Bu çalışma, Johns Hopkins ALS Araştırma Packard Merkezi (ADG), NIH Müdürü Yenilikçisi Ödülü 1DP2OD004417-01 (ADG), NIH R01 NS065317 (ADG), Rita Allen Vakfı Scholar Ödülü bir hibe ile desteklenmiştir. ADG Pew Charitable Ortaklıkları tarafından desteklenen Biyomedikal Bilimler Pew Scholar.

1. Maya dönüşüm için hazırlıklar Bu protokol on 96-kuyucuğu için tasarlanmıştır ancak bu doğrultuda yukarı veya aşağı ölçeklendirilebilir. Biz bu protokolü, dönüşüm yuvarlak başına yirmiden fazla 96 kuyucuğu için iyi olmadığını bulduk. Tüm dönüşüm prosedürü (adım I.3) yaklaşık sekiz saat sürer. <ol><li> Kısım yuvarlak bir alt her kuyuya Biorobot RapidPlate sıvı işleyici 96-plaka Maya FLEXGene ORF kütüphane plazmid DNA (100 ng / ul) 5-10μL. Place kuru geceleme temiz tezgah davlumbaz 96-kuyucuğu ortaya çıkardı. Biz CEN ve 2μ maya plazmid ile benzer dönüşüm verimliliği bulduk. </li><li> Maya pepton dekstroz (YPD) medya (10g / L maya özü, 20g / L pepton, 20g / L glukoz) ve 200 ml 500ml şaşkın Erlenmeyer şişesinin sorgu suşu ile inoküle edin. Sallayarak (200 rpm) ile 30 ° C&#39;de bir gece inkübe edin. Gerekirse, seçici medya bu starter kültürler için de kullanılabilir. </li><li> Ertesi sabah, sorgu gerginlik OD 600 = 0.1 YPD, 2L sulandırmak. 30 ° C (200 rpm) kültürü OD 600 = 0.8-1.0 ulaşıncaya kadar 4-6 saat çalkalanır. Optimum büyüme için, iki ayrı 2.8 L şaşkın şişeler 1L kültürler büyüyorlar. Gerekirse, seçici medya da YPD yerine kullanılan olabilir. </li></ol> 2. Maya dönüşüm <ol><li> Hasat hücreler santrifüj yoluyla. 5 dakika masaüstü santrifüj (Eppendorf 5810R) 3000rpm (~ 1800 xg) maya kültürü ve spin ile dört tek 225mL konik tüpler doldurun. Süpernatant dökün ve tüm hücreleri hasat kadar tekrarlayın. </li><li> Otoklavlanmış distile H 2 O. 200ml hücrelerin yıkayın Sallayarak veya vorteks yeniden süspanse hücre pelletini. 225mL konik bir tüp içinde hücreleri birleştirin. 5 dakika 3000rpm dönerler. Süpernatantı. </li><li> 0.1MLiOAc/1XTE çözüm (0.1M LiOAc, 10mM Tris, 1mm EDTA, otoklavlanmış distile su, pH 8) hazırlayın. 100 ml 0.1MLiOAc/1XTE hücrelerin yıkayın. 5 dakika 3000rpm dönerler. Süpernatantı. </li><li> 70mL 0.1M LiOAc/1xTE yeniden süspanse hücre pelletini. Shake ve / veya vorteks pelet tamamen yeniden süspanse sağlamak için. </li><li> 30 ° C&#39;de 30 dakika (200 rpm) sallayarak inkübe edin. </li><li> 0.1M için β-mercaptoethanol ekleyin. 30 ° C&#39;de 30 dakika (200 rpm) sallayarak inkübe edin. Not: beta-mercaptoethanol duyarlı maya suşu kullanılarak, bu adım atlanabilir. Biz bu adımı başarılı bir dönüşüm için zorunlu olduğunu bulduk, ancak dönüşüm verimliliği artırmak. </li><li> Sonicated somon sperm DNA (10mg/ml) 2mL 15 dakika boyunca kaynatın ve ardından buz üzerinde soğutun. </li><li> 2mL haşlanmış somon sperm DNA hücre karışımı ekleyin. Dikkat: hücre karışımı somon sperm DNA eklenmesi halinde bir çökelti. Pipetman ve 200 ul ucu kullanarak, dikkatli bir şekilde bu mansap pipetleme engel olabilir çünkü formları herhangi bir çökelti kaldırın. </li><li> 96-plaka BioRobot Rapidplate kullanarak her bir kuyunun hücre karışımı kısım 50μL (aynı zamanda çok kanallı pipet kullanabilirsiniz). Karışmaz. </li><li> Sallayarak 30 dakika oda sıcaklığında inkübe edin. </li><li> 200ml 40% PEG-3350/10% DMSO/0.1M LiOAc (160mL% 50 PEG-3350, 20ml DMSO, 20ml 1M LiOAc) hazırlayın. Kullanımdan hemen önce çözüm hazırlayın. </li><li> Her iyi PEG / LioAc / DMSO çözüm 125μL ekleyin. RapidPlate ile sekiz kez aspire ve hücre