Towards Synthetic Life: Establishing a Minimal Segrosome for the Rational Design of Biomimetic Systems

DNA segregation is a fundamental life process, crucial for renewal, reproduction and propagation of all forms of life. Hence, a dedicated segregation machinery, a segrosome, must function reliably also in the context of a minimal cell. Conceptionally, the development of such a minimal cell follows a minimalistic approach, aiming at engineering a synthetic entity only consisting of the essential key elements necessary for a cell to survive. In this thesis, various prokaryotic segregation systems were explored as possible candidates for a minimal segrosome. Such a minimal segrosome could be applied for the rational design of biomimetic systems including, but not limited to, a minimal cell. DNA segregation systems of type I (ParABS) and type II (ParMRC) were compared for ensuring genetic stabilities in vivo using vectors derived from the natural secondary chromosome of Vibrio cholerae. The type II segregation system R1-ParMRC was chosen as the most promising candidate for a minimal segrosome, and it was characterized and reconstituted in vitro. This segregation system was encapsulated into biomimetic micro-compartments and its lifetime prolonged by coupling to ATP-regenerating as well as oxygen-scavenging systems. The segregation process was coupled to in vitro DNA replication using DNA nanoparticles as a mimic of the condensed state of chromosomes. Furthermore, another type II segregation system originating from the pLS20 plasmid from Bacillus subtilis (Alp7ARC) was reconstituted in vitro as a secondary orthogonal segrosome. Finally, a chimeric RNA segregation system was engineered that could be applied for an RNA-based protocell. Overall, this work demonstrates successful bottom-up assemblies of functional molecular machines that could find applications in biomimetic systems and lead to a deeper understanding of living systems

Biomimetic membranes as a technology platform: Challenges and opportunities

Biomimetic membranes are attracting increased attention due to the huge potential of using biological functional components and processes as an inspirational basis for technology development. Indeed, this has led to several new membrane designs and applications. However, there are still a number of issues which need attention. Here, I will discuss three examples of biomimetic membrane developments within the areas of water treatment, energy conversion, and biomedicine with a focus on challenges and applicability. While the water treatment area has witnessed some progress in developing biomimetic membranes of which some are now commercially available, other areas are still far from being translated into technology. For energy conversion, there has been much focus on using bacteriorhodopsin proteins, but energy densities have so far not reached sufficient levels to be competitive with state-of-the-art photovoltaic cells. For biomedical (e.g., drug delivery) applications the research focus has been on the mechanism of action, and much less on the delivery ‘per se’. Thus, in order for these areas to move forward, we need to address some hard questions: is bacteriorhodopsin really the optimal light harvester to be used in energy conversion? And how do we ensure that biomedical nano-carriers covered with biomimetic membrane material ever reach their target cells/tissue in sufficient quantities? In addition to these area-specific questions the general issue of production cost and scalability must also be treated in order to ensure efficient translation of biomimetic membrane concepts into reality

Roadmap on semiconductor-cell biointerfaces.

This roadmap outlines the role semiconductor-based materials play in understanding the complex biophysical dynamics at multiple length scales, as well as the design and implementation of next-generation electronic, optoelectronic, and mechanical devices for biointerfaces. The roadmap emphasizes the advantages of semiconductor building blocks in interfacing, monitoring, and manipulating the activity of biological components, and discusses the possibility of using active semiconductor-cell interfaces for discovering new signaling processes in the biological world

Quantum scale biomimicry of low dimensional growth: An unusual complex amorphous precursor route to TiO2 band confinement by shape adaptive biopolymer-like flexibility for energy applications

Crystallization via an amorphous pathway is often preferred by biologically driven processes enabling living species to better regulate activation energies to crystal formation that are intrinsically linked to shape and size of dynamically evolving morphologies. Templated ordering of 3-dimensional space around amorphous embedded non-equilibrium phases at heterogeneous polymer-metal interfaces signify important routes for the genesis of low-dimensional materials under stress-induced polymer confinement. We report the surface induced catalytic loss of P=O ligands to bond activated aromatization of C-C C=C and Ti=N resulting in confinement of porphyrin-TiO(2 )within polymer nanocages via particle attachment. Restricted growth nucleation of TiO2 to the quantum scale (˂= 2 nm) is synthetically assisted by nitrogen, phosphine and hydrocarbon polymer chemistry via self-assembly. Here, the amorphous arrest phase of TiO, is reminiscent of biogenic amorphous crystal growth patterns and polymer coordination has both a chemical and biomimetic significance arising from quantum scale confinement which is atomically challenging. The relative ease in adaptability of non-equilibrium phases renders host structures more shape compliant to congruent guests increasing the possibility of geometrical confinement. Here, we provide evidence for synthetic biomimicry akin to bio-polymerization mechanisms to steer disorder-to-order transitions via solvent plasticization-like behaviour. This challenges the rationale of quantum driven confinement processes by conventional processes. Further, we show the change in optoelectronic properties under quantum confinement is intrinsically related to size that affects their optical absorption band energy range in DSSC.This work was supported by the National Research Foundation of Korea (NRF) grant funded by Korea government (MEST) NRF-2012R1A1A2008196, NRF 2012R1A2A2A01047189, NRF 2017R1A2B4008801, 2016R1D1A1A02936936, (NRF-2018R1A4A1059976, NRF-2018R1A2A1A13078704) and NRF Basic Research Programme in Science and Engineering by the Ministry of Education (No. 2017R1D1A1B03036226) and by the INDO-KOREA JNC program of the National Research Foundation of Korea Grant No. 2017K1A3A1A68. We thank BMSI (A*STAR) and NSCC for support. SJF is funded by grant IAF25 PPH17/01/a0/009 funded by A* STAR/NRF/EDB. CSV is the founder of a spinoff biotech Sinopsee Therapeutics. The current work has no conflicting interests with the company. We would like to express our very great appreciation to Ms. Hyoseon Kim for her technical expertise during HRTEM imaging

