This work was supported by the Australian Research Council [DP11010246].

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The authors declare that they have no competing financial interests.

During chromosome duplication, the replication machinery will encounter various impediments that prevent its progress. To ensure the entire single-origin chromosome is replicated, bacteria have numerous pathways for repair of the DNA that then enables the replisome to be reloaded20,21. Lesions, single stranded breaks, double stranded breaks and proteins tightly bound to the DNA may each be dealt with using a dedicated pathway, although there is likely to be significant overlap in these pathways. The most commo...

DNA複製時には、レプリソームは、その進行を阻害し、DNA上の障害に直面しています。病変とのギャップだけでなく、異常な構造を含むDNA損傷は、1を進むからレプリソームを防ぐことができます。最近、DNAに結合したタンパク質は、フォークの進行2を複製する障害の最も一般的な供給源であることが見出されました。核ブロックでレプリソームの出会いを次のイベントの知識は以前から知られている場所で生きている細胞の染色体にそのようなブロックを誘導することができないことによって制限されてきた。 インビトロ分析は、速度論的挙動の理解を強化しましたそれは核閉塞3、ならびにレプリソーム自体4,5のメカニズムの詳細を満たしているアクティブレプリソームの。レプリケーションの修理の現在の理解は、一般的に、インビボ 6-8 にプラスミドDNAを用いて、損傷剤としてのUVで行われ、研究されています</s&gt;アップ。それはin vivoで核ブロックに遭遇した後、DNAの修復に関与することができるタンパク質は、一般的に複製ブロックの明確な原因による修復経路内の分子イベントにばらつきがあるかどうか、これらの研究から理解されているがまだ残っています決断される。 ここでは、核のブロックは蛍光プレッサーオペレータシステム（FROS）を使用して、染色体の特定の場所に確立することを可能にするシステムを説明します。私たちは、Eの歪みを利用します9番染色体に組み込まれた240 のtetO部位の配列を持っていた大腸菌 。アレイ内のそれぞれのtetO部位は、アレイ内のRecA媒介性の再結合を防止することにより、配列の安定性を高めるために、それに隣接する10塩基対のランダムな配列を有します。この配列、およびそれの変形は、元々 E.を理解するために使用しました。 大腸菌染色体ダイナミクス10,11が、その後preveに適応させましたntのin vivoでの複製12。アレイは安定に維持されるとのTetR 10,12によって結合されたときに複製フォークの100％に近いブロックすることが見出されています。 in vitroでの類似のlacO配列の使用は、この短い配列は生体内 13 にはあまり効果的であったが、22のサイトは、複製の90％を阻止するのに十分であったとして、いくつかのを発見しました。核の閉塞を作成するために、アレイを適応させるために、リプレッサータンパク質は非常にそれがその後、バリケードを作成するために、配列に特異的に結合する最適化された条件の下で過剰生産されなければなりません。 Tetリプレッサーの蛍光タグ付けされた変異体が使用される場合、閉塞の形成、およびその後の放出は、蛍光顕微鏡を使用することによって監視することができます。各セルの複製のステータスが単一焦点が配列のコピーは1つだけのセル内に存在すると、複数の病巣がアクティブ複製の指標である意味見病巣の数によって示されています。このアクティブ再核の閉塞がレプリソームは、配列を続行するために十分なオペレーター部位のためのTetRの結合親和性を低下させる無償誘導物質の添加により逆転されたときに襞が有効になっています。リプレッサータンパク質は、まだアレイの現在複数のコピーを可視化することができる十分に高い親和性でDNAに結合することができます。 核の閉塞でのイベントのより複雑な詳細は中立中立的な2次元アガロースゲル電気泳動及びサザンハイブリダイゼーション14-16を使用して検出することができます。これらの技術は、集団を横切るDNA構造の分析を可能にします。複製イベントの間に形成される中間体、および潜在的に修復されないままであるが、可視化することができます。複製フォークが17,18を退行する際に利用制限酵素およびプローブを変化させることにより、中間体は、アレイ領域ではなく、アレイの上流だけでなく可視化することができます。ザ回帰はレプリソーム解離に続いて行われます。先頭とラギング新生鎖は4ウェイDNA構造（ホリデイジャンクショ​​ン）で得られたテンプレート鎖同時に再アニールとして互いにテンプレート鎖とアニールとは別。 このシステムを使用して、このブロック18を検出したときに複製フォークが安定しないことが示されています。また、レプリソーム部品の温度に敏感な誘導体は、それが崩壊した後、複製フォークの再読み込みを防止するために利用することができます。ブロックが確立されると、歪みがレプリソームの同期失活及びリロードの制御予防を確実にするために、非許容温度にシフトすることができます。この温度誘導不活性化は、所定の時間で崩壊した集団内のフォークのすべてを確実にし、レプリソームは、DNAがどのように処理されるか、崩壊、そして何を再する必要があるときに何が起こるかの評価を可能にしますDNA複製のプロセスを開始します。 ここで説明するシステムの利点は、核のブロックが完全に可逆的であるということです。従って、核ブロックから回復する細胞の能力を追跡することが可能です。細胞へのアンヒドロを添加すると、複製フォークが通過進行させる、タイトなリプレッサーの結合を軽減し、細胞は生存能力を取り戻すために。閉塞の緩和は5分後、10分以内に顕微鏡によって中性中性二次元アガロースゲル電気泳動により可視化することができます。また、生存率分析は、複製閉塞から回復する株の能力を明らかにし、増殖し続けることができます。 ここに記載の実験手順で使用される菌株の遺伝的背景を変更することによって、閉塞のこのタイプの修復経路を解明することができます。