süspansiyonu dağıtım karıştırın. </li><li> Sallayarak 30 dakika oda sıcaklığında inkübe edin. </li><li> Isı şok hücreleri 42 ° C 15 dakika boyunca kuru bir inkübatör. Plakalar koymayın. Not: Bu ısı şok adım başarılı bir dönüşüm için gerekli olduğunu bulduk. Bazı maya suşları (örneğin, ısıya duyarlı mutantlar), ısı şok zararlı. Daha önce bir dakika ısı şok süresini azalttı ve bu protokolü kullanarak ypt1 ısıya duyarlı mutasyon barındıran bir maya suşu başarıyla dönüştürmek mümkün olmuştur. </li><li> 96-kuyucuğu karşılamak için santrifüj adaptörleri kullanarak, 5 dakika için 2500rpm (~ 1100 xg) plakaları Spin. Atık kovası üzerinde tersini plakaları ile PEG çözüm çıkarın. Kalan sıvı kaldırmak için kağıt havlu üzerine ters plaka Blot (hücreleri kuyu altında kalacaktır). </li><li> RapidPlate her iyi minimal medya 200μL ekleyerek (örneğin SD /-Ura) hücreleri durulayın. </li><li> 2500rpm az 5 dakika boyunca plakalar Spin ve tersini üzerinde atık kovası ve kağıt havlu üzerinde blot süpernatant kaldırmak. </li><li> RapidPlate ile her kuyuya 200μL minimal medya (örneğin SD /-Ura) ekleyin. </li><li> Titreme olmadan 2 gün boyunca 30 ° C&#39;de inkübe edin. 2 gün sonra, hücre başarıyla iyi dönüştürülmüş her altındaki küçük koloniler oluşturmak için başlamalıdır. </li></ol> 3. Tahlil Spotting <ol><li> Her kuyuya raffinose tabanlı minimal medya (örneğin SRaf /-Ura) 200μL Uygulamayeni bir düz-alt 96-plaka. </li><li> Rapidplate ile sekiz kez aspire ve hücre süspansiyonu dağıtım dönüşüm plaka her iyi karıştırın. </li><li> Dönüşüm plaka hücre karışımı 5μL ile SRaf plakalarını aşılamak. </li><li> Bir gün için 30 ° C&#39;de inkübe edin. Küçük kolonileri bir gün sonra, her bir kuyunun dibinde mevcut olmalıdır. </li><li> SD / Ura ve kaputu SGal /-Ura plakaları Spot koloniler. 96-cıvata çoğaltıcı (Frogger) alev tarafından sterilize edin. Frogger ile seçici besiyerleri plakaları lekelenme önce koloniler karıştırın. Lekelenme sonra, tabak, 5-10 dakika kaputu kurumasını bekleyin. </li><li> 2-3 gün boyunca 30 ° C&#39;de inkübe edin. </li></ol> Dijital kamera ile fotoğraf plakaları ve görsel SGal /-Ura plakaları sorgu gerginlik toksisite (hızlı büyüme / daha yoğun koloniler) (daha yavaş büyüme / daha az yoğun koloniler) ya da bastırılmış gelişmiş olduğu koloniler bulmak için kolonilerin büyüme karşılaştırmak. 4. Temsilcisi sonuçları: <img src="/files/ftp_upload/2836/2836fig1.jpg" alt="Şekil 1"/> Şekil 1. Maya plazmid aşırı ekspresyonu TDP-43 toksisite bastırıcılarının ve arttırıcılar tanımlamak için ekran. TDP-43 Amyotrofik lateral skleroz (Lou Gehrig hastalığı) patogenezinde sorumlu olan bir insan proteini. TDP-43 Sitoplazmik agrega ALS hastası 17 beyin ve omurilik nöronlarının birikir. Agregasyonu ve sitotoksisite 18 maya hücreleri sonuçları ifade TDP-43. Biz TDP-43 toksisite 19,20 mekanizmaları tanımlamak için bu model sistem kullandık. Gösterilen bizim maya TDP-43 toksisite değiştirici ekran plaka repesentative örnekler. Bu plakalar FLEXGene ORF ifade kütüphane plazmid ile entegre bir galaktoz-indüklenebilir TDP-43 plazmid ve aynı zamanda dönüştürülmüş koloniler gösterilecek. Soldaki plaka TDP-43 veya FLEXGene plazmid ifade bastıran glikoz içerir. Sağdaki plaka TDP-43 ekspresyonu ve FLEXGene plazmid Orfs neden olur galaktoz içerir. Yeşil ok ucu TDP-43 toksisite bastırır bir plazmid ile dönüştürülmüş bir koloni gösterir. Kırmızı ok ucu TDP-43 toksisitesini arttırır bir plazmid ile dönüştürülmüş bir koloni gösterir.

