Bu malzeme Office deniz Araştırma Ödülü sayıya N00014-13-1-0074 (V.N.) tarafından desteklenen çalışma dayanır, insan Sınır Bilim Programı hibe numarası RGP0037/2015 (V.N. için) ve iki uluslu Bilim Vakfı (V.N.) 2014400 verin.

<ol>
	<li>Carlson, E. D., Gan, R., Hodgman, C. E., Jewett, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-free+protein+synthesis:+Applications+come+of+age.">Cell-free protein synthesis: Applications come of age.</a> Biotechnol Adv. , (2011).</li><li>Hodgman, C. E., Jewett, M. 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H., Kwon, Y. C., Jewett, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-standard+amino+acid+incorporation+into+proteins+using+Escherichia+coli+cell-free+protein+synthesis.">Non-standard amino acid incorporation into proteins using Escherichia coli cell-free protein synthesis.</a> Frontiers in Chemistry. 2, (2014).</li><li>Noireaux, V., Bar-Ziv, R., Libchaber, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Principles+of+cell-free+genetic+circuit+assembly.">Principles of cell-free genetic circuit assembly.</a> Proc Natl Acad Sci U S A. 100 (22), 12672-12677 (2003).</li><li>Sun, Z. Z., Yeung, E., Hayes, C. A., Noireaux, V., Murray, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Linear+DNA+for+Rapid+Prototyping+of+Synthetic+Biological+Circuits+in+an+Escherichia+coli+Based+TX-TL+Cell-Free+System.">Linear DNA for Rapid Prototyping of Synthetic Biological Circuits in an Escherichia coli Based TX-TL Cell-Free System.</a> Acs Synthetic Biology. 3 (6), 387-397 (2014).</li><li>Takahashi, M. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterizing+and+prototyping+genetic+networks+with+cell-free+transcription-translation+reactions.">Characterizing and prototyping genetic networks with cell-free transcription-translation reactions.</a> Methods. , (2015).</li><li>Shin, J., Jardine, P., Noireaux, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome+Replication,+Synthesis,+and+Assembly+of+the+Bacteriophage+T7+in+a+Single+Cell-Free+Reaction.">Genome Replication, Synthesis, and Assembly of the Bacteriophage T7 in a Single Cell-Free Reaction.</a> ACS Synthetic Biology. 1 (9), 408-413 (2012).</li><li>Garamella, J., Marshall, R., Rustad, M., Noireaux, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+All+E.+coli+TX-TL+Toolbox+2.0:+A+Platform+for+Cell-Free+Synthetic+Biology.">The All E. coli TX-TL Toolbox 2.0: A Platform for Cell-Free Synthetic Biology.</a> ACS Synth Biol. , (2016).</li><li>Karzbrun, E., Shin, J., Bar-Ziv, R. H., Noireaux, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coarse-grained+dynamics+of+protein+synthesis+in+a+cell-free+system.">Coarse-grained dynamics of protein synthesis in a cell-free system.</a> Phys Rev Lett. 106 (4), 048104 (2011).</li><li>Daube, S. S., Arad, T., Bar-Ziv, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-free+co-synthesis+of+protein+nanoassemblies:+tubes,+rings,+and+doughnuts.">Cell-free co-synthesis of protein nanoassemblies: tubes, rings, and doughnuts.</a> Nano Lett. 7 (3), 638-641 (2007).</li><li>Chen, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+immunoblot+assay+reveals+that+bacteriophage+T4+thymidylate+synthase+and+dihydrofolate+reductase+are+not+virion+proteins.">An immunoblot assay reveals that bacteriophage T4 thymidylate synthase and dihydrofolate reductase are not virion proteins.</a> J Virol. 69 (4), 2119-2125 (1995).</li><li>Sun, Z. Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protocols+for+implementing+an+Escherichia+coli+based+TX-TL+cell-free+expression+system+for+synthetic+biology.">Protocols for implementing an Escherichia coli based TX-TL cell-free expression system for synthetic biology.</a> J Vis Exp. (79), e50762 (2013).</li><li>Shin, J. N. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+cell-free+expression+with+the+endogenous+E.+coli+RNA+polymerase+and+sigma+factor+70.">Efficient cell-free expression with the endogenous E. coli RNA polymerase and sigma factor 70.</a> J Biol Eng. , (2010).</li><li>Caschera, F., Noireaux, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+amino+acid+mixtures+for+cell-free+expression+systems.">Preparation of amino acid mixtures for cell-free expression systems.</a> Biotechniques. 58 (1), 40-43 (2015).</li><li>Minton, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+can+biochemical+reactions+within+cells+differ+from+those+in+test+tubes.">How can biochemical reactions within cells differ from those in test tubes.</a> J Cell Sci. 119, 2863-2869 (2006).</li><li>Minton, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Implications+of+macromolecular+crowding+for+protein+assembly.">Implications of macromolecular crowding for protein assembly.</a> Curr Opin Struct Biol. 10 (1), 34-39 (2000).</li><li>Zimmerman, S. B., Minton, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macromolecular+crowding:+biochemical,+biophysical,+and+physiological+consequences.">Macromolecular crowding: biochemical, biophysical, and physiological consequences.</a> Annu Rev Biophys Biomol Struct. 22, 27-65 (1993).</li></ol>

