Immobilization of Proteins of Cell Extract to Hydrogel Networks Enhances the Longevity of Cell-Free Protein Synthesis and Supports Gene Networks

Abstract

Herein, we constructed a new type of hydrogel based artificial cells supporting long-lived protein synthesis, posttranslational modification, and gene networks. We constructed the artificial cells by immobilizing the transcription and translation system from E. coli cytoplasmic extract onto the polyacrylamide hydrogel. With the continuous supply of energy and nutrition, the artificial cells were capable of stable protein expression for at least 30 days. Functional proteins which were difficult to produce in vivo, including colicin E1 and urokinase, were synthesized in the artificial cells with high bioactivity. Furthermore, we constructed a sigma factor based genetic oscillator in the artificial cells. The artificial cells not only provide a powerful platform for continuous protein synthesis and convenient design and testing of genetic networks, but also hold great promise for the development of metabolic engineering, drug delivery, and biosensors.

Constructing Cell-Free Expression Systems for Low-Cost Access

“… 47 However, protein synthesis yields can be low due to the endogenous exonuclease activity present in the cell extracts and degradation of DNA templates. To address this problem, strategies such as the use of Chi sequences, 25 GamS, 21 , 47 , 48 and polymerase chain reaction (PCR) products with long flanks 3 have been used to stabilize linear DNAs. To determine whether the addition of lactose to either of our two fresh formulations ( Tables S1 and S2 ) can improve the protein yields in cell-free reactions based on linear DNA templates, we tested the two best lactose concentrations from our previous experiments with fresh samples ( Figures 2 D,I and S2I ).…”

“…Since the discovery of the functional relationship between mRNA and protein by Nirenberg and Matthaei in the early 1960s, cell-free systems (CFSs) have become powerful tools with broad applications now in synthetic biology, ranging from the study of artificial gene circuits and synthetic cells to protein production. − The technology relies on in vitro transcription–translation systems that employ cell extracts as a source of ribosomes and auxiliary transcriptional factors, or reconstitution of purified cell components in the case of the PURE system. , In addition to transcription–translation machinery, CFS requires an adequate supply of key elements such as amino acids, crowding reagents, salts, nucleotide triphosphates (NTPs), homeostatic environment, and an ATP regeneration system . There have been numerous reports of improvements in the way extracts are generated and fed.…”

Synthetic biology enables field-deployable biosensors for water contaminants

“…1b). Additionally, recent advances in synthetic biology has offered safer alternative chassis to living cells such as chromosome-free simple cells (SimCells) that cannot replicate and have a limited lifetime before run out of energy [40], and artificial cells which are constructed from bottom-up chemistry-based approaches and loaded with cellular extract or specified cellular components [41]. Yet, these technical routes are still at an early development phase with further optimization required towards field-deployable biosensing applications.…”

Cell Free Expression in Proteinosomes Prepared from Native Protein‐PNIPAAm Conjugates

“…[8] Notwithstanding lipid vesicles, there have been a variety of different compartments which can support cell free expression from water-in-water membrane bound compartments such as polymersomes [12,13] to water-in-oil systems such as surfactant stabilized droplets and droplet interface bilayers (DIBS), [14] inorganic colloidosomes, [15] and membrane free systems such as coacervates [16] and hydrogel based systems. [23] These synthetic cellular platforms have been important for increasing compartment stability via polymer stabilization; [17] for enhancing properties such as intercellular communication and for integrating higher order cellular architectures [18][19][20] that can be critical for integrating specific cellular properties into nonliving matter.…”

“…To this end, proteinosomes are alternative membrane-bound compartments prepared from protein-polymer conjugates [19,20] with membranes that are elastic with tunable permeability to enzymes or DNA molecules. [21][22][23][24] Water-in-water proteinosomes have been shown to support reaction cascades; [25] DNA strand displacement reactions and PEN DNA reactions.…”