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Burada maya yüksek verimli bir plazmid aşırı ekspresyonu ekran gerçekleştirmek için bir protokol mevcut. Bu yaklaşım, birçok farklı hücre fenotipleri genetik nitelemeler için hızlı ve tarafsız bir tarama sağlar. Bu yaklaşımı kullanarak, bir araştırmacı, birkaç hafta içinde maya genomu önemli bir bölümünü ekrana. Bu tarafsız bir yaklaşım aynı zamanda, önceki bulgulara göre tahmin edilmiş olabilir değiştirici belirlenmesi için olanak sağlar. Biz insan nörodejeneratif hastalık pro...

<table border="1"><tbody><tr><th> Reaktif Adı </th><th> Şirket </th><th> Katalog numarası </th></tr><tr><td> BioRobot RapidPlate </td><td> Qiagen </td><td> 9000490 </td></tr><tr><td> 96 cıvata çoğaltıcı (Frogger) </td><td> V &amp; P Bilimsel </td><td> VP404 </td></tr><tr><td> FLEXGene ORF Kütüphanesi </td><td> Proteomik Enstitüsü, Harvard Tıp Fakültesi </td><td></td></tr><tr><td> Masa üstü santrifüj </td><td> Eppendorf </td><td> 5810R </td></tr><tr><td> 500ml şaşkın şişesi </td><td> Bellco </td><td> 2543-00500 </td></tr><tr><td> 2.8L üçlü şaşkın FERNBACH şişesi </td><td> Bellco </td><td> 2551-02800 </td></tr><tr><td> 100μL Rapidplate pipet uçları </td><td> Axygen </td><td> ZT-100-RS </td></tr><tr><td> 200μL Rapidplate pipet uçları </td><td> Axygen </td><td> ZT-200-RS </td></tr></tbody></table>

