Team:Vilnius-Lithuania/Description
Description
Describe the Impossible
Cell-free systems are becoming an increasingly popular in vitro tool to study biological processes as it is accompanied by less intrinsic and extrinsic noise. Relying on fundamental concepts of synthetic biology, we apply a bottom-up forward engineering approach to create a novel cell-free system for unorthodox protein-evolution. The core of this system is cell-sized liposomes that serve as excellent artificial membrane models. By encapsulating genetic material and full in vitro protein transcription and translation systems within the liposomes, we create reliable and incredibly efficient nanofactories for the production of target proteins. Even though there are many alternative proteins that can be synthesized, our main focus is directed towards membrane proteins, which occupy approximately one third of living-cells’ genomes. Considering their significance, membrane proteins are spectacularly understudied since synthesis and thus characterization of them remain prevailing obstacles to this day. We aim to utilize liposomes as nanofactories for directed evolution of membrane proteins. Furthermore, by means of directed membrane protein-evolution, a universal exposition system will be designed in order to display any protein of interest on the surface of the liposome. This way, a system is built where a phenotype of a particular protein is expressed on the outside while containing its genotype within the liposome. To prove the concept, small antibody fragments will be displayed to create a single-chain variable fragment (scFv) library for rapid screening of any designated target.
Fig. 1 Schematic overview of the SynDrop
“What I cannot create, I do not understand”
R. Feynmann, February 1988
Brief overview of the SynDrop - Synthetic Droplets for Membrane Protein Research.
SynDrop started from the idea of working towards developing a minimal synthetic cell. However it was soon realized that synthetic life is not something that will be made in one go - it will be the culmination of all the small, separate systems that will come together and work in unison. As such, it was understood that these systems need to be independently functional and well described first, before more complex systems are built on top of them. One of the most fundamental differences between life and synthetic systems is the responsivity and communication with the surrounding environment. This function in living cells is mostly performed by membrane proteins. We quickly realized that in order to make a significant impact on synthetic life development, membrane proteins are that understudied field that holds great potential for future applications in synthetic biology. Fig. 1 beautifully summarizes the workflow, complexity and at the same time minimalism of SynDrop. We utilized the emerging technology of microfluidics to synthesize cell-sized liposomes and provide them with the minimal set of all the necessary tools and machineries for the successful synthesis of membrane proteins. These fully equipped liposomes form the core of the SynDrop. Within them are encapsulated purified BAM complex proteins and the chaperone SurA which facilitate beta-barrel bearing protein assembly. Liposomes also contain genetically engineered membrane-associating ribosomes which increase the yields of target protein expression. SynDrop liposomes contain an in vitro transcription-translation system and custom DNA. Their inner aqueous environment is suitable for molecular reactions to occur. Finally, SynDrop provides a novel platform for protein display, whether they were antibodies, single chain fragments, globular proteins, or peptides. It is a huge step forward in membrane protein research and perhaps another resolved puzzle towards the creation of synthetic cell.
Background
Synthetic biologists have come a long way since 1912 Stéphane Leduc’s La Biologie Synthétique. Throughout the course of synthetic biology, there were many stepping stones that lead to greater things, such as the discovery of restriction enzymes which lead to the simplified construction of recombinant DNA molecules and arrangements of new genes, or the creation of synthetic biological circuit devices by combining different genes within E. coli. Through these canonical inventions we acquired not only better tools, but also greater ambitions. From understanding how genes work and that they can be modified or replaced, to programming cellular behavior with external impulses, we have reached a point, where no longer the singular modal elements like switches, cascades, pulse generators or oscillators matter. We have reached a point where scientists are ready to face the biggest challenge of synthetic biology - creating an artificial cell.
However designing complex, several layered circuitries resembling the behavior of a natural cell is still an overwhelming challenge due to many limitations like crosstalk, mutations, ambiguous intracellular and extracellular conditions, and biological noise. Therefore we propose to start from something simpler and more minimal.. Although the journey of creating a synthetic minimal cell has already begun, we hoped to contribute to this ultimate goal as well by investing our time and effort. This year we are engineering liposomes, lipid-coated vesicles, that are perfect models to study the initial steps for creating synthetic cells. Liposomes can offer a system with fully controllable experimental parameters and only the exact elements for our custom circuit design without the need to ever worry about the crosstalk and noise. We believe that most of the future synthetic biology applications will rely on bottom-up engineering solutions. Having mastered some hard-core bottom-up liposome engineering, we won’t take long to create the first synthetic cell.
Keeping that in mind we raised a question - what is the trivial difference between completely artificial systems like liposomes and living cells? The answer has pushed us to develop the SynDrop system the way it is being presented today. That major difference between synthetic and natural systems is the capacity to develop an active interface between outside and inside, which is vital for communication, transport, signaling, growth, and proliferation. Most of this communication is mediated by integral membrane proteins. Surprisingly, though membrane proteins are key players in many cellular functions and even human health and disease, they are greatly understudied when compared to e.g. globular proteins. Membrane protein integration into the membranes in vitro is still a particularly delicate issue that halts more rapid scientific development in this field. Having realized this, we knew that our goal this year was going to be the creation of an in vitro membrane protein synthesis system in liposomes in order to not only broaden the research possibilities of integral proteins but also to move one step further in building a minimal cell, which with what SynDrop offers - now will be capable of executing at least minimal .communication between its inside (genotype) and surrounding environment (display phenotype).
