Name: Cell-Free Protein Synthesis in Deployable Hydrogels
Uploaded: 2023-06-23T14:20:00
Description: Watch this Scientific Journal Video about Cell-Free Protein Synthesis in Deployable Hydrogels at JoVE.com

cell-free protein synthesis in deployable hydrogels

Incorporamento di reazioni CFPS in una varietà di matrici di idrogel

Synthetic biology integrates diverse engineering disciplines to design and engineer biologically based parts, devices, and systems that can perform functions that are not found in nature. Most synthetic biology approaches are still bound to living cells. By contrast, cell-free synthetic biology systems facilitate unprecedented levels of control and freedom in design, enabling increased flexibility and a shortened time for engineering biological systems while eliminating many of the constraints of traditional cell-based gene expression methods1,2,3. CFPS is being used in an increasing number of applications across numerous disciplines, including constructing artificial cells, prototyping genetic circuits, developing biosensors, and producing metabolites4,5,6. CFPS has also been particularly useful for producing recombinant proteins that cannot be readily expressed in living cells, such as aggregation-prone proteins, transmembrane proteins, and toxic proteins6,7,8.
CFPS is typically performed in liquid reactions. This, however, may restrict their deployment in some situations, as any liquid cell-free device must be contained within a reaction vessel. The rationale for the development of the methods presented here was to provide robust protocols for embedding cell-free synthetic biology devices into hydrogels, not as a protein production platform per se but, instead, to allow the use of hydrogels as a physical chassis for the deployment of cell-free devices beyond the laboratory. The use of hydrogels as CFPS chassis has several advantages. Hydrogels are polymeric materials that, despite a high water content (sometimes in excess of 98%), possess solid properties9,10,11. They have uses as pastes, lubricants, and adhesives and are present in products as diverse as contact lenses, wound dressings, marine adhesive tapes, soil improvers, and baby diapers9,11,12,13,14. Hydrogels are also under active investigation as drug delivery vehicles9,15,16,17. Hydrogels may also be biocompatible, biodegradable, and possess some stimuli responses of their own9,18,19,20. Therefore, the goal here is to create a synergy between molecular biology-derived functionality and materials science. To this end, efforts have been made to integrate cell-free synthetic biology with a range of materials, including collagen, laponite, polyacrylamide, fibrin, PEG-peptide, and agarose11,21,22, as well as to coat surfaces of glass, paper, and cloth11,23,24 with CFPS devices. The protocols presented here demonstrate two methods for embedding CFPS reactions in macro-scale (i.e., &#62;1 mm) hydrogel matrices, using agarose as the exemplar material. Agarose was chosen due to its high water absorption capacity, controlled self-gelling properties, and tuneable mechanical properties11,24,25,26. Agarose also supports functional CFPS, is cheaper than many other hydrogel alternatives, and is biodegradable, making it an attractive choice as an experimental model system. These methods have, however, been previously demonstrated as appropriate for embedding CFPS in a range of alternative hydrogels11. Considering the wide range of applications of hydrogels and the functionality of CFPS, the methods demonstrated here can provide a basis from which researchers are able to develop biologically enhanced hydrogel materials suited to their own ends.
In previous studies, microgel systems with a size range of 1 &#181;m to 400 &#181;m have been used to perform CFPS in hydrogels submerged in reaction buffer23,27,28,29,30,31. The requirement to submerge hydrogels within CFPS reaction buffers, however, limits opportunities for their deployment as materials in their own right. The protocols presented here allow CFPS reactions to occur within hydrogels without the need to submerge the gels in reaction buffers. Second, the use of macroscale gels (between 2 mm and 10 mm in size) allows the study of the physical interaction between hydrogels and cell-free gene expression. For example, with this technique, it is possible to study how the hydrogel matrix affects CFPS reactions11 and how CFPS reactions can impact the hydrogel matrix31. Larger sizes of hydrogels also allow for the development of novel, bio-programmable materials32. Finally, by embedding CFPS reactions into hydrogels, there is also a potential reduction in the requirement for plastic reaction vessels. For the deployment of cell-free sensors, this has clear advantages over devices dependent on plasticware. Taken together, embedding CFPS reactions in hydrogels provides several advantages for the deployment of cell-free devices beyond the laboratory.
The overall goal of the methods presented here is to allow the operation of CFPS reactions within hydrogel matrices. Two different methods are demonstrated for embedding cell-free protein production reactions in macro-scale hydrogel materials (Figure 1). In Method A, CFPS components are introduced to lyophilized agarose hydrogels to form an active system. In Method B, molten agarose is mixed with CFPS reaction components to form a complete CFPS hydrogel system, which is then lyophilized and stored until needed. These systems can be rehydrated with a volume of water or buffer and analyte to commence the reaction.
This study uses Escherichia coli cell lysate-based systems. These are some of the most popular experimental CFPS systems, as E. coli cell lysate preparation is simple, inexpensive, and achieves high protein yields. The cell lysate is complemented with the macromolecular components needed to perform transcription and translation, including ribosomes, tRNAs, aminoacyl-tRNA synthetases, and initiation, elongation, and termination factors. Specifically, this paper demonstrates the production of eGFP and mCherry in agarose hydrogels using E. coli cell lysates and monitors the appearance of fluorescence using a plate reader and confocal microscopy. Representative results for the microtiter plate reader can be seen in Whitfield et al.31, and the underlying data are publicly available33. Further, the expression of fluorescent proteins throughout the gels is confirmed using confocal microscopy. The two protocols demonstrated in this paper allow for the assembly and storage of CFPS-based genetic devices in materia with the ultimate goal of creating a suitable physical environment for the distribution of cell-free gene circuitry in a manner supportive of field deployment.