high-throughput yeast plasmid overexpression screen

Saccharomyces cerevisiae, tomurcuklanan bir maya, insan hastalığı ile doğrudan ilgili olanlar da dahil olmak üzere birçok önemli hücresel süreçlerin temel mekanizmaları, tanımlamak için güçlü bir model sistem. Çünkü, kısa üretim süresi ve iyi karakterize genom, maya model sistem büyük bir deneysel avantajı, belirli bir süreç içinde yer genler ve yolları belirlemek için genetik ekranlar gerçekleştirmek için yeteneğidir. Son otuz yıl içinde bu tür genetik ekranlar hücre döngüsü, salgı yolağı ve ökaryotik hücre biyolojisi 1-5 çok daha yüksek oranda korunmuş yönlerini aydınlatmak için kullanılmaktadır . Son birkaç yıl içinde, maya suşları ve plazmid birkaç genomewide kütüphaneler 6-10 oluşturuldu. Bu koleksiyon, kazanç ve kayıp fonksiyon-yaklaşımları kullanarak 11-16 genin fonksiyonu ile sistematik bir sorgulama için izin verir. Burada dizilmiş 5.500 maya plazmid bir kütüphaneyi kullanarak, plazmid aşırı ekspresyonu ekranı gerçekleştirmek için bir sıvı taşıma robotu ile yüksek verimli bir maya dönüşüm protokolü kullanımı için ayrıntılı bir protokol sağlar. Biz kümeleme eğilimli insan nörodejeneratif hastalık proteinlerin birikimi ile ilişkili toksisite genetik nitelemeler tanımlamak için bu ekranlar kullanıyoruz. Burada sunulan yöntemler diğer ilgi hücre fenotipleri çalışma kolayca uyarlanabilir.

Watch this Scientific Journal Video about High-throughput Yeast Plasmid Overexpression Screen at JoVE.com

High-throughput Yeast Plasmid Overexpression Screen

Burada bir plazmid aşırı ekspresyonu ekran tarif<em&gt; Saccharomyces cerevisiae</em&gt;, Bir sıvı taşıma robotu ile dizilmiş plazmid bir kütüphane ve yüksek verimli bir maya dönüşüm protokolü kullanarak.

Yüksek verim Maya Plazmid overekspresyonu Ekran

The budding yeast, Saccharomyces cerevisiae, is a powerful model system for defining fundamental mechanisms of many important cellular processes, ...

high-throughput-yeast-plasmid-overexpression-screen

Research

JoVE Journal

Biology

16.3K Görüntüleme.  University of Pennsylvania School of Medicine. Burada bir plazmid aşırı ekspresyonu ekran tarif Saccharomyces cerevisiae, Bir sıvı taşıma robotu ile dizilmiş plazmid bir kütüphane ve yüksek verimli bir maya dönüşüm protokolü kullanarak.

Video: High-throughput Yeast Plasmid Overexpression Screen

Mating-based Overexpression Library Screening in Yeast

Budding yeast has been widely used as a model in studying proteins associated with human diseases. Genome-wide genetic screening is a powerful tool commonly used in yeast studies. The expression of a number of neurodegenerative disease-associated proteins in yeast causes cytotoxicity and aggregate formation, recapitulating findings seen in patients with these disorders. Here, we describe a method for screening a yeast model of the Amyotrophic Lateral Sclerosis-associated protein FUS for modifiers of its toxicity. Instead of using transformation, this new screening platform relies on the mating of yeast to introduce an arrayed library of plasmids into the yeast model. The mating method has two clear advantages: first, it is highly efficient; second, the pre-transformed arrayed library of plasmids can be stored for long-term as a glycerol stock, and quickly applied to other screens without the labor-intensive step of transformation into the yeast model each time. We demonstrate how this method can successfully be used to screen for genes that modify the toxicity of FUS.

