(162q) Construction of Artificial Biological Nucleus System via the Volume Phase Transition of Thermal-responsive Hydrogels

A recent frontier in the bottom-up synthetic biology is the construction of artificial cells, which could help to determine the essential compounds and processes for sustaining life. These synthetic cells also show great potential for drug delivery, gene therapy, and the study of life origin. So far, they have been proved to be capable of hosting some biochemical reactions, thus mimicking certain functions of living cells, such as self-replication, protein synthesis, and coupled transcription and translation.

The construction of artificial cells mainly draws support from the encapsulation of cell-free protein synthesis (CFPS) system, in which transcription and translation process are enabled outside cells by the reasonable arrangement of key factors such as amino acids, salts, nutrients, etc. The CFPS system could facilitate the construction mainly due to the vast simplification compared with natural cell systems and their high level of protein expression.

Although there has been great progress in the construction of artificial cells, these were mostly created to mimic and improve one or more functions of the whole living cell. The intrinsic characteristics of the cell have been more or less neglected. For example, a smaller confinement particle has been proved to facilitate the protein expression. Nevertheless, it has not been systematically explored whether the distribution of templates or other substrates could affect the final performance of artificial cells. In fact, genetic materials generally exist in the form of aggregation in the nucleus or nucleoid, and the high local concentration could help to improve the bioreaction efficiency.

Here we abandoned the construction of a complete artificial cell, but focused on a nucleus-mimic system to create an aggregation state for the genetic materials during the process of cell-free protein synthesis. Therefore, the main issue was the construction of nucleus-mimic particles, or called artificial nuclei. Here the thermal responsive hydrogel poly (N-isopropylacrylamide) (PNIPAM) was selected to encapsulate the plasmids, as it could experience a reversible volume phase transition (VPT) with the change of ambient temperature. Based on this characteristic, the plasmids embedded inside the particles could be gathered on the porous surface to mimic the aggregation state. The PNIPAM aqueous droplets were prepared with the microfluidic system. The prepared hydrogels were added to the plasmid solution at 37℃ for wrinkling and then placed at 4℃. The surrounding plasmids would be absorbed into the hydrogels to get the artificial nuclei because of the re-expansion of the hydrogels. The artificial nuclei were supplied to the cell-free system to test their performance with the report protein. In this situation, we discovered persistent and effective protein expression. We also found that the artificial nuclei had a carrying capacity, which should owe to the saturated state of the porous structure of the hydrogels, for the best expression level.

We believe this study has provided a new approach to support sustainable and efficient protein synthesis by introducing the nucleus-mimic particles. As the volume phase transition property of PNIPAM hydrogel helped to encapsulate templates in a flexible and harmless way, the artificial nucleus system might become instructive for the construction of a configurable artificial cell or a complicated living system, also more available and applicable for the cell-free synthesis in mass production.