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Cell-free Systems

Cell-free systems allow for the reliable and consistent expression of recombinant proteins outside of a living cell, bypassing issues with genetic regulation and cellular noise [1].

Such systems are advantageous over cell-based synthetic biology due to the:

• - Capability of tolerating toxins normally detrimental to the cell
• - Ability to direct all energy resources to the application, increasing the freedom of design
• - Inherent feature of reduced bio-contamination, as components do not replicate mutate or evolve
• - Easy control of transcription and translation in an open environment
• - Easy incorporation of unnatural amino acids
• - Capacity to modulate the environment for optimal protein expression
• - Rapid design-build-test cycle
• - Proficiency of using both linear and circular template DNA

Applications

Emerging as a new platform for synthetic biology, cell-free systems have shown potential for use in a variety of applications exemplifying the utility of such systems [2,3].

Types of Cell-free Systems

Cell-free systems include all of the necessary biomachinery for protein production, utilizing cell extracts or a purified reconstituted system.

Cell Lysates

Purified Reconstituted

Our System

It is our goal to make a completely customizable and accessible cell-free system, that is inherently safe and user-friendly. To accomplish this, we developed Next Vivo, a standardized and modular system that contains all of the necessary biomachinery for protein production.

We designed, in BioBrick standard, an open-source collection of parts for cell-free protein synthesis. Next Vivo allows individual user groups to select which components they want in their system and leave out any additional factors of their choosing. As such, future modules can be added to the system with ease, creating a wide variety of customizable TX-TL kits.

Specifically, we aim to over-express all TX-TL components simultaneously and pool the resulting cell lysates for co-purification, providing a simple method for producing the system. Using our approach, the tRNA and ribosomes will bind to an MS2 coat protein/nickel complex, and the TX-TL proteins will bind directly to the nickel affinity chromatography resin. In this way, components can be purified in batch and a tailored cell-free system can be created on demand.

Biocontainment

A modular and accessible cell-free platform enables the development of re-coding technologies leading to the creation of a modified codon table. Re-coded systems are unable to be horizontally transferred to living system, providing an intrinsic form of biocontainment and preventing accidental environmental release. We have created a nucleic acid sequence modification tool that will produce re-coded sequences to support such developments.

Biosecurity

With the development of re-coded systems comes additional consideration of the impact on biosecurity and the potential for synthesizing compounds undetected by current screening methods. We propose a solution to this problem by providing software tools to combat the potential misuse of our system and highlight the significant implications of cell-free synthetic biology.

Education

Next Vivo provides a useful learning tool.Taking advantage of the non-proliferating nature, we have developed simplified protocols for the educational system to teach new users the basic concepts of synthetic biology.

Recent Advancements

Recent work by Shephard et al. [23] published in August of 2017 developed a 30-cistron translation factor module to provide an affordable and scalable method for obtaining a reconstituted TX-TL system. Focusing only on 30 of the 31 translation factors needed for protein synthesis, other necessary factors such as tRNA and ribosomes were not included in their design. Our work improves on this by providing a strategy for purifying and obtaining tRNA and ribosomes, in addition to all of the factors required for transcription and translation.

References

• [1] Hodgman, C.E. and M.C. Jewett, Cell-Free Synthetic Biology: Thinking Outside the Cell. Metabolic Engineering, 2012. 14(3): p. 261-269.
• [2] Chong, S., Overview of cell-free protein synthesis: historic landmarks, commercial systems, and expanding applications. Curr Protoc Mol Biol, 2014. 108: p. 16.30.1-11.
• [3] Smith, M.T., et al., The emerging age of cell-free synthetic biology. FEBS Letters, 2014. 588(17): p. 2755-2761.
• [4] Caschera, F. and V. Noireaux, Compartmentalization of an all-E. coli Cell-Free Expression System for the Construction of a Minimal Cell. Artificial Life, 2016. 22(2): p. 185-195.
• [5] Jia, H., et al., Cell-free protein synthesis in micro compartments: building a minimal cell from biobricks. N Biotechnol, 2017. 39(Pt B): p. 199-205.
• [6] France, S.P., et al., Constructing Biocatalytic Cascades: In Vitro and in Vivo Approaches to de Novo Multi-Enzyme Pathways. ACS Catalysis, 2017. 7(1): p. 710-724.
• [7] Jewett, M.C., et al., An integrated cell-free metabolic platform for protein production and synthetic biology. Mol Syst Biol, 2008. 4: p. 220.
• [8] Pardee, K., et al., Paper-Based Synthetic Gene Networks. Cell. 159(4): p. 940-954.
• [9] Karig, D.K., Cell-free synthetic biology for environmental sensing and remediation. Curr Opin Biotechnol, 2017. 45: p. 69-75.
• [10] Wang, L., Genetically encoding new bioreactivity. New Biotechnology, 2017. 38(Part A): p. 16-25.
• [11] Kawakami, T., et al., Directed Evolution of a Cyclized Peptoid-Peptide Chimera against a Cell-Free Expressed Protein and Proteomic Profiling of the Interacting Proteins to Create a Protein-Protein Interaction Inhibitor. ACS Chem Biol, 2016. 11(6): p. 1569-77.
• [12] Sun, Z.Z., et al., Linear DNA for rapid prototyping of synthetic biological circuits in an Escherichia coli based TX-TL cell-free system. ACS Synth Biol, 2014. 3(6): p. 387-97.
• [13] Nagumo, Y., et al., PURE mRNA display for in vitro selection of single-chain antibodies. J Biochem, 2016. 159(5): p. 519-26.
• [14] Elani, Y., R.V. Law, and O. Ces, Vesicle-based artificial cells: recent developments and prospects for drug delivery. Ther Deliv, 2015. 6(5): p. 541-3.
• [15] Menezes, A.A., et al., Grand challenges in space synthetic biology. J R Soc Interface, 2015. 12(113): p. 20150803.
• [16] Carlson, E.D., et al., Cell-Free Protein Synthesis: Applications Come of Age. Biotechnology advances, 2012. 30(5): p. 1185-1194.
• [17] Shin, J. and V. Noireaux, An E. coli Cell-Free Expression Toolbox: Application to Synthetic Gene Circuits and Artificial Cells. ACS Synthetic Biology, 2012. 1(1): p. 29-41.
• [18] Garamella, J., et al., The All E. coli TX-TL Toolbox 2.0: A Platform for Cell-Free Synthetic Biology. ACS Synthetic Biology, 2016. 5(4): p. 344-355.
• [19] Tarui, H., et al., Establishment and characterization of cell-free translation/glycosylation in insect cell (Spodoptera frugiperda 21) extract prepared with high pressure treatment. Appl Microbiol Biotechnol, 2001. 55(4): p. 446-53.
• [20] Katzen, F., G. Chang, and W. Kudlicki, The past, present and future of cell-free protein synthesis. Trends Biotechnol, 2005. 23(3): p. 150-6.
• [21] Mikami, S., et al., A human cell-derived in vitro coupled transcription/translation system optimized for production of recombinant proteins. Protein Expr Purif, 2008. 62(2): p. 190-8.
• [22] Shimizu, Y., et al., Cell-free translation reconstituted with purified components. Nature biotechnology, 2001. 19(8): p. 751-755.
• [23] Shepherd, T.R., et al., De novo design and synthesis of a 30-cistron translation-factor module. Nucleic Acids Research, 2017. 45(18): p. 10895-10905.