This article presents a mating-based method to facilitate overexpression screening in budding yeast using an arrayed plasmid library.

Maya Overexpression çiftleşme tabanlı Kütüphane tarama

Budding yeast has been widely used as a model in studying proteins associated with human diseases. Genome-wide genetic screening is a powerful tool ...

Genetik

A Yeast 2-Hybrid Screen in Batch to Compare Protein Interactions

Screening for protein-protein interactions using the yeast 2-hybrid assay has long been an effective tool, but its use has largely been limited to the discovery of high-affinity interactors that are highly enriched in the library of interacting candidates. In a traditional format, the yeast 2-hybrid assay can yield too many colonies to analyze when conducted at low stringency where low affinity interactors might be found. Moreover, without a comprehensive and complete interrogation of the same library against different bait plasmids, a comparative analysis cannot be achieved. Although some of these problems can be addressed using arrayed prey libraries, the cost and infrastructure required to operate such screens can be prohibitive. As an alternative, we have adapted the yeast 2-hybrid assay to simultaneously uncover dozens of transient and static protein interactions within a single screen utilizing a strategy termed DEEPN (Dynamic Enrichment for Evaluation of Protein Networks), which incorporates high-throughput DNA sequencing and computation to follow the evolution of a population of plasmids that encode interacting partners. Here, we describe customized reagents and protocols that allow a DEEPN screen to be executed easily and cost-effectively.

Batch processing of yeast 2-hybrid screens allows for direct comparison of the interaction profiles of multiple bait proteins with a highly complex set of prey fusion proteins. Here, we describe refined methods, new reagents, and how to implement their use for such screens.

Bir Maya 2-melez ekran Protein etkileşimleri karşılaştırmak için bir toplu iş

Screening for protein-protein interactions using the yeast 2-hybrid assay has long been an effective tool, but its use has largely been limited to the ...

Biyokimya

A Comparative Approach to Characterize the Landscape of Host-Pathogen Protein-Protein Interactions

Significant efforts were gathered to generate large-scale comprehensive protein-protein interaction network maps. This is instrumental to understand the pathogen-host relationships and was essentially performed by genetic screenings in yeast two-hybrid systems. The recent improvement of protein-protein interaction detection by a Gaussia luciferase-based fragment complementation assay now offers the opportunity to develop integrative comparative interactomic approaches necessary to rigorously compare interaction profiles of proteins from different pathogen strain variants against a common set of cellular factors.
This paper specifically focuses on the utility of combining two orthogonal methods to generate protein-protein interaction datasets: yeast two-hybrid (Y2H) and a new assay, high-throughput Gaussia princeps protein complementation assay (HT-GPCA) performed in mammalian cells.
A large-scale identification of cellular partners of a pathogen protein is performed by mating-based yeast two-hybrid screenings of cDNA libraries using multiple pathogen strain variants. A subset of interacting partners selected on a high-confidence statistical scoring is further validated in mammalian cells for pair-wise interactions with the whole set of pathogen variants proteins using HT-GPCA. This combination of two complementary methods improves the robustness of the interaction dataset, and allows the performance of a stringent comparative interaction analysis. Such comparative interactomics constitute a reliable and powerful strategy to decipher any pathogen-host interplays.

This article focuses on the identification of high-confident interaction datasets between host and pathogen proteins using a combination of two orthogonal methods: yeast two-hybrid followed by a high-throughput interaction assay in mammalian cells called HT-GPCA.

Ana-Patojen protein-protein etkileşimleri ve Peyzaj karakterize etmek Karşılaştırmalı Bir Yaklaşım

Significant efforts were gathered to generate  large-scale comprehensive protein-protein interaction network maps. This is  instrumental to understand the ...

