The physical interaction between the cytoskeleton and the cell membrane is essential in defining the morphology of living organisms. In this study, we use a synthetic approach to polymerize bacterial MreB filaments inside phospholipid vesicles. When the proteins MreB and MreC are expressed inside the liposomes, the MreB cytoskeleton structure develops at the inner membrane. Furthermore, when purified MreB is used inside the liposomes, MreB filaments form a 4-10 μm rigid bundle structure and deform the lipid vesicles in physical contact with the vesicle inner membrane. These results indicate that the fibrillation of MreB filaments can take place either in close proximity of deformable lipid membrane or in the presence of associated protein. Our finding might be relevant for the self-assembly of cytoskeleton filaments toward the construction of artificial cell systems.

This article describes the state and the development of an artificial cell project. We discuss the experimental constraints to synthesize the most elementary cell-sized compartment that can self-reproduce using synthetic genetic information. The original idea was to program a phospholipid vesicle with DNA. Based on this idea, it was shown that in vitro gene expression could be carried out inside cell-sized synthetic vesicles. It was also shown that a couple of genes could be expressed for a few days inside the vesicles once the exchanges of nutrients with the outside environment were adequately introduced. The development of a cell-free transcription/translation toolbox allows the expression of a large number of genes with multiple transcription factors. As a result, the development of a synthetic DNA program is becoming one of the main hurdles. We discuss the various possibilities to enrich and to replicate this program. Defining a program for self-reproduction remains a difficult question as nongenetic processes, such as molecular self-organization, play an essential and complementary role. The synthesis of a stable compartment with an active interface, one of the critical bottlenecks in the synthesis of artificial cell, depends on the properties of phospholipid membranes. The problem of a self-replicating artificial cell is a long-lasting goal that might imply evolution experiments.

Cell-free protein synthesis is becoming a serious alternative to cell-based protein expression. Cell-free systems can deliver large amounts of cytoplasmic recombinant proteins after a few hours of incubation. Recent studies have shown that membrane proteins can be also expressed in cell-free reactions and directly inserted into phospholipid membranes. In this work, we present a quantitative method to study in real time the concurrent cell-free expression and insertion of membrane proteins into phospholipid bilayers. The pore-forming protein α-hemolysin, fused to the reporter protein eGFP, was used as a model of membrane protein. Cell-free expression of the toxin in solution and inside large synthetic phospholipid vesicles was measured by fluorometry and fluorescence microscopy respectively. A quartz crystal microbalance with dissipation was used to characterize the interaction of the protein with a supported phospholipid bilayer. The cell-free reaction was directly incubated onto the bilayer inside the microbalance chamber while the frequency and the dissipation signals were monitored. The presence of pores in the phospholipid bilayer was confirmed by atomic force microscopy. A model is presented which describes the kinetics of adsorption of the expressed protein on the phospholipid bilayer. The combination of cell-free expression, fluorescence microscopy and quartz crystal microbalance-dissipation is a new quantitative approach to study the interaction of membrane proteins with phospholipid bilayers.

A large amount of recombinant proteins can be synthesized in a few hours with Escherichia coli cell-free expression systems based on bacteriophage transcription. These cytoplasmic extracts are used in many applications that require large-scale protein production such as proteomics and high throughput techniques. In recent years, cell-free systems have also been used to engineer complex informational processes. These works, however, have been limited by the current available cell-free systems, which are not well adapted to these types of studies. In particular, no method has been proposed to increase the mRNA inactivation rate and the protein degradation rate in cell-free reactions. The construction of in vitro informational processes with interesting dynamics requires a balance between mRNA and protein synthesis (the source), and mRNA inactivation and protein degradation (the sink).

Escherichia coli cell-free expression systems use bacteriophage RNA polymerases, such as T7, to synthesize large amounts of recombinant proteins. These systems are used for many applications in biotechnology, such as proteomics. Recently, informational processes have been reconstituted in vitro with cell-free systems. These synthetic approaches, however, have been seriously limited by a lack of transcription modularity. The current available cell-free systems have been optimized to work with bacteriophage RNA polymerases, which put significant restrictions to engineer processes related to biological information. The development of efficient cell-free systems with broader transcription capabilities is required to study complex informational processes in vitro.

For work involving proteins that require disulfide bond formation we recommend our myTXTL Antibody/DS Kit. It is possible that some proteins with just 1 disulfide bond, such as VHH/Nanobodies, may express and show activity in the myTXTL Pro Kit, but generally speaking disulfide bond containing proteins will have the highest yields and best activity in the myTXTL Antibody/DS Kit.

If you were using any of our 3 original myTXTL kits, those will be discontinued in August 2024. The myTXTL Pro Kit is now the kit that will serve your needs as it has all the properties of the old kits combined into one: it supports linear and plasmid DNA as well as all E. coli promoters and also enables T7 promoter-based expression through the addition of the Pro Helper Plasmid that expresses the T7 RNA polymerase. All Pro Kits include the Pro Helper Plasmid and a T7 deGFP Positive Control Plasmid.

If you are interested in expressing proteins that contain disulfide bonds, the myTXTL Antibody/DS Kit is what you need.

Yes, myTXTL reactions have been conducted from 2 to 100 uL volume, but above 50 uL we recommend shaking and/or switching to a reaction vessel with higher surface:volume ratio to allow proper oxygenation of the reaction mix. myTXTL reactions are very sensitive to the amount of dissolved oxygen. If using over 50 uL volume, a flat-bottomed ELISA, deep well plate or tissue culture plate may be advised along with shaking at 650 RPM. We advise testing such a setup with one of the positive control plasmids, such as T7 deGFP. The key is to balance oxygenation and avoid the reactions drying out due to too much surface area. Please refer to the appropriate kit manual for additional guidance on scaling up reaction volume.

As the myTXTL platform relies on the endogenous transcription and translation machinery of E. coli, a functional gene cassette must contain a promoter that can be transcribed by E. coli RNA polymerase and associated transcription factors (primarily Sigma 70) or by a T7/T3 RNA polymerase if those polymerases are expressed from a helper plasmid (available in our Toolkit). The ribosomal binding site should also be compatible with E. coli translation machinery. For more general advice on how to construct a functional gene cassette, please refer to the current myTXTL handbook.

myTXTL supports all promoters used in E. coli protein expression, including promoters that rely on the endogenous E. coli transcription machinery and those that require a separate RNA polymerase such as T7. All kits come with a Helper plasmid to express the T7 RNA polymerase and enable transcription from T7 promoters. If a promoter is used that is recognized by the E. coli RNA polymerase, then this helper plasmid is not needed. If using a plasmid with an inducible promoter, such as the pET vectors, you need to add your inducer IPTG at 1 mM to the reaction and you may want to explore plasmid concentration in the range of y your inducer like IPTG if it is an inducible promoter.