Yazarlar aşağıdaki rakip mali interest(s) ilan: Noireaux laboratuvar MYcroarray, MYtxtl protein boş hücre sağma seti distribütörü araştırma fonları alır.

Chen, et al. teknikte takip 14 SPMC için önemli bir adım superinfection için uygun koşullar belirlerken ulaşılır. En yakın bir ana yük'ın yeteneği superinfection dayanacak şekilde denetleyen parametre sık ilk bulaşmasını fajının bölgedir. Konak hücreleri Logaritmik büyüme aşamasında ilk enfeksiyon fajının çok küçük bir miktar ile önce olması gerekir. Sonunda, fajının Ayrıca Logaritmik büyüme ulaşacak ve bırakmak fajının her konak hücrede birden ?...

Son on yıl içinde ifade boş hücre teknolojisi acil çok disiplinli araştırma alanları ile ilgili sentetik ve nicel Biyolojide adres roman uygulamaları için tasarlanmış. Aslında proteinler yaşayan bir organizma bağımsız ifade etmek için kullanılan, önemli ölçüde bu teknoloji kapsamını genişleterek her iki temel ve Uygulamalı Bilimler1,2, yeni boş hücre TXTL sistemleri geliştirilmiştir. TXTL platformları yeni nesil kullanıcı dostu, olmak için tasarlanmış daha verimli (2 mg/mL toplu iş modu3protein sentezi, daha ulaşan) transkripsiyon4düzeyde çok yönlü ve böylece olarak için modüler kolayca roman doğal entegre veya varolan biyolojik sistemleri5,6yeteneklerini genişletmek sentetik işlevleri. Özellikle, boş hücre TXTL sistemleri tasarım yapı testin azaltarak, düzenleyici elemanlarının veya küçük genetik devreleri7,8,9, gibi genetik programların hızlı prototipleme için kullanışlı hale gelmiştir bir kaç gün için döngüsü. Dikkat çekici, yeni TXTL da coliphages10,11güçlü aktif genomik sulandırma desteklemek için yeterli performans gösteren, tam sentezi gibi büyük DNA programları işleyebilme yeteneği olan sistemlerdir DNA canlı varlıklar kodlanmış.
TXTL sistemleri geleneksel vitro yapıcı biyokimyasal deneyleri için karşılaştırıldığında birçok teknik avantajları mevcut. Boş hücre TXTL gen ekspresyon sürecin nihai ürün yaşam hücrenin karmaşık sitoplazma aksine, azaltılmış ve açık bir ortamda bağlar. TXTL DNA, modern DNA montaj teknikleri ile uygun fiyatlı ve titiz protein saflaştırma adımlar gerektirmeyen yanı sıra hızlı olan biyokimyasal sistemleri vitro, yeniden oluşturmak için kullanır. İfade boş hücre bileşenlerinin biyokimyasal reaksiyonlarda böylece moleküler etkileşimleri12daha derin bir diseksiyon izin doğrudan erişim sağlar. TXTL tepki olarak bir yaşam hücrede neredeyse imkansız olacaktır, biyokimya ve biyofizik ayarlarını değiştirebilirsiniz. Bu avantajları ve son gelişmeleri göz önüne alındığında, TXTL teknoloji sentetik ve nicel Biyoloji için alternatif bir platform olarak daha popüler hale geliyor. TXTL standart bir teknoloji olan Biyomühendislik hale geliyor ve TXTL kullanarak araştırma topluluğu hızla büyüyor olsa da, nasıl TXTL reaksiyonlar ve için icra etmek ile ilgili yeterli yöntemler geliştirmek için böyle platformlar kullanılacağını anlamak önemlidir sonuçların yorumlanması.