İmmünoloji ve Enfeksiyon

Identification of Host Pathways Targeted by Bacterial Effector Proteins using Yeast Toxicity and Suppressor Screens

Intracellular bacteria secrete virulence factors called effector proteins into the host cytosol that act to subvert host proteins and/or their associated biological pathways to the benefit of the bacterium. Identification of putative bacterial effector proteins has become more manageable due to advances in bacterial genome sequencing and the advent of algorithms that allow in silico identification of genes encoding secretion candidates and/or eukaryotic-like domains. However, identification of these important virulence factors is only an initial step. Naturally, the goal is to determine the molecular function of effector proteins and elucidate how they interact with the host. In recent years, techniques like the yeast two-hybrid screen and large-scale immunoprecipitations coupled with mass spectrometry have aided in the identification of protein-protein interactions. Although identification of a host binding partner is the crucial first step toward elucidating the molecular function of a bacterial effector protein, sometimes the host protein is found to have multiple biological functions (e.g., actin, clathrin, tubulin), or the bacterial protein may not physically bind host proteins, depriving the researcher of crucial information about the precise host pathway being manipulated. A modified yeast toxicity screen coupled with a suppressor screen has been adapted to identify host pathways impacted by bacterial effector proteins. The toxicity screen relies on a toxic effect in yeast caused by the effector protein interfering with the host biological pathways, which often manifests as a growth defect. Expression of a yeast genomic library is used to identify host factors that suppress the toxicity of the bacterial effector protein and thus identify proteins in the pathway that the effector protein targets. This protocol contains detailed instructions for both the toxicity and suppressor screens. These techniques can be performed in any lab capable of molecular cloning and cultivation of yeast and Escherichia coli.

Bacterial pathogens secrete proteins into the host that target crucial biological processes. Identifying the host pathways targeted by bacterial effector proteins is key to addressing molecular pathogenesis. Here, a method using a modified yeast suppressor and toxicity screen to elucidate host pathways targeted by toxic bacterial effector proteins is described.

Maya Toksisitesi ve Bastırıcı Ekranlar Kullanılarak Bakteriyel Efektör Proteinlertarafından Hedeflenen Konak Yollarının Belirlenmesi

Intracellular bacteria secrete virulence factors called effector proteins into the host cytosol that act to subvert host proteins and/or their associated ...

Unravelling the Function of a Bacterial Effector from a Non-cultivable Plant Pathogen Using a Yeast Two-hybrid Screen

Unravelling the molecular mechanisms of disease manifestations is important to understand pathologies and symptom development in plant science. Bacteria have evolved different strategies to manipulate their host metabolism for their own benefit. This bacterial manipulation is often coupled with severe symptom development or the death of the affected plants. Determining the specific bacterial molecules responsible for the host manipulation has become an important field in microbiological research. After the identification of these bacterial molecules, called &#34;effectors,&#34; it is important to elucidate their function. A straightforward approach to determine the function of an effector is to identify its proteinaceous binding partner in its natural host via a yeast two-hybrid (Y2H) screen. Normally the host harbors numerous potential binding partners that cannot be predicted sufficiently by any in silico algorithm. It is thus the best choice to perform a screen with the hypothetical effector against a whole library of expressed host proteins. It is especially challenging if the causative agent is uncultivable like phytoplasma. This protocol provides step-by-step instructions for DNA purification from a phytoplasma-infected woody host plant, the amplification of the potential effector, and the subsequent identification of the plant&#39;s molecular interaction partner with a Y2H screen. Even though Y2H screens are commonly used, there is a trend to outsource this technique to biotech companies that offer the Y2H service at a cost. This protocol provides instructions on how to perform a Y2H in any decently equipped molecular biology laboratory using standard lab techniques.

Bacterial effector proteins are important for establishing successful infections. This protocol describes the experimental identification of proteinaceous binding partners of a bacterial effector protein in its natural plant host. Identifying these effector interactions via yeast two-hybrid screens has become an important tool in unravelling molecular pathogenicity strategies.

