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

다양한 하이드로겔 매트릭스에 CFPS 반응 임베딩

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.

저자 스포트라이트: 하이드로겔에 cell-free 단백질 합성 반응을 포함하기 위한 새로운 접근법 

거시적 규모의 하이드로겔에 cell-free 단백질 합성 반응을 임베딩하는 방법

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  

배치 가능한 하이드로겔에서 Cell-Free 단백질 합성

얼음 위의 1.5 밀리리터 마이크로 원심 분리 튜브에서 10 마이크로 리터의 무 세포 추출물과 4 마이크로 그램의 플라스미드 DNA 및 25 마이크로 리터의 2X 무 세포 단백질 합성 완충액을 결합합니다. 이중 증류수로 총 부피 50 마이크로리터를 만들어 무세포 단백질 합성 용액을 제조한다. 0.75 그램의 아가로스를 칭량하고, 이를 100 밀리리터의 이중-증류수 완충액에 첨가하여 0.75% 아가로스를 제조한다.마이크로파는 0.75% 아가로스를 고출력에서 30초 동안 파열하고, 용융된 아가로스의 50 마이크로리터를 1.5 밀리리터 마이크로원심분리 튜브 또는 원하는 모양의 몰드에 피펫팅한다. 용융된 아가로스를 섭씨 50도로 설정된 열 블록 상에 놓고, 아가로스가 냉각되나 중합되지 않도록 한다. 이어서, 용융된 아가로스를 무세포 단백질 합성 용액과 피펫팅하고 피펫 팁으로 교반하여 혼합한다.겔을 실온으로 식히고 약 2분 동안 중합합니다. 중합된 아가로스를 주걱으로 1.5-밀리리터 마이크로원심분리 튜브로 옮기고 액체 질소에서 플래시 동결시킨다. 급속 냉동 하이드로겔을 섭씨 영하 80도의 보관 장소에 1시간 동안 보관합니다.그런 다음 미세 원심 분리기 튜브 뚜껑을 제거하십시오. 튜브를 왁스 필름으로 덮고 필름을 뚫어 수분이 건조되도록 합니다. 동결 건조기의 온도를 섭씨 영하 20도, 압력을 0.1밀리바로 설정하고 무세포 단백질 합성 장치를 18시간 또는 하룻밤 동안 동결 건조합니다.동결건조 장치를 과량의 액체 없이 50 마이크로리터의 이중 증류수로 30분 동안 재수화시키고, 주걱을 사용하여 겔을 흑색 384-웰 마이크로타이터 플레이트로 옮긴다. 마지막으로 마이크로타이터 플레이트를 플레이트 리더에 놓고 형광 검출 및 분석을 위해 화면에 표시된 플레이트 리더 설정을 사용합니다. E.coli 세포 용해물을 사용한 하이드로겔에서 eGFP 및 mCherry의 cell-free 단백질 합성이 이 그림에 나와 있습니다.0.75% 아가로스 겔은 4마이크로그램의 eGFP 또는 mCherry 템플릿 또는 4마이크로그램의 eGFP 및 mCherry 템플릿을 사용하여 DNA 템플릿 없이 제조되었습니다. 두 채널의 오버레이도 표시되며 오버레이에는 차동 간섭 대비 이미지가 포함됩니다. 그 결과 아가로스에서 mCherry와 eGFP의 동시 발현이 확인되었습니다.

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. ...