Bu makalede, biz nasıl sentez, one-pot tepkiler onların genom11, MS2 gibi bacteriophages için bütün bir E. coli TXTL sistemi kullanılacağını açıklar (RNA, 3.4 kb), ΦΧ174 (ssDNA, 5.4 kb) ve T7 (dsDNA, 40 kb). Biz nasıl phages miktarı ile ilgili olarak bazı reaksiyonlar (magnezyum ve potasyum konsantrasyonu) biyokimyasal ayarları değişiklikleri sentezlenmiş gösterir. Moleküler kalabalık, taklit bir PEG aralığı 8000 konsantrasyonları ile büyüklükte birkaç emir fajının sentezi üzerinde dramatik bir etkisi vardır. Aynı anda transkripsiyon, çeviri, süreçleri özetlemek ve kendinden montajlı Biyoloji ve biyofizik ile ilgili temel sorulara adresleme için ilginç tek tüp reaksiyonlar büyük tür biyokimyasal sistemlerde gerçekleşme 10 (gen düzenlemesi, kendinden montajlı), yanı sıra yeni nanoyapıların13oluşturmak için fajının işlevleri repurposing gibi uygulamalar, geliştirmek gibi. TXTL üzerinde bir pratik ek olarak, biz yöntemleri fajının amplifikasyon, genom çıkarma ve arıtma ve FAJ miktar için plak tahlil tarafından sağlar. Bu el yazması sunulan E. coli dayalı özü boş hücre sistemleri kullanan ve bacteriophages içinde ilgilenen araştırmacılar için uygun yöntemlerdir.
Bu çalışmada sunulan protokolleri takip özetlenebilir: 1) fajının amplifikasyon (gün 1: aşılama hazırlamak hücreleri, 2. gün: tek plak, birden çok fajının büyüme ve konsantrasyon ve 3. gün: FAJ arıtma), 2) çift sarmallı genomu DNA ekstraksiyon (fenol/kloroform çıkarma) ve 3) boş hücre fajının tepki ve titresi deney (gün 1: konak hücreleri plaka ve Ağar Kaplamalar, yapmak 2. gün: öncesi kültür ve gün 3 hücre hücre-Alerjik reaksiyon ve ana bilgisayar: konak hücre kültür ve FAJ titresi).

<table><tbody><tr><td>Ultracentrifugation tubes</td><td>Beckman Coulter</td><td>344057</td><td></td></tr><tr><td>Conical tubes</td><td>Falcon</td><td>352070</td><td></td></tr><tr><td>Gradient maker</td><td>BioComp Gradient Master</td><td>see Anal Biochem. 1985 Jul;148(1):254-9.</td><td></td></tr><tr><td>Syringe and Blunt Cannula</td><td>Monoject</td><td>8881513918 and 888202017</td><td></td></tr><tr><td>Wide-bore pipette tips</td><td>Fischerbrand</td><td>02-707-134</td><td></td></tr><tr><td>Plaque counter</td><td>New Brunswik Scientific</td><td>Colony Counter Model C-110</td><td></td></tr><tr><td>Culture tubes</td><td>Fischerbrand</td><td>14-961-33</td><td></td></tr><tr><td>Cell-free system</td><td>Mycroarray Inc</td><td>Mytxtl</td><td></td></tr><tr><td>BioComp Gradient Master</td><td>BioComp Instruments</td><td>Model 105ME</td><td></td></tr><tr><td>LB agar plate recipe</td><td></td><td></td><td>25 g/L Luria-Bertani medium (LB Broth, Miller - Fisher BioReagents product number BP1426) and 15 g/L Bacto-Agar solid (Brenton, Dickenson and Company - product number 214010).</td></tr></tbody></table>

synthesis of infectious bacteriophages in an e. coli-based cell-free expression system