Bir Maya İki hibrid Ekran kullanarak Olmayan ekilebilir Bitki Patojen bir Bakteriyel Efektör Fonksiyonu aydınlatmak

Unravelling the molecular mechanisms of disease manifestations is important to understand pathologies and symptom development in plant science. Bacteria ...

Competitive Genomic Screens of Barcoded Yeast Libraries

By virtue of advances in next generation sequencing technologies, we have access to new genome sequences almost daily. The tempo of these advances is accelerating, promising greater depth and breadth. In light of these extraordinary advances, the need for fast, parallel methods to define gene function becomes ever more important. Collections of genome-wide deletion mutants in yeasts and E. coli have served as workhorses for functional characterization of gene function, but this approach is not scalable, current gene-deletion approaches require each of the thousands of genes that comprise a genome to be deleted and verified. Only after this work is complete can we pursue high-throughput phenotyping. Over the past decade, our laboratory has refined a portfolio of competitive, miniaturized, high-throughput genome-wide assays that can be performed in parallel. This parallelization is possible because of the inclusion of DNA 'tags', or 'barcodes,' into each mutant, with the barcode serving as a proxy for the mutation and one can measure the barcode abundance to assess mutant fitness. In this study, we seek to fill the gap between DNA sequence and barcoded mutant collections. To accomplish this we introduce a combined transposon disruption-barcoding approach that opens up parallel barcode assays to newly sequenced, but poorly characterized microbes. To illustrate this approach we present a new Candida albicans barcoded disruption collection and describe how both microarray-based and next generation sequencing-based platforms can be used to collect 10,000 - 1,000,000 gene-gene and drug-gene interactions in a single experiment.

We have developed comprehensive, unbiased genome-wide screens to understand gene-drug and gene-environment interactions. Methods for screening these mutant collections are presented.

Barkodlu Maya Kütüphaneler Rekabet Genomik Ekranlar

By virtue of advances in next generation sequencing technologies, we have access to new genome sequences almost daily.  The tempo of these advances is ...

Biyoloji

High Throughput Yeast Strain Phenotyping with Droplet-Based RNA Sequencing

The powerful tools available to edit yeast genomes have made this microbe a valuable platform for engineering. While it is now possible to construct libraries of millions of genetically distinct strains, screening for a desired phenotype remains a significant obstacle. With existing screening techniques, there is a tradeoff between information output and throughput, with high-throughput screening typically being performed on one product of interest. Therefore, we present an approach to accelerate strain screening by adapting single cell RNA sequencing to isogenic picoliter colonies of genetically engineered yeast strains. To address the unique challenges of performing RNA sequencing on yeast cells, we culture isogenic yeast colonies within hydrogels and spheroplast prior to performing RNA sequencing. The RNA sequencing data can be used to infer yeast phenotypes and sort out engineered pathways. The scalability of our method addresses a critical obstruction in microbial engineering.

A bottleneck in the &#8216;design-build-test&#8217; cycle of microbial engineering is the speed at which we can perform functional screens of strains. We describe a high-throughput method for strain screening applied to hundreds to thousands of yeast cells per experiment that utilizes droplet-based RNA sequencing.

Damlacık Tabanlı RNA Sıralamaile Yüksek EldeLi Maya Zorlanma Fenotilik

The powerful tools available to edit yeast genomes have made this microbe a valuable platform for engineering. While it is now possible to construct ...

