation into lipidic vesicles can be used to partially restore spatial organization. (C) In an attempt to restore homeostasis and adaptive behaviors, porous membranes or phospholipid vesicles can be used to supply the CFS reaction with substrate while rejecting waste to prevent deleterious accumulation. (D) An alternative way to achieve this goal is to use microfluidic devices.