Tuning Photocurrent Responses from Photosystem I via Microenvironment Alterations: Effect of Plasmonic Electric Fields and Membrane Confinements

Robust photoelectrochemical activities of PSI make it an ideal candidate for bio-hybrid photovoltaic and optoelectronic devices. This dissertation focuses on role of microenvironment alterations around PSI in tuning its photocurrent responses when assembled with tailored plasmonic metal nanostructures and biomimetic lipid interfaces. To this end, a series of systematic studies aimed at tuning the plasmon enhanced photocurrent responses from PSI assembled with gold and silver metal nanopatterns tailored for different plasmonic absorption wavelengths. The experimental observation of plasmon-induced photocurrent enhancements in PSI is investigated using Fischer patterns of silver nanopyramids (Ag-NPs) wherein the resonant peaks were tuned to match the PSI absorption peaks at ~450 and ~680 nm. A conservative estimate for the enhancement factors were found to be ~ 5.8 – 6.5 when compared to PSI on planar Ag substrate assemblies. Furthermore, spatially localized and spectrally resolved wavelength-dependent plasmon-enhanced photocurrents from PSI are investigated by specifically assembling the protein units in regions around highly ordered Au (AuND) and Ag (AgND) nano-discs where the dipolar plasmon resonance modes from the respective NDs are tuned to the wavelengths of ~680 nm and ~560 nm, respectively. Specifically, we report plasmon-enhancement factors of ~6.8 and ~17.5 for the PSI photocurrents recorded under the excitation wavelengths of ~680 nm and ~565 nm respectively as compared to PSI assembled on planar ITO substrates. The results indicate: 1) direct correlations between the photocurrent enhancement spectra from the PSI assemblies and the plasmonic resonance modes for the respective nanopatterned substrates, and 2) broadband photocurrent enhancements due to plasmon-coupled photoactivation in the otherwise blind chlorophyll regions of the native PSI absorption spectra. In our continuing efforts to investigate the alterations in the photoexcitation/dissipation pathways in PSI due to characteristic changes in their optical and structural properties under biomimetic membrane confinements, , the PSI complexes are reconstituted in synthetic lipid membranes of 1,2-diphytanoyl-sn-glycero-3-phospho-(1ʹ-rac-glycerol) (DPhPG) and 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC). The results presented here from absorption, fluorescence and circular dichroism indicate unique changes around the carotenoid/chlorophyll spectral bands leading to attainment of broad-band light harvesting via enhanced absorption in the otherwise non-absorptive green region (500 – 580 nm) of unconfined PSI absorption spectra

Biomimetic microscale platforms for the visualization of biological processes : from GUVs towards artificial cells