Boş hücre transkripsiyon-çeviri (TXTL) sistemleri, bir daha fazla çok yönlülük ve modülerlik, temel ve Uygulamalı Bilimler test tüpü tepkileri gerçekleştirmek için yeni yetenekleri sağlamak için tasarlanmış yeni bir nesil. Son on yıl içinde boş hücre TXTL roman çok disiplinli araştırma alanları ile ilgili nicel ve sentetik Biyoloji geniş bir yelpazesi için güçlü bir teknik haline gelmiştir. Yeni TXTL platformlar oluşturmak ve sentetik veya doğal gen devreleri yürütülmesini biyokimyasal sistemlerde sorgulamak özellikle yararlıdır. Vitro TXTL uygun hızla prototip düzenleyici elemanlarının ve biyolojik ağlara de özetlemek için kendinden montajlı mekanizmaları oturma sistemlerinde bulunan moleküler kanıtlamıştır. Bu makalede, tarif biz nasıl bulaşıcı bacteriophages, gibi MS2 (RNA), ΦΧ174 (ssDNA) ve T7 (dsDNA), tamamen kendi genom kullanarak tüm Escherichia coli, TXTL sistem boş hücre bir pot reaksiyonlarda gelen sentez. Üç coliphages sentezi plak tahlil kullanarak sayılabilir. Gösterdiğimiz nasıl sentezlenmiş fajının verim reaksiyonlar biyokimyasal ayarlarına bağlıdır. Moleküler kalabalık, taklit 8000 PEG, kontrollü bir konsantrasyon büyüklükleri emriyle sentezlenmiş phages miktarını etkiler. Biz de phages yükseltmek ve onların genleri arındırmak anlatan. İletişim kuralları ve sonuçları bu çalışmada sunulan bir dizi boş hücre sentetik Biyoloji ve Biyomühendislikte multidisipliner araştırmacılar dahil ilgi olmalıdır.

Biz dört temsilcisi sonuçları göster. Şekil 1' de, biz boş hücre TXTL sistemi ve FAJ DNA stokları yaşam ile kontamine değil olduğunu emin olmak için negatif denetimler kümesi mevcut E. coli hücreleri. Boş hücre TXTL sistem sağlam E. coli ücretsiz olduğundan emin olun hücre kirlenme genomik DNA (şekil 1A veB şekil 1) geçersiz b...

Watch this Scientific Journal Video about Synthesis of Infectious Bacteriophages in an E. coli-based Cell-free Expression System at JoVE.com

Synthesis of Infectious Bacteriophages in an E. coli-based Cell-free Expression System

Bir E. coli -bulaşıcı Bacteriophages sentezi ifade sistem boş hücre tabanlı

Boş hücre transkripsiyon-çeviri platformları yeni nesil Gen devreleri yürütülmesi ile biyokimyasal sistemleri vitro oluşturmak için tasarlanmış. Bu makalede, nasıl bacteriophages MS2, ΦΧ174 ve T7, gibi bir bütün E. coli boş hücre TXTL sistemini kullanarak kendi genom sentezlenmiş açıklayın.

A new generation of cell-free transcription-translation (TXTL) systems, engineered to have a greater versatility and modularity, provide novel ...

synthesis-infectious-bacteriophages-an-e-coli-based-cell-free

Research

JoVE Journal

Bioengineering

Synthesis of Infectious Bacteriophages in an E. coli-based Cell-free Expression System

14.5K Görüntüleme.  University of Minnesota. Boş hücre transkripsiyon-çeviri platformları yeni nesil Gen devreleri yürütülmesi ile biyokimyasal sistemleri vitro oluşturmak için tasarlanmış. Bu makalede, nasıl bacteriophages MS2, ΦΧ174 ve T7, gibi bir bütün E. coli boş hücre TXTL sistemini kullanarak kendi genom sentezlenmiş açıklayın.

