protocols-for-implementing-an-escherichia-coli-based-tx-tl-cell-free

Protocols for Implementing an <em>Escherichia coli</em> Based TX-TL Cell-Free Expression System for Synthetic Biology

Ideal cell-free expression systems can theoretically emulate an in vivo cellular environment in a controlled in vitro platform.1 This is useful for ...

University of Minnesota

We consider the problem of optimizing a liposomal drug formulation: a complex chemical system with many components (e.g., elements of a lipid library) that interact nonlinearly and synergistically in ways that cannot be predicted from first principles.

Automated discovery of novel drug formulations using predictive iterated high throughput experimentation.

The fusion between synthetic vesicles is an interesting mechanism for the stepwise construction of vesicle compartments for origins of life models and synthetic biology. In this communication, we report an innovative study on the not well-known case of fusion between oppositely charged vesicles, in particular by using fatty acid vesicles and DDAB as cationic surfactant. By combining fluorescence, turbidity vs. time profiles and vesicle size distribution obtained by dynamic light scattering, we show that POPC/oleate 1/4 mol/mol anionic vesicles can be fused with POPC/DDAB 1/1 mol/mol cationic vesicles with about 20% yield. Other non-fusion processes also occur, vesicle fusion being more effective by reducing the ionic strength of the buffer. This study also contributes to clarify the term "vesicle fusion", which is not always properly used in describing reactivity among vesicles.

Reactivity and fusion between cationic vesicles and fatty acid anionic vesicles.

We have developed a method to evaluate the fusion process of giant vesicles using a fluorescence-activated cell sorter (FACS). Three fluorescent markers and FACS technology were used to evaluate the extent of association and fusion of giant vesicles. Two fluorescent markers encapsulated in different vesicle populations were used as association markers; when these vesicles associate, the two independent markers should be observed simultaneously in a single detection event. The quenched fluorescent marker and the dequencher, which were encapsulated in separate vesicle populations, were used as the fusion marker. When the internal aqueous solutions mix, the quenched marker is liberated by the dequencher and emits the third fluorescent signal. Although populations of pure POPC vesicles showed no detectable association or fusion, the same populations, oppositely charged by the exogenous addition of charged amphiphiles, showed up to 50% association and 30% fusion upon population analysis of 100,000 giant vesicles. Although a substantial fraction of the vesicles associated in response to a small amount of the charged amphiphiles (5% mole fraction compared to POPC alone), a larger amount of the charged amphiphiles (25%) was needed to induce vesicle fusion. The present methodology also revealed that the association and fusion of giant vesicles was dependent on size, with larger giant vesicles associating and fusing more frequently.

Detection of association and fusion of giant vesicles using a fluorescence-activated cell sorter.

Biological systems contain complex metabolic pathways with many nonlinearities and synergies that make them difficult to predict from first principles. Protein synthesis is a canonical example of such a pathway. Here we show how cell-free protein synthesis may be improved through a series of iterated high-throughput experiments guided by a machine-learning algorithm implementing a form of evolutionary design of experiments (Evo-DoE). The algorithm predicts fruitful experiments from statistical models of the previous experimental results, combined with stochastic exploration of the experimental space. The desired experimental response, or evolutionary fitness, was defined as the yield of the target product, and new experimental conditions were discovered to have âˆ¼ 350% greater yield than the standard. An analysis of the best experimental conditions discovered indicates that there are two distinct classes of kinetics, thus showing how our evolutionary design of experiments is capable of significant innovation, as well as gradual improvement.

Coping with complexity: machine learning optimization of cell-free protein synthesis.

The membrane properties of phospholipid vesicles can be manipulated to both regulate and initiate encapsulated biochemical reactions and networks. We present evidence for the inhibition and activation of reactions encapsulated in vesicles by the exogenous addition of charged amphiphiles. While the incorporation of cationic amphiphile exerts an inhibitory effect, complementation of additional anionic amphiphiles revitalize the reaction. We demonstrated both the simple hydrolysis reaction of Î²-glucuronidase and the in vitro gene expression of this enzyme from a DNA template. Furthermore, we show that two vesicle populations decorated separately with positive and negative amphiphiles can fuse selectively to supply feeding components to initiate encapsulated reactions. This mechanism could be one of the rudimentary but effective means to regulate and maintain metabolism in dynamic artificial cell models.

Filippo Caschera

School of Physics and Astronomy

University of Minnesota

Protocols for Implementing an Escherichia coli Based TX-TL Cell-Free Expression System for Synthetic Biology

Filippo Caschera

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University of Minnesota

Protocols for Implementing an Escherichia coli Based TX-TL Cell-Free Expression System for Synthetic Biology

School of Physics and Astronomy