<table><tbody><tr><td>Tryptone</td><td>Sigma-Aldrich</td><td>16922</td><td>Growth media component</td></tr><tr><td>Sodium Chloride</td><td>VWR</td><td>27810.364</td><td>Growth media component</td></tr><tr><td>Yeast extract</td><td>Sigma-Aldrich</td><td>92144</td><td>Growth media component</td></tr><tr><td>Potassium phosphate monobasic</td><td>Sigma-Aldrich</td><td>P9791</td><td>Growth media component; Potassium buffer component</td></tr><tr><td>Potassium phosphate dibasic</td><td>Sigma-Aldrich</td><td>P3786</td><td>Growth media component; Potassium buffer component</td></tr><tr><td>L-Arabinose</td><td>Sigma-Aldrich</td><td>A3256</td><td>For induction of TetR-YFP production</td></tr><tr><td>Anhydrotetracycline hydrochloride</td><td>Sigma-Aldrich</td><td>37919</td><td>Release of replication bloackage</td></tr><tr><td>Axioskop 2 Fluorescence microscope</td><td>Zeiss</td><td>452310</td><td>Visualization of cells</td></tr><tr><td>eYFP filter set</td><td>Chroma Technology</td><td>41028</td><td>Visualization of YFP</td></tr><tr><td>CCD camera</td><td>Hamamatsu</td><td>Orca-AG</td><td>Visualization of cells</td></tr><tr><td>MetaMorph&amp;nbsp; Software (Molecular Devices)</td><td>SDR Scientific</td><td>31282</td><td>Version 7.8.0.0 used in the preparation of this manuscript</td></tr><tr><td>Agarose</td><td>Bioline</td><td>BIO-41025</td><td>For agarose plugs and gel electrophoresis</td></tr><tr><td>Original Glass Water Repellent (Rain-X)</td><td>Autobarn</td><td>DIO1470</td><td>For agarose plug manufacture</td></tr><tr><td>TRIS</td><td>VWR</td><td>VWRC103157P</td><td>TE, TBE buffer component</td></tr><tr><td>Ethylene diaminetetraacetic acid</td><td>Ajax Finechem</td><td>AJA180</td><td>0.5 M EDTA disodium salt solution adjusted to pH 8.0 with NaOH.</td></tr><tr><td>Sodium azide</td><td>Sigma-Aldrich</td><td>S2002</td><td>Bacteriostatic agent</td></tr><tr><td>Hydrochloric acid</td><td>Sigma-Aldrich</td><td>258148</td><td>TE buffer component</td></tr><tr><td>Sodium deoxycholate</td><td>Sigma-Aldrich</td><td>D6750</td><td>Cell lysis buffer component</td></tr><tr><td>N-Lauroylsarcosine sodium salt (Sarkosyl)</td><td>Sigma-Aldrich</td><td>L5125</td><td>Cell lysis and ESP buffer component</td></tr><tr><td>Rnase A</td><td>Sigma-Aldrich</td><td>R6513</td><td>Cell lysis buffer component</td></tr><tr><td>Lysozyme</td><td>Amresco</td><td>6300</td><td>Cell lysis buffer component</td></tr><tr><td>Proteinase K</td><td>Amresco</td><td>AM0706</td><td>ESP buffer component</td></tr><tr><td>Sub-Cell Model 192 Cell</td><td>BioRad</td><td>1704507</td><td>Electrophoresis system</td></tr><tr><td>UV transilluminator 2000</td><td>BioRad</td><td>1708110</td><td>Visualization of DNA</td></tr><tr><td>Ethidium Bromide</td><td>BioRad</td><td>1610433</td><td>Visualization of DNA</td></tr><tr><td>Boric acid</td><td>VWR</td><td>PROL20185.360</td><td>TBE component</td></tr><tr><td>Hybond-XL nylon memrbane</td><td>Amersham</td><td>RPN203S</td><td>Zeta-Probe Memrbane (BioRad 1620159) can also be used</td></tr><tr><td>3MM Whatman chromatography paper</td><td>GE Healthcare Life Sciences</td><td>3030690</td><td>Southern blotting</td></tr><tr><td>HL-2000 Hybrilinker</td><td>UVP</td><td>95-0031-01/02</td><td>Crosslinking of DNA and hybridization</td></tr><tr><td>Deoxyribonucleic acid from salmon sperm</td><td>Sigma-Aldrich</td><td>31149</td><td>Hybridization buffer component</td></tr><tr><td>Sodium hydroxide</td><td>Sigma-Aldrich</td><td>S5881</td><td>Denaturation buffer component</td></tr><tr><td>Trisodium citrate dihydrate</td><td>VWR</td><td>PROL27833.363</td><td>Transfer buffer</td></tr><tr><td>Sodium dodecyl sulphate (SDS)</td><td>Amresco</td><td>227</td><td>Wash buffer component</td></tr><tr><td>Bovine serum albumin</td><td>Sigma-Aldrich</td><td>A7906</td><td>Hybridization buffer component</td></tr><tr><td>Random Hexamer Primers</td><td>Bioline</td><td>BIO-38028</td><td></td></tr><tr><td>Klenow fragment</td><td>New England BioLabs</td><td>M0212L</td><td></td></tr><tr><td>dNTP Set</td><td>Bioline</td><td>BIO-39025</td><td></td></tr><tr><td>Adenosine 5&amp;rsquo;-triphosphate-&lt;sup&gt;32&lt;/sup&gt;P-ATP</td><td>PerkinElmer</td><td>BLU502A</td><td></td></tr><tr><td>Storage Phosphor Screen</td><td>GE Healthcare Life Sciences</td><td>GEHE28-9564-76</td><td>BAS-IP MS 3543 E multipurpose standard 35 cm x 43 cm screen</td></tr><tr><td>Typhoon FLA 7000</td><td>GE Healthcare Life Sciences</td><td>28-9558-09</td><td>Visualization of blot</td></tr><tr><td>Hybridization bottle</td><td>UVP</td><td>07-0194-02</td><td>35 mm x 300 mm</td></tr></tbody></table>