Video: Synthesis of Infectious Bacteriophages in an E. coli-based Cell-free Expression System

Following Cell-fate in E. coli After Infection by Phage Lambda

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the simultaneous infection of the cell by a number of phages, one of two pathways is chosen: lytic (virulent) or lysogenic (dormant)3,4. We recently developed a method for fluorescently labeling individual phages, and were able to examine the post-infection decision in real-time under the microscope, at the level of individual phages and cells5. Here, we describe the full procedure for performing the infection experiments described in our earlier work5. This includes the creation of fluorescent phages, infection of the cells, imaging under the microscope and data analysis. The fluorescent phage is a "hybrid", co-expressing wild- type and YFP-fusion versions of the capsid gpD protein. A crude phage lysate is first obtained by inducing a lysogen of the gpD-EYFP (Enhanced Yellow Fluorescent Protein) phage, harboring a plasmid expressing wild type gpD. A series of purification steps are then performed, followed by DAPI-labeling and imaging under the microscope. This is done in order to verify the uniformity, DNA packaging efficiency, fluorescence signal and structural stability of the phage stock. The initial adsorption of phages to bacteria is performed on ice, then followed by a short incubation at 35&deg;C to trigger viral DNA injection6. The phage/bacteria mixture is then moved to the surface of a thin nutrient agar slab, covered with a coverslip and imaged under an epifluorescence microscope. The post-infection process is followed for 4 hr, at 10 min interval. Multiple stage positions are tracked such that ~100 cell infections can be traced in a single experiment. At each position and time point, images are acquired in the phase-contrast and red and green fluorescent channels. The phase-contrast image is used later for automated cell recognition while the fluorescent channels are used to characterize the infection outcome: production of new fluorescent phages (green) followed by cell lysis, or expression of lysogeny factors (red) followed by resumed cell growth and division. The acquired time-lapse movies are processed using a combination of manual and automated methods. Data analysis results in the identification of infection parameters for each infection event (e.g. number and positions of infecting phages) as well as infection outcome (lysis/lysogeny). Additional parameters can be extracted if desired.

Following Cell-fate in E. coli After Infection by Phage Lambda

This article describes the procedure for preparing a fluorescently-labeled version of bacteriophage lambda, infection of E. coli bacteria, following the infection outcome under the microscope, and analysis of infection results.

Hücre-kaderi ardından<em&gt; E. coli</em&gt; Faj Lambda tarafından Enfeksiyon Sonrası

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the ...

İmmünoloji ve Enfeksiyon

Large Insert Environmental Genomic Library Production

The vast majority of microbes in nature currently remain inaccessible to traditional cultivation methods. Over the past decade, culture-independent environmental genomic (i.e. metagenomic) approaches have emerged, enabling researchers to bridge this cultivation gap by capturing the genetic content of indigenous microbial communities directly from the environment. To this end, genomic DNA libraries are constructed using standard albeit artful laboratory cloning techniques. Here we describe the construction of a large insert environmental genomic fosmid library with DNA derived from the vertical depth continuum of a seasonally hypoxic fjord. This protocol is directly linked to a series of connected protocols including coastal marine water sampling [1], large volume filtration of microbial biomass [2] and a DNA extraction and purification protocol [3]. At the outset, high quality genomic DNA is end-repaired with the creation of 5 -phosphorylated blunt ends. End-repaired DNA is subjected to pulsed-field gel electrophoresis (PFGE) for size selection and gel extraction is performed to recover DNA fragments between 30 and 60 thousand base pairs (Kb) in length. Size selected DNA is purified away from the PFGE gel matrix and ligated to the phosphatase-treated blunt-end fosmid CopyControl vector pCC1 (EPICENTRE <a href="http://www.epibio.com/item.asp?ID=385">http://www.epibio.com/item.asp?ID=385</a>). Linear concatemers of pCC1 and insert DNA are subsequently headfull packaged into phage particles by lambda terminase, with subsequent infection of phage-resistant E. coli cells. Successfully transduced clones are recovered on LB agar plates under antibiotic selection and archived in 384-well plate format using an automated colony picking robot (Qpix2, GENETIX). The current protocol draws from various sources including the CopyControl Fosmid Library Production Kit from EPICENTRE and the published works of multiple research groups [4-7]. Each step is presented with best practice in mind. Whenever possible we highlight subtleties in execution to improve overall quality and efficiency of library production. The whole process of fosmid library production and automated colony picking takes at least 7-10 days as there are many incubation steps included. However, there are several stopping points possible which are mentioned within the protocol.