Biyomühendislik

Rapid Assembly of Multi-Gene Constructs using Modular Golden Gate Cloning

The Golden Gate cloning method enables the rapid assembly of multiple genes in any user-defined arrangement. It utilizes type IIS restriction enzymes that cut outside of their recognition sites and create a short overhang. This modular cloning (MoClo) system uses a hierarchical workflow in which different DNA parts, such as promoters, coding sequences (CDS), and terminators, are first cloned into an entry vector. Multiple entry vectors then assemble into transcription units. Several transcription units then connect into a multi-gene plasmid. The Golden Gate cloning strategy is of tremendous advantage because it allows scar-less, directional, and modular assembly in a one-pot reaction. The hierarchical workflow typically enables the facile cloning of a large variety of multi-gene constructs with no need for sequencing beyond entry vectors. The use of fluorescent protein dropouts enables easy visual screening. This work provides a detailed, step-by-step protocol for assembling multi-gene plasmids using the yeast modular cloning (MoClo) kit. We show optimal and suboptimal results of multi-gene plasmid assembly and provide a guide for screening for colonies. This cloning strategy is highly applicable for yeast metabolic engineering and other situations in which multi-gene plasmid cloning is required.

The goal of this protocol is to provide a detailed, step-by-step guide for assembling multi-gene constructs using the modular cloning system based on Golden Gate cloning. It also gives recommendations on critical steps to ensure optimal assembly based on our experiences.

Modüler Altın Kapı Klonlama Kullanılarak Çok Genli Yapıların Hızlı Montajı

The Golden Gate cloning method enables the rapid assembly of multiple genes in any user-defined arrangement. It utilizes type IIS restriction enzymes that ...

<meta charset="utf8"></head><body>Maya Yüzey Görüntüleme Teknikleri ile Protein Fonksiyonlarının Optimize Edilmesi

Protein Engineering by Yeast Surface Display

Protein engineering enables the improvement of existing functions of a given protein or the generation of novel functions. One of the most widely used and versatile tools in the protein engineering field is yeast surface display, where a pool of randomized proteins is expressed on the surface of yeast. The linkage of phenotype (e.g., binding of the yeast-displayed protein to the antigen of interest) and genotype (the plasmid encoding for the protein variant) enables selection of this library for desired properties and subsequent sequencing of enriched variants. By combining magnetic bead selection with flow cytometric sorting, protein variants with enhanced binding to a target antigen can be selected and enriched. Notably, in addition to affinity maturation, binding to a target can also be achieved without any initial binding affinity. Here, we provide a step-by-step protocol that covers all essential parts of a yeast surface display selection campaign and gives examples of typical yeast surface display results. We demonstrate that yeast surface display is a broadly applicable and robust method that can be established in any molecular biology laboratory with access to flow cytometry.

<meta charset="utf8"></head><body>Maya Yüzey Ekranı ile Protein Mühendisliği

This protocol describes the essential steps for conducting yeast surface display selection campaigns to enrich protein variants binding to an antigen of interest.

Maya Yüzey Ekranı ile Protein Mühendisliği

Protein engineering enables the improvement of existing functions of a given protein or the generation of novel functions. One of the most widely used and ...

Yüksek verim Maya Plazmid overekspresyonu Ekran

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Maya Overexpression çiftleşme tabanlı Kütüphane tarama

Bir Maya 2-melez ekran Protein etkileşimleri karşılaştırmak için bir toplu iş

Ana-Patojen protein-protein etkileşimleri ve Peyzaj karakterize etmek Karşılaştırmalı Bir Yaklaşım

Maya Toksisitesi ve Bastırıcı Ekranlar Kullanılarak Bakteriyel Efektör Proteinlertarafından Hedeflenen Konak Yollarının Belirlenmesi

Bir Maya İki hibrid Ekran kullanarak Olmayan ekilebilir Bitki Patojen bir Bakteriyel Efektör Fonksiyonu aydınlatmak

Barkodlu Maya Kütüphaneler Rekabet Genomik Ekranlar

Damlacık Tabanlı RNA Sıralamaile Yüksek EldeLi Maya Zorlanma Fenotilik

Modüler Altın Kapı Klonlama Kullanılarak Çok Genli Yapıların Hızlı Montajı

Maya Yüzey Ekranı ile Protein Mühendisliği

Maya Yüzey Ekranı ile Protein Mühendisliği