inducing a site specific replication blockage in e. coli using a fluorescent repressor operator system

密結合タンパク質と様々な病変を含むDNA上に存在する障害物は、深刻な細胞の複製機構の進行を阻害することができます。レプリソームの失速は、複製フォークの崩壊につながる、一部または全部のいずれか、染色体からの解離につながることができます。この崩壊からの回復は、細胞に対して正確に完全な染色体重複、その後分割の必要性です。崩壊が発生したときしたがって、細胞はDNAのフォークを復元し、レプリケーションが高い忠実度で完了することができるようにするために行われる多様な機構を進化させました。以前は、細菌におけるこれらの複製修復経路は、既知の部位に局在化されていないという欠点を有するUVダメージを用いて研究されています。この原稿は、foは複製の失速と崩壊を誘導することができる部位特異的なタンパク質のブロックを作成するために、蛍光プレッサーオペレータシステム（FROS）を利用するシステムを説明します大腸菌でRKS。複製の状態は、蛍光顕微鏡およびDNA複製中間体を用いて、単一の生細胞内で可視化することができる方法のプロトコルの詳細は、2次元のアガロースゲル電気泳動によって分析することができます。レプリソーム構成要素（ 例えば DnaBts）の温度感受性変異体は、複製フォークの同期崩壊を誘導するためにシステムに組み込むことができます。さらに、これらのプロセスに関与している組換えタンパク質およびヘリカーゼの役割は、このシステム内での遺伝子ノックアウトを用いて研究することができます。

FROSは、生きた細胞12,18で可視化することが複製中間体を可能に誘導、サイト固有の核ブロックです。細胞をサンプリングするための一般的な実験設計は、図1に示されている。遺伝的背景におけるサンプリングおよび変化のタイミングは、このようなブロックの修復を研究するための汎用性の高いシステムにします。概略的には、18以前に...