Construction of a fosmid library with environmental genomic DNA isolated from the vertical depth continuum of a seasonally hypoxic fjord is described. The resulting clone library is picked into 384-well plates and archived for downstream sequencing and functional screening by the application of an automated colony picking system.

Büyük Ekle Çevre Genomik Kütüphane Üretim

The vast majority of microbes in nature currently remain inaccessible to traditional cultivation methods. Over the past decade, culture-independent ...

Biyoloji

Escherichia coli-Based Cell-Free Protein Synthesis: Protocols for a robust, flexible, and accessible platform technology

Over the last 50 years, Cell-Free Protein Synthesis (CFPS) has emerged as a powerful technology to harness the transcriptional and translational capacity of cells within a test tube. By obviating the need to maintain the viability of the cell, and by eliminating the cellular barrier, CFPS has been foundational to emerging applications in biomanufacturing of traditionally challenging proteins, as well as applications in rapid prototyping for metabolic engineering, and functional genomics. Our methods for implementing an E. coli-based CFPS platform allow new users to access many of these applications. Here, we describe methods to prepare extract through the use of enriched media, baffled flasks, and a reproducible method of tunable sonication-based cell lysis. This extract can then be used for protein expression capable of producing 900 &#181;g/mL or more of super folder green fluorescent protein (sfGFP) in just 5 h from experimental setup to data analysis, given that appropriate reagent stocks have been prepared beforehand. The estimated startup cost of obtaining reagents is $4,500 which will sustain thousands of reactions at an estimated cost of $0.021 per &#181;g of protein produced or $0.019 per &#181;L of reaction. Additionally, the protein expression methods mirror the ease of the reaction setup seen in commercially available systems due to optimization of reagent pre-mixes, at a fraction of the cost. In order to enable the user to leverage the flexible nature of the CFPS platform for broad applications, we have identified a variety of aspects of the platform that can be tuned and optimized depending on the resources available and the protein expression outcomes desired.

Escherichia coli-Based Cell-Free Protein Synthesis: Protocols for a robust, flexible, and accessible platform technology

This protocol details the steps, costs, and equipment necessary to generate E. coli-based cell extracts and implement in vitro protein synthesis reactions within 4 days or less. To leverage the flexible nature of this platform for broad applications, we discuss reaction conditions that can be adapted and optimized.

Escherichia coli-boş hücre Protein sentezi dayalı: sağlam, esnek ve erişilebilir platform teknolojisi için iletişim kuralları

Over the last 50 years, Cell-Free Protein Synthesis (CFPS) has emerged as a powerful technology to harness the transcriptional and translational capacity ...

Kimya

<meta charset="utf8"></head><body>Mikro Bölmeli Sentetik Hücreler için CFPS Protokolleri

Cell-Free Protein Synthesis System for Building Synthetic Cells

The Cell-Free Protein Synthesis (CFPS) system has been widely employed to facilitate the bottom-up assembly of synthetic cells. It serves as the host for the core machinery of the Central Dogma, standing as an optimal chassis for the integration and assembly of diverse artificial cellular mimicry systems. Despite its frequent use in the fabrication of synthetic cells, establishing a tailored and robust CFPS system for a specific application remains a nontrivial challenge. In this methods paper, we present a comprehensive protocol for the CFPS system, routinely employed in constructing synthetic cells. This protocol encompasses key stages in the preparation of the CFPS system, including the cell extract, template preparation, and routine expression optimization utilizing a fluorescent reporter protein. Additionally, we show representative results by encapsulating the CFPS system within various micro-compartments, such as monolayer droplets, double-emulsion vesicles, and chambers situated atop supported lipid bilayers. Finally, we elucidate the critical steps and conditions necessary for the successful assembly of these CFPS systems in distinct environments. We expect that our approach will facilitate the establishment of good working practices among various laboratories within the continuously expanding synthetic cell community, thereby accelerating progress in the field of synthetic cell development.