First in order to explore the possibilities of building a synthetic artificial cell following a biomimetic approach, we engineered synthetic polymer-based giant unilamellar vesicles (GUVs) with selective membrane permeability. Since the membranes of polymeric GUVs present a high impermeability compared to natural lipid membranes, membranes are selectively permeabilized in a biomimetic approach by the insertion of the small pore-forming peptide gramicidin (gA) as gA biopores are known to allow the transport of protons and monovalent ions. Whilst gA has been inserted in lipid membranes in numerous research studies, the challenge of inserting the bacterial pore into polymer membranes is greater because of the significant difference between the pore length and the thickness of the polymer membrane (more than 3.5 times). Confocal laser scanning microscopy (CLSM) was used to show that neither the size, nor the morphology of the GUVs was affected by successful insertion of gA and further to visualize the pH change inside the cavity of GUVs in real-time by recording videos. In order to demonstrate the successful insertion of gA, a pH-sensitive dye is encapsulated inside the cavity of GUVs and proton gradients between the environment of GUVs and their inner cavity serves to assess the exchange of protons across the membrane upon gA insertion. The results showed that gA was successfully inserted and remained functional in polymer membranes with thickness of 9.2–12.1 nm. Larger membrane thicknesses did not allow gA insertion, and 12.1 nm represents a limit for the mismatch between the pore length and the membrane thickness. Our gA-GUVs are therefore pH-regulating and maintain their integrity in different pH conditions in a cell-like manner. This bio-mimetic approach to use ion channels with specific selectivity for insertion in polymer membranes is an elegant strategy to develop mimics of biomembranes or for supporting the design of bioreactors. Next, a functional cell mimetic compartment is developed by the insertion of the bacterial membrane protein (OmpF) in thick synthetic polymer membranes of an artificial GUV compartment that encloses the oxidative enzyme horseradish peroxidase. In this manner a simple and robust cell mimic is designed, that supports a rudimental form of metabolism. The biopore serves as a gate, which allows substrates to enter the cavities of the GUVs, where they are converted into the resorufin-like products by the encapsulated enzyme, and then released in the environments of GUVs. Our bio-equipped GUVs facilitate the control of specific catalytic reactions in confined micro-scale spaces mimicking cell size and architecture and thus provide a straightforward approach serving to obtain deeper insights in the real-time of biological processes inside cells. This elegant strategy of equipping both GUV membranes and GUV cavities with biomolecules, opens the way towards cell-like compartments as novel materials with bio-functionality is the combination of synthetic micrometer-sized giant unilamellar vesicles (GUVs) with biomolecules because it enables studying the behavior of biomolecules and processes within confined cavities. Finally a visionary strategy for creating the first bioinspired molecular factory with functionality as a real cell-mimic based on micrometer-sized giant plasma membrane vesicles (GPMVs) is addressed. GPMVs are cell-derived giant vesicles consisting of an outer compartment architecture (membrane) and an inner composition, which both directly mirror the composition of cells from which they originate except the larger organelles like for example nuclei and Golgi apparatus making measurements easier and are the closest cell-mimic available on the market up to now. In a step towards the development of bioinspired molecular factories with functionality as cell mimics, we generate the next generation of cell mimics by the production of sophisticated hybrid molecular factories based on GPMVs, which are equipped with a synthetic molecular machinery inside their cavities that provides functionality. Such a hierarchical approach in compartmentalization allows the lower-level synthetic functional compartments encapsulated within the cavity of the GPMV to act as independent anatomically discreet units that specialize in their own function, making them nanoscale versions of nature own organelles. Towards the first bioinspired molecular factory enzyme-equipped polymersomes with a reconstituted membrane protein (OmpF) are encapsulated inside the GPMVs as enzymatic nanocompartment spaces, where they retain their structure and functionality. When substrates were added to the outer solution of the GPMVs, it was shown that they could penetrate both the membrane of the GPMVs and the inner compartment membranes of the synthetic nanoreactors equipped with OmpF pores. In this respect the equipment of the catalytic nanocompartment spaces with OmpF was essential as it allowed the enzyme to perform in the inner cavities. Successful substrate conversion was visualized by following the fluorescent product of the enzymatic reaction (resorufin-like product), which could leave the polymersome and diffuse inside the GPMV cavity. Finally we demonstrate that equipped GPMVs can act as artificial cell mimics – retaining their membrane and inner composition if they are injected into multicellular organisms – Zebrafish embryos. To the best of our knowledge, this is the first time that a molecular factory functioning as a cell-like mimic has been be constructed by using a top down-bottom up approach and has been tested in vivo by taking advantage of the fundamental nature of GPMVs

Construction of membrane-bound artificial cells using microfluidics: a new frontier in bottom-up synthetic biology

The quest to construct artificial cells from the bottom-up using simple building blocks has received much attention over recent decades and is one of the grand challenges in synthetic biology. Cell mimics that are encapsulated by lipid membranes are a particularly powerful class of artificial cells due to their biocompatibility and the ability to reconstitute biological machinery within them. One of the key obstacles in the field centres on the following: how can membrane-based artificial cells be generated in a controlled way and in high-throughput? In particular, how can they be constructed to have precisely defined parameters including size, biomolecular composition and spatial organization? Microfluidic generation strategies have proved instrumental in addressing these questions. This article will outline some of the major principles underpinning membrane-based artificial cells and their construction using microfluidics, and will detail some recent landmarks that have been achieved

Photoactivated biological processes as quantum measurements.

We outline a framework for describing photoactivated biological reactions as generalized quantum measurements of external fields, for which the biological system takes on the role of a quantum meter. By using general arguments regarding the Hamiltonian that describes the measurement interaction, we identify the cases where it is essential for a complex chemical or biological system to exhibit nonequilibrium quantum coherent dynamics in order to achieve the requisite functionality. We illustrate the analysis by considering measurement of the solar radiation field in photosynthesis and measurement of the earth's magnetic field in avian magnetoreception