Watch this Scientific Journal Video about Inducing a Site Specific Replication Blockage in E. coli Using a Fluorescent Repressor Operator System at JoVE.com

Inducing a Site Specific Replication Blockage in E. coli Using a Fluorescent Repressor Operator System

We describe here a system utilizing a site-specific, reversible in vivo protein block to stall and collapse replication forks in Escherichia coli. The establishment of the replication block is evaluated by fluorescence microscopy and neutral-neutral 2-dimensional agarose gel electrophoresis is used to visualize replication intermediates.

サイトを誘導する具体的なレプリケーション閉塞中<i&gt; E。大腸菌</i&gt;蛍光プレッサーオペレータシステムの使用

Obstacles present on DNA, including tightly-bound proteins and various lesions, can severely inhibit the progression of the cell&#39;s replication ...

inducing-site-specific-replication-blockage-e-coli-using-fluorescent

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

Inducing a Site Specific Replication Blockage in E. coli Using a Fluorescent Repressor Operator System

8.9K ビュー.  University of Newcastle. We describe here a system utilizing a site-specific, reversible in vivo protein block to stall and collapse replication forks in Escherichia coli. The establishment of the replication block is evaluated by fluorescence microscopy and neutral-neutral 2-dimensional agarose gel electrophoresis is used to visualize replication intermediates.

ビデオ: Inducing a Site Specific Replication Blockage in E. coli Using a Fluorescent Repressor Operator System

Determination of the Optimal Chromosomal Location(s) for a DNA Element in Escherichia coli Using a Novel Transposon-mediated Approach

The optimal chromosomal position(s) of a given DNA element was/were determined by transposon-mediated random insertion followed by fitness selection. In bacteria, the impact of the genetic context on the function of a genetic element can be difficult to assess. Several mechanisms, including topological effects, transcriptional interference from neighboring genes, and/or replication-associated gene dosage, may affect the function of a given genetic element. Here, we describe a method that permits the random integration of a DNA element into the chromosome of Escherichia coli and select the most favorable locations using a simple growth competition experiment. The method takes advantage of a well-described transposon-based system of random insertion, coupled with a selection of the fittest clone(s) by growth advantage, a procedure that is easily adjustable to experimental needs. The nature of the fittest clone(s) can be determined by whole-genome sequencing on a complex multi-clonal population or by easy gene walking for the rapid identification of selected clones. Here, the non-coding DNA region DARS2, which controls the initiation of chromosome replication in E. coli, was used as an example. The function of DARS2 is known to be affected by replication-associated gene dosage; the closer DARS2 gets to the origin of DNA replication, the more active it becomes. DARS2 was randomly inserted into the chromosome of a DARS2-deleted strain. The resultant clones containing individual insertions were pooled and competed against one another for hundreds of generations. Finally, the fittest clones were characterized and found to contain DARS2 inserted in close proximity to the original DARS2 location.

Determination of the Optimal Chromosomal Locations for a DNA Element in Escherichia coli Using a Novel Transposon-mediated Approach

Here, the power of a transposon-mediated random insertion of a non-coding DNA element was used to resolve its optimal chromosomal position.

新規トランスポゾンを介したアプローチを使用してエシェリヒア属大腸菌DNA 要素の最適な染色体の場所の決定

The optimal chromosomal position(s) of a given DNA element was/were determined by transposon-mediated random insertion followed by fitness selection. In ...