<meta charset="utf8"></head><body>Yazar Spotlight: Sentetik Hücre Yapısı için CFPS Sistemlerini Optimize Etme

This protocol describes the Cell-Free Protein Synthesis (CFPS) system used in constructing synthetic cells. It outlines key stages with representative results in different micro-compartments. The protocol aims to establish best practices for diverse laboratories in the synthetic cell community, advancing progress in synthetic cell development.

Sentetik hücreler oluşturmak için hücresiz protein sentez sistemi

The Cell-Free Protein Synthesis (CFPS) system has been widely employed to facilitate the bottom-up assembly of synthetic cells. It serves as the host for ...

Biyokimya

Functional Surface-immobilization of Genes Using Multistep Strand Displacement Lithography

Immobilization of genes on lithographically structured surfaces allows the study of compartmentalized gene expression processes in an open microfluidic bioreactor system. In contrast to other approaches towards artificial cellular systems, such a setup allows for a continuous supply with gene expression reagents and simultaneous draining of waste products. This facilitates the implementation of cell-free gene expression processes over extended periods of time, which is important for the realization of dynamic gene regulatory feedback systems. Here we provide a detailed protocol for the fabrication of genetic biochips using a simple-to-use lithographic technique based on DNA strand displacement reactions, which exclusively uses commercially available components. We also provide a protocol on the integration of compartmentalized genes with a polydimethylsiloxane (PDMS)-based microfluidic system. Furthermore, we show that the system is compatible with total internal reflection fluorescence (TIRF) microscopy, which can be used for the direct observation of molecular interactions between DNA and molecules contained in the expression mix.

We describe a simple lithographic procedure for the immobilization of gene-length DNA molecules on a surface, which can be used to perform cell-free gene expression experiments on biochips.

Genlerin Multistep Strand deplasman litografi kullanarak işlevsel yüzey immobilizasyon

Immobilization of genes on lithographically structured surfaces allows the study of compartmentalized gene expression processes in an open microfluidic ...

Biyomühendislik

Preparing Protein Producing Synthetic Cells using Cell Free Bacterial Extracts, Liposomes and Emulsion Transfer

The bottom-up assembly approach for construction of synthetic cells is an effective tool for isolating and investigating cellular processes in a cell mimicking environment. Furthermore, the development of cell-free expression systems has demonstrated the ability to reconstitute the protein production, transcription and translation processes (DNA&#8594;RNA&#8594;protein) in a controlled manner, harnessing synthetic biology. Here we describe a protocol for preparing a cell-free expression system, including the production of a potent bacterial lysate and encapsulating this lysate inside cholesterol-rich lipid-based giant unilamellar vesicles (GUVs) (i.e., stable liposomes), to form synthetic cells. The protocol describes the methods for preparing the components of the synthetic cells including the production of active bacterial lysates, followed by a detailed step-by-step preparation of the synthetic cells based on a water-in-oil emulsion transfer method. These facilitate the production of millions of synthetic cells in a simple and affordable manner with a high versatility for producing different types of proteins. The obtained synthetic cells can be used to investigate protein/RNA production and activity in an isolated environment, in directed evolution, and also as a controlled drug delivery platform for on-demand production of therapeutic proteins inside the body.

This protocol describes the method, materials, equipment and steps for bottom-up preparation of RNA and protein producing synthetic cells. The inner aqueous compartment of the synthetic cells contained the S30 bacterial lysate encapsulated within a lipid bilayer (i.e., stable liposomes), using a water-in-oil emulsion transfer method.

