Semi-permeable artificial cells and their applications in synthetic biology
Creators: name: Van Raad, Damian
Publisher: name: The Australian National University
Year: 2023

description: Biotechnology depends on our ability to produce proteins rapidly, in large purity and at large scale. In-vivo protein production in bacterial cells is generally the preferred choice to economically produce large quantities of protein, and cell-free protein synthesis is applied for specialized applications or when proteins are required quickly. This thesis is the development of the encapsulated cell-like structures (eCells), which can be considered a hybrid approach at the intersection of in-vivo and in-vitro protein expression. Chapter 2 of this thesis introduces a method for efficiently producing encapsulated bacterial lysate which can be applied to cell-free protein synthesis for recombinant protein expression. This facile method produces eCells using layer-by-layer polymer assembly to coat bacterial cells prior to lysis of their cell wall. As a result, eCells maintain many of the advantages of living cells while at the same time allow full control over the chemical environment. Individual eCells are separated by FACS which highlights the femto-litre size and demarcation of unique protein features housed in a semipermeable polymer capsule. In chapter 2: Site specific non-canonical amino acid incorporation (ncAA) is utilised in eCells, with amino acyl-tRNA synthetases such as the pyrrolysyl-tRNA synthetase which is typically inactive when purified and added to standard cell-free protein synthesis. Using PylRS synthetase, photocaged-cysteine and N(epsilon)-(tert-butoxycarbonyl)-L-lysine was incorporated into Peptidylprolyl cis-trans isomerase B (PpiB) using eCells. eCells are not living entities, although amino acid anabolism has been shown to be functional. Chapter 3 and 4 demonstrate the application of eCells to isotope label proteins atom selectively and reduce the cost of isotope labelling proteins for NMR spectroscopy. The use of eCells, the strategically chosen precursor and the omission of the amino acid from the reaction mixture allow the production of proteins with residue and atom specific isotope labelling patterns, increasing the sensitivity of 13C HSQC spectra with the elimination of one-bond 13C-13C scalar couplings. Chapter 3 introduces new approaches to selectively label methyl groups of valine, leucine, isoleucine and alanine residues in proteins. In addition, stereospecific labelling of Pro-S signals of leucine and valine resides has been achieved in eCells using the precursor 2-13C-methylacetolactate. Chapter 4 explores labelling approaches for aromatic residues for NMR spectroscopy. Starting from 3-13C-pyruvate, the proteins PpiB and ubiquitin were selectively labelled in the epsilon and delta positions of Phe/Tyr and analysed by NMR. eCells provide advantages over cell-free protein synthesis and in-vivo expression. In Chapter 5 (i) The ability to influence protein folding and change the redox environment is an attractive feature of cell-free protein synthesis. Human protein disulfide isomerase (PDI) which is expressed prior to encapsulation, helps proteins fold correctly during cell-free protein synthesis into a correct form and allows for expression of soluble disulfide bonded proteins. Using eCells and PDI, two proteins that previously could not be expressed actively in bacterial cells, neurotrophic growth factor neuritin and active light-chain enterokinase, were expressed. Chapter 5 (ii) highlights that eCells have shown to be effective regarding bioremediation and (iii) biosensing. Using this system in conjunction with biocatalytic enzymes for an organophosphate pesticide, Paraoxon-ethyl, the contaminant can be inactivated and made harmless, eliminating it from the natural environment. eCells are also able to detect heavy metals such as Hg2+ with a lower limit of detection and higher sensitivity than in-vivo biosensing. Chapter 5 (iv) introduces that eCells can be made with other derivatives of E. coli and using eCells, site specific introduction of phosphorylated groups can be achieved.