Autore in primo piano: un nuovo approccio per l'incorporazione di reazioni di sintesi proteica cell-free negli idrogel 

Metodi per incorporare reazioni di sintesi proteica cellulo-free in idrogel su macroscala

We know living systems continuously integrate information from many different sources and that these signals trigger complex and subtle responses. Much of this information processing occurs at the molecular level. We're interested in operating these processes within materials, but without cells.The purpose is to give materials novel functionality. Challenges in this work are primarily associated with the cell-free protein synthesis reaction, rather than working with hydrogel. Cell reaction require high quality cell lysate as well as high quality and purity DNA to function well.This protocol allows researchers to embed molecular biology reactions into a physical biodegradable material that can be taken outside of the laboratory. Also, because the reaction output interacts directly with the material chassis, there is potential for the development of materials with novel sensing and response capabilities. In short-term, we see these methods as providing a novel route to the deployment of cell-free diagnostic devices.This protocol allows a number of devices to be created and spatially organized in a manner that is not possible for liquid reactions. Our future interests are twofold. First, we are interested in expanding the nature of the hydrogels we use to bring more material functionality to the work.Second, we are interested in increasing the complexity and performance of the gene networks we operate in gels, expanding the biological functionality of these systems.

Watch this Scientific Journal Video about Cell-Free Protein Synthesis in Deployable Hydrogels at JoVE.com

Cell-Free Protein Synthesis in Deployable Hydrogels  

Sintesi proteica priva di cellule in idrogel dispiegabili

In una provetta da microcentrifuga da 1,5 millilitri su ghiaccio, combina 10 microlitri di estratto privo di cellule con quattro microgrammi di DNA plasmidico e 25 microlitri di tampone di sintesi proteica 2X privo di cellule. Preparare fino a un volume totale di 50 microlitri con acqua bidistillata per preparare la soluzione di sintesi proteica priva di cellule. Pesare 0,75 grammi di agarosio e aggiungerlo a 100 millilitri di tampone di acqua bidistillata per preparare lo 0,75% di agarosio.Cuocere a microonde l'agarosio allo 0,75% in raffiche di 30 secondi ad alta potenza e pipettare 50 microlitri di agarosio fuso in provette da microcentrifuga da 1,5 millilitri o in stampi della forma desiderata. Metti l'agarosio fuso su un blocco termico impostato a 50 gradi Celsius e lascia raffreddare l'agarosio ma non polimerizzare. Quindi, miscelare l'agarosio fuso con la soluzione di sintesi proteica priva di cellule pipettando e mescolando con il puntale della pipetta.Lasciare raffreddare i gel a temperatura ambiente e polimerizzare per circa due minuti. Trasferire l'agarosio polimerizzato in provette da microcentrifuga da 1,5 millilitri con una spatola e congelare in azoto liquido. Metti gli idrogel congelati a meno 80 gradi Celsius per un'ora.Successivamente, rimuovere i coperchi delle provette per microcentrifuga. Coprire i tubi con una pellicola di cera e forare la pellicola per consentire all'umidità di asciugarsi. Impostare la temperatura del liofilizzatore a meno 20 gradi Celsius e la pressione a 0,1 millibar e liofilizzare i dispositivi di sintesi proteica senza cellule per 18 ore o per tutta la notte.Reidratare i dispositivi liofilizzati per 30 minuti con 50 microlitri di acqua bidistillata senza liquidi in eccesso e trasferire i gel su una piastra nera per microtitolazione a 384 pozzetti utilizzando una spatola. Infine, posizionare la piastra per microtitolazione su un lettore di piastre e utilizzare le impostazioni del lettore di piastre mostrate sullo schermo per il rilevamento e l'analisi della fluorescenza. In questa figura è mostrata la sintesi proteica priva di cellule di eGFP e mCherry in un idrogel utilizzando lisati cellulari di E.coli.I gel di agarosio allo 0,75% sono stati preparati senza stampo di DNA con quattro microgrammi di eGFP o mCherry o con quattro microgrammi di eGFP e mCherry. Viene inoltre mostrata una sovrapposizione dei due canali e la sovrapposizione include l'immagine del contrasto di interferenza differenziale. I risultati hanno confermato la co-espressione di mCherry ed eGFP nell'agarosio.

Research

JoVE Journal

Biology

Cell-Free Protein Synthesis in Deployable Hydrogels

Methods for Embedding Cell-Free Protein Synthesis Reactions in Macro-Scale Hydrogels

Synthetic gene networks provide a platform for scientists and engineers to design and build novel systems with functionality encoded at a genetic level. ...