Hücre Serbest Bakteriyel Özler, Lipozomlar ve Emülsiyon Transferi Kullanarak Protein Üreten Sentetik Hücreler Hazırlama

The bottom-up assembly approach for construction of synthetic cells is an effective tool for isolating and investigating cellular processes in a cell ...

From Cells to Cell-Free Protein Synthesis within 24 Hours Using Cell-Free Autoinduction Workflow

Cell-free protein synthesis (CFPS) has grown as a biotechnology platform that captures transcription and translation machinery in vitro. Numerous developments have made the CFPS platform more accessible to new users and have expanded the range of applications. For lysate based CFPS systems, cell extracts can be generated from a variety of organisms, harnessing the unique biochemistry of that host to augment protein synthesis. Within the last 20 years, Escherichia coli (E. coli) has become one of the most widely used organisms for supporting CFPS due to its affordability and versatility. Despite numerous key advances, the workflow for E. coli cell extract preparation has remained a key bottleneck for new users to implement CFPS for their applications. The extract preparation workflow is time-intensive and requires technical expertise to achieve reproducible results. To overcome these barriers, we previously reported the development of a 24 hour cell-free autoinduction (CFAI) workflow that reduces user input and technical expertise required. The CFAI workflow minimizes the labor and technical skill required to generate cell extracts while also increasing the total quantities of cell extracts obtained. Here we describe that workflow in a step-by-step manner to improve access and support the broad implementation of E. coli based CFPS.

This work describes the preparation of cell extract from Escherichia coli (E. coli) followed by cell-free protein synthesis (CFPS) reactions in under 24 hours. Explanation of the cell-free autoinduction (CFAI) protocol details improvements made to reduce researcher oversight and increase quantities of cell extract obtained.

Hücresiz Otomatik İndüksiyon İş Akışını Kullanarak 24 Saat İçinde Hücrelerden Hücresiz Protein Sentezine

Cell-free protein synthesis (CFPS) has grown as a biotechnology platform that captures transcription and translation machinery in vitro. Numerous ...

Protocols for Implementing an Escherichia coli 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 expressing proteins and genetic circuits in a controlled manner as well as for providing a prototyping environment for synthetic biology.2,3 To achieve the latter goal, cell-free expression systems that preserve endogenous Escherichia coli transcription-translation mechanisms are able to more accurately reflect in vivo cellular dynamics than those based on T7 RNA polymerase transcription. We describe the preparation and execution of an efficient endogenous E. coli based transcription-translation (TX-TL) cell-free expression system that can produce equivalent amounts of protein as T7-based systems at a 98% cost reduction to similar commercial systems.4,5 The preparation of buffers and crude cell extract are described, as well as the execution of a three tube TX-TL reaction. The entire protocol takes five days to prepare and yields enough material for up to 3000 single reactions in one preparation. Once prepared, each reaction takes under 8 hr from setup to data collection and analysis. Mechanisms of regulation and transcription exogenous to E. coli, such as lac/tet repressors and T7 RNA polymerase, can be supplemented.6 Endogenous properties, such as mRNA and DNA degradation rates, can also be adjusted.7 The TX-TL cell-free expression system has been demonstrated for large-scale circuit assembly, exploring biological phenomena, and expression of proteins under both T7- and endogenous promoters.6,8 Accompanying mathematical models are available.9,10 The resulting system has unique applications in synthetic biology as a prototyping environment, or "TX-TL biomolecular breadboard."

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

This five-day protocol outlines all steps, equipment, and supplemental software necessary for creating and running an efficient endogenous Escherichia coli based TX-TL cell-free expression system from scratch. With reagents, the protocol takes 8 hours or less to setup a reaction, collect, and process data.

Bir uygulama için Protokoller<em&gt; Escherichia coli</emSentetik Biyoloji&gt; Tabanlı TX-TL Hücre-Özgür İfade Sistemi

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

Bir E. coli -bulaşıcı Bacteriophages sentezi ifade sistem boş hücre tabanlı

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Genlerin Multistep Strand deplasman litografi kullanarak işlevsel yüzey immobilizasyon

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Hücresiz Otomatik İndüksiyon İş Akışını Kullanarak 24 Saat İçinde Hücrelerden Hücresiz Protein Sentezine

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