SynDrop - Synthetic Droplets for Membrane Protein Research
Current methods for studying MPs are mostly based on cellular systems, which are really not the best environment to study membrane proteins. Large intrinsic and extrinsic cellular noise makes it awfully difficult to characterize discrete MPs as well as other limitations like toxicity and limited yields. Only a few self-integrating membrane proteins have been utilized for research as the majority of them aggregate or are toxic to the host. The capacity to vary parameters is too restrictive for an in-depth investigation of a complete spatiotemporal arrangement of a particular protein or mechanism.
As mentioned above, to untangle these problems we decided not to try to decipher and modulate natural cells. We chose something much more simple - a cell-free synthetic system with well defined, easily controlled parameters, that could be engineered from scratch. We called it SynDrop - Synthetic Droplets for Membrane Protein Research. SynDrop edges not only cellular systems, but also other current cell-free systems in terms of membrane protein research and studying their behavior, including folding and membrane insertion.
Thus SynDrop is a novel bottom-up liposome-based platform created to empower the manipulation of membrane proteins. It is carefully though and bares the exact minimum components to successfully perform its function and not become too unpredictable or metabolically unstable. Transcriptional and translational machineries together with the plasmid DNA, chaperones, energy regeneration systems, and cellular MP insertion facilitators are encapsulated within the liposomes, enabling synthesis, integration, and display of target membrane proteins. It is also equipped with synthetic tools that regulate, attenuate and modulate the whole system.
Applications
As our project focuses on a novel platform for membrane protein research it offers various future applications.
1. Membrane protein characterization
Since liposome systems contain no unknown variables, SynDrop could be used to characterize membrane proteins and their biogenesis. This could possibly unlock new and innovative breakthrough in proteomics.
2. Membrane protein-lipid interactions
Microfluidics allow to synthetize liposomes with modifying their lipid composition rather simply. By using SynDrop we can quickly get huge quantities of liposomes with various lipid compositions. Further, membrane proteins translated in these liposomes will interact accordingly with these lipids. This characterization of membrane protein interactions with custom lipids can be modelled and then implemented in in vivo systems.
3. Membrane protein-membrane protein interactions
Expanding even further of the membrane protein interactions. The amount of genetic material that gets encapsulated in the liposome can be regulated. Therefore multiple genes coding for different membrane proteins can be translated inside liposomes. It yields multiple membrane protein-protein interactions that can be measured and modelled accordingly.
4. Molecular evolution of the exposed particle
Liposomes could be effectively used in molecular evolution. We constructed a few membrane proteins that proved to display a protein particle on the surface of bacteria. By incorporating a few membrane protein integration helpers, we believe that liposome exposition could serve as a better alternative to current molecular evolution methods.
5. Molecular evolution of the membrane protein
Molecular evolution could be also applied not only for the exposed protein particle, but also for the membrane protein itself. Since liposomes have no additional noise and contains a changeable lipid bilayer, it could be one of the most feasible methods of membrane protein evolution.
6. Membrane protein biogenesis regulation
DNA or RNA that gets encapsulated inside liposomes can be altered. Therefore we can insert modifications that change the rate and volume of membrane protein biogenesis. For example, modifications might include thermoswitches that we used in our project to regulate the rate of translation inside the liposomes, so that one membrane protein has the priority to get translated over the other. These or similar modifications might lead to a fully functional one or more genetic circuit incorporation into liposomes that regulate the biogenesis of membrane proteins.
7. Liposome communication
Microfluidic field enables the production of similar or different composition liposomes. Theoretically, these liposomes could cross-react with each other or even share signals. This could create an autonomous artificial ecosystem and could lead towards the creation of artificial life.
Background of other display systems
An extensive literature search was performed to analyze every display system type that currently exists in order to be sure about the necessity and applicability of our project.
Phage display
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Library size |
107 |
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Transformation required |
Yes
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Mechanism |
Phage display systems can be grouped into two classes: true phage vectors and phagemid vectors. In both cases, the protein to be displayed is fused to the capsid protein. |
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Evolution |
Typically the phage library screening entails several (usually three) consecutive rounds of panning and phage amplification before the selected phage and the polypeptide that they present are individually analyzed. |
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Protein displayed |
Fv, scFv or Fab fragments |
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Proteins to be displayed |
Soluble, non-toxic, compatible with crossing membranes |
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Surface anchorage |
Capsid proteins |
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Properties of protein enhanced |
Affinity, enzymatic activity, stability, folding, selection from cDNA libraries |
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Stability |
Stable |
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Applications |
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Advantages |
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Disadvantages |
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Membrane protein research |
Compatible Nogo-66 - monotopic membrane protein (7,5 kDa) |
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References |
1. Imai, S. et al. Development of an antibody proteomics system using a phage antibody library for efficient screening of biomarker proteins. Biomaterials 32, 162-169 (2011). 3. Paschke, M. Phage display systems and their applications. Applied Microbiology and Biotechnology 70, 2-11 (2005). |
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