遺伝学

Testing the Role of Multicopy Plasmids in the Evolution of Antibiotic Resistance

Multicopy plasmids are extremely abundant in prokaryotes but their role in bacterial evolution remains poorly understood. We recently showed that the increase in gene copy number per cell provided by multicopy plasmids could accelerate the evolution of plasmid-encoded genes. In this work, we present an experimental system to test the ability of multicopy plasmids to promote gene evolution. Using simple molecular biology methods, we constructed a model system where an antibiotic resistance gene can be inserted into Escherichia coli MG1655, either in the chromosome or on a multicopy plasmid. We use an experimental evolution approach to propagate the different strains under increasing concentrations of antibiotics and we measure survival of bacterial populations over time. The choice of the antibiotic molecule and the resistance gene is so that the gene can only confer resistance through the acquisition of mutations. This &#34;evolutionary rescue&#34; approach provides a simple method to test the potential of multicopy plasmids to promote the acquisition of antibiotic resistance. In the next step of the experimental system, the molecular bases of antibiotic resistance are characterized. To identify mutations responsible for the acquisition of antibiotic resistance we use deep DNA sequencing of samples obtained from whole populations and clones. Finally, to confirm the role of the mutations in the gene under study, we reconstruct them in the parental background and test the resistance phenotype of the resulting strains.

Here we present an experimental method to test the role of multicopy plasmids in the evolution of antibiotic resistance.

抗生物質耐性の進化のマルチコピー プラスミドのロールのテスト

Multicopy plasmids are extremely abundant in prokaryotes but their role in bacterial evolution remains poorly understood. We recently showed that the ...

Engineering Adherent Bacteria by Creating a Single Synthetic Curli Operon

The method described here consists in redesigning E. coli adherence properties by assembling the minimum number of curli genes under the control of a strong and metal-overinducible promoter, and in visualizing and quantifying the resulting gain of bacterial adherence. This method applies appropriate engineering principles of abstraction and standardization of synthetic biology, and results in the BBa_K540000 Biobrick (Best new Biobrick device, engineered, iGEM 2011).
The first step consists in the design of the synthetic operon devoted to curli overproduction in response to metal, and therefore in increasing the adherence abilities of the wild type strain. The original curli operon was modified in silico in order to optimize transcriptional and translational signals and escape the "natural" regulation of curli. This approach allowed to test with success our current understanding of curli production. Moreover, simplifying the curli regulation by switching the endogenous complex promoter (more than 10 transcriptional regulators identified) to a simple metal-regulated promoter makes adherence much easier to control.
The second step includes qualitative and quantitative assessment of adherence abilities by implementation of simple methods. These methods are applicable to a large range of adherent bacteria regardless of biological structures involved in biofilm formation. Adherence test in 24-well polystyrene plates provides a quick preliminary visualization of the bacterial biofilm after crystal violet staining. This qualitative test can be sharpened by the quantification of the percentage of adherence. Such a method is very simple but more accurate than only crystal violet staining as described previously 1 with both a good repeatability and reproducibility. Visualization of GFP-tagged bacteria on glass slides by fluorescence or laser confocal microscopy allows to strengthen the results obtained with the 24-well plate test by direct observation of the phenomenon.

The design of a synthetic operon encoding both the secretory apparatus and the structural monomers of curli fibers is described. Overproduction of these amyloids and adherent polymers allows a measurable gain of adherence of the E. coli chassis1. Easy ways to visualize and quantify adherence are explained.

単一の合成Curliオペロンを作成して付着細菌を工学

The  method described here consists in redesigning E. coli adherence properties by assembling the minimum number of  curli genes under the control of a ...

生物工学

Producing Gene Deletions in Escherichia coli by P1 Transduction with Excisable Antibiotic Resistance Cassettes

A first approach to study the function of an unknown gene in bacteria is to create a knock-out of this gene. Here, we describe a robust and fast protocol for transferring gene deletion mutations from one Escherichia coli strain to another by using generalized transduction with the bacteriophage P1. This method requires that the mutation be selectable (e.g., based on gene disruptions using antibiotic cassette insertions). Such antibiotic cassettes can be mobilized from a donor strain and introduced into a recipient strain of interest to quickly and easily generate a gene deletion mutant. The antibiotic cassette can be designed to include flippase recognition sites that allow the excision of the cassette by a site-specific recombinase to produce a clean knock-out with only a ~100-base-pair-long scar sequence in the genome. We demonstrate the protocol by knocking out the tamA gene encoding an assembly factor involved in autotransporter biogenesis and test the effect of this knock-out on the biogenesis and function of two trimeric autotransporter adhesins. Though gene deletion by P1 transduction has its limitations, the ease and speed of its implementation make it an attractive alternative to other methods of gene deletion.

Producing Gene Deletions in Escherichia coