Noireaux Lab
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What, Why and How?
My lab has developed a cell-free transcription-translation (TXTL) platform to construct biochemical systems in vitro by executing synthetic gene circuits. Unlike the other cell-free expression systems, our platform is based on an E. coli extract that uses the endogenous TX and TL machineries. The circuits (plasmids or linear DNA) are executed in a cell-free TXTL mix entirely prepared in our lab. This cell-free system is available under the name myTXTL at Arbor Biosciences. Our research is based on this unique system and includes: (I) prototyping regulatory elements and circuits, (II) quantitative biology of self-assembly with phages as models, (III) bottom-up construction of a minimal cell, (IV) application to biotechnologies and medicine. Our work is both fundamental and applied and covers the research areas of synthetic biology and quantitative biology such as biological physics.
Cell-free expression systems
Cell-free protein synthesis was developed in the 60s to understand the process of protein synthesis in living organisms. In vitro protein synthesis had an immediate impact with the elucidation of the genetic code (1). In the 70s, DNA-dependent cell-free expression became a research tool to analyze gene products and to unravel the regulation of natural genetic elements such as the E. coli lactose (2) and tryptophan (3) operons. The development of highly efficient hybrid cell-free expression systems in the early 90s marked a turning point for this technology (4). Cell-free TXTL systems, optimized for large-scale protein synthesis as an alternative to the recombinant protein technology (5, 6), are used in an increasing number of applications in biotechnology, industry and proteomics (7-9).
With the emergence of synthetic biology, a new generation of cell-free TXTL systems has been engineered. The construction of biological systems in test tubes using DNA programs provides a means to study biochemical processes in isolation, with a greater level of control and a greater freedom of design compared to in vivo. In addition to increasing our knowledge of the molecular repertoire found in biology, constructing information-based biochemical systems in vitro offers the possibility of expanding the capabilities of existing biological systems (10). Elementary gene circuits (11, 12), pattern formation (13) and prototypes of artificial cells (14, 15) have been engineered with cell-free TXTL systems. Cell-free synthetic biology is a rapidly growing research area.

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(I) Prototyping



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First, we demonstrated that cell-free TXTL using the E. coli endogenous TX machinery (core RNAP + housekeeping sigma factor 70) is as efficient as the conventional T7 bacteriophage systems (16). Methods to tune the mRNA and protein degradation rates were added to this system (17) so as to change the dynamics of expression. A model of cell-free protein synthesis was published (18), in collaboration with the Bar-Ziv lab at the Weizmann Institute of Sciences. We then developed a platform that recapitulates the entire transcription scheme of E. coli (12). The primary sigma factor 70 is used to cascade any of the six other sigma factors 19, 24, 28, 32, 38, 54-NtrC, as well the T7 and T3 RNA polymerases. Hundreds of circuit parts are available from E. coli to design, build and test synthetic circuits in vitro. We developed new metabolisms to energize TXTL up to 2 mg/ml in batch mode (19) and published an improved TXTL toolbox 2.0 (20). The cell-free TXTL toolbox 2.0 is used to prototype regulatory elements (such as riboregulators or CRISPR guide RNAs) and gene circuits in vitro, such as the multiple stage cascade shown in Figure 1.

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Figure 1: Left: a switch was constructed with bacteriophage promoters using a commercial kit. The circuit is composed of 3 plasmids and two stages. In the first stage, the phage T7 RNA polymerase and the E. coli lac repressor are expressed from SP6 promoters (The phage SP6 RNA polymerase is added to the TXTL reaction). In the second stage, the expression of the luminescent reporter protein firefly Luciferase is activated by the T7 RNA polymerase and/or repressed by the lac repressor. The repression can be inhibited by addition of IPTG, an inhibitor of the lac repressor. This circuit was published in (11). Right: a multiple stage cascade composed of 5 stages. This circuit was constructed with the new all E. coli cell-free TXTL system specifically developed for cell-free synthetic biology. The solid arrows show the cascades, the dotted lines represent the negative feedback due to the competition between sigma factors. This circuit was published in (12) and (20).

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(II) Self-Assembly

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Recently, we challenged the system by testing large natural DNA programs. First, we determined that the maximum size of the genetic DNA program that can be executed in a test tube is about 150-250 genes, based on the toolbox 2.0 performances (20). We expressed the T7 phage (40 kbp, about 60 genes) and observed its complete synthesis. In addition we showed that the phage DNA genome is replicated (21). It is the first time that a living entity was entirely synthesized in vitro from the expression of its genome. This work is not limited to the bacteriophage T7, the phages MS2 and phiX174 can also be synthesized in vitro (20).
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Figure 2: Left: a schematic that recapitulates the steps of phage expression and synthesis in a test tube. The phage T7 has its own RNA polymerase and its own DNA polymerase. We demonstrated that the phage T7 genome was replicated concurrently with its synthesis. This work was published in (20) and (21). Right: an electron microscopy image of T7 phages synthesized in cell-free TXTL reactions


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(III) Synthetic cell
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One of the most challenging goals in cell-free synthetic biology is the bottom up construction minimal cells. Different types of cell analogs have been proposed. Our approach consists of encapsulating the TXTL system into cell-sized synthetic liposomes. The liposomes are programmed with gene circuits towards self-reproduction, by achieving cell functions such as membrane permeability (14) and cytoskeleton (22). This approach helps us understand the links between information, self-assembly and metabolism (23).
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Figure 3: Left: cell-free TXTL was used to express the toxin alpha-hemolysin into cell-sized liposomes. The reporter protein eGFP was fused to the toxin to visualized the interaction of the pore-forming protein with the phospholipid membrane. The toxin forms a membrane channel of 1.3 nm diameter that allows exchanges of small nutrients and reaction byproducts between the liposome and the external medium, which results in the extension of TXTL expression inside the liposomes. This work was described in (14) and (20). Right: the all E. coli toolbox was used to express the MreB and MreC cytoskeletal protein inside liposomes at the same time. MreB polymerizes at the membrane through its interaction with MreC, a membrane protein. These observations were published in (19).

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(IV) Biotechnology and Medicine
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In collaboration with other labs and companies, we are using our TXTL system to develop molecules and devices, such as solid state probes, for application in biotechnologies and medicine. We use the fast prototyping capabilities of TXTL to test and select peptides and proteins with therapeutic functions.
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1.     Nirenberg, M. (2004) Historical review: Deciphering the genetic code--a personal account, Trends Biochem Sci 29, 46-54.
2.     Chambers, D. A., and Zubay, G. (1969) The stimulatory effect of cyclic cAMP on DNA-directed synthesis of beta-galactosidase in a cell-free system, Proc Natl Acad Sci U S A 63, 118-122.
3.     Zalkin, H., Yanofsky, C., and Squires, C. L. (1974) Regulated in vitro synthesis of Escherichia coli tryptophan operon messenger ribonucleic acid and enzymes, J Biol Chem 249, 465-475.
4.     Nevin, D. E., and Pratt, J. M. (1991) A coupled in vitro transcription-translation system for the exclusive synthesis of polypeptides expressed from the T7 promoter, FEBS Let 291, 259-263.
5.     Kigawa, T., Yabuki, T., Yoshida, Y., Tsutsui, M., Ito, Y., Shibata, T., and Yokoyama, S. (1999) Cell-free production and stable-isotope labeling of milligram quantities of proteins, FEBS Let 442, 15-19.
6.     Jewett, M. C., and Swartz, J. R. (2004) Rapid expression and purification of 100 nmol quantities of active protein using cell-free protein synthesis, Biotechnol Prog 20, 102-109.
7.     Spirin, A. S. (2004) High-throughput cell-free systems for synthesis of functionally active proteins, Trends Biotechnol 22, 538-545.
8.     Katzen, F., Chang, G., and Kudlicki, W. (2005) The past, present and future of cell-free protein synthesis, Trends Biotechnol 23, 150-156.
9.     Swartz, J. (2006) Developing cell-free biology for industrial applications, J Ind Microbiol Biotechnol 33, 476-485.
10.   Hodgman, C. E., and Jewett, M. C. (2012) Cell-free synthetic biology: Thinking outside the cell, Metab Eng, 14(3) 261-269
11.   Noireaux, V., Bar-Ziv, R., and Libchaber, A. (2003) Principles of cell-free genetic circuit assembly, Proc Natl Acad Sci U S A 100, 12672-12677.
12.   Shin, J., Noireaux, V. (2011) An E. coli cell-free expression toolbox: application to synthetic gene circuits and artificial cells., ACS Synthetic Biology 1, 29-41.
13.   Isalan, M., Lemerle, C., and Serrano, L. (2005) Engineering gene networks to emulate Drosophila embryonic pattern formation, PLoS Biol 3(3), e64.
14.   Noireaux, V., and Libchaber, A. (2004) A vesicle bioreactor as a step toward an artificial cell assembly, Proc Natl Acad Sci U S A 101, 17669-17674.
15.   Ishikawa, K., Sato, K., Shima, Y., Urabe, I., and Yomo, T. (2004) Expression of a cascading genetic network within liposomes, FEBS Lett 576, 387-390.
16.   Shin, J., Noireaux, V. (2010) Efficient cell-free expression with the endogenous E. Coli RNA polymerase and sigma factor 70, J. Biol. Eng. 4:8.
17.   Shin, J., Noireaux, V. (2010) Study of messenger RNA inactivation and protein degradation in an Escherichia coli cell-free expression system, J. Biol. Eng. 4:9.
18.   Karzbrun, E., Shin, J., Bar-Ziv, R., Noireaux, V. (2011) Coarse grained dynamics of protein synthesis in a cell-free system, PRL 106(4), 048104.
19.   Caschera, F. and Noireaux, V. (2013) Synthesis of 2.3 mg/ml of protein with an all Escherichia coli cell-free transcription-translation system. Biochimie 99, 162-168.
20.   Garamella, J., Marshall, R., Rustad, M., Noireaux, V. (2016) The all E. coli Cell-free TX-TL Toolbox 2.0: a platform for cell-free synthetic biology. ACS Synthetic Biology DOI: 10.1021/acssynbio.5b00296.
21.   Shin, J., Jardine, P., Noireaux, V. (2012) Genome replication, synthesis and assembly of the bacteriophage T7 in a single cell-free reaction, ACS Synthetic Biology 1(9), 408-413.
22.   Maeda, Y., Nakadai, T., Shin, J., Uryu, K., Noireaux, V., Libchaber, A. (2012) Assembly of MreB Filaments on Vesicular Membranes: A Synthetic Biology Approach. ACS Synthetic Biology 1(2), 53-59.
23.   Noireaux, V., Maeda, Y., Libchaber, A. (2011) Development of an artificial cell, from self-organization to computation and self-reproduction. Proc. Nat. Acad. Sci. USA 108(9), 3473-3480.


Noireaux lab is or was sponsored by: DARPA, ONR, NSF, BSF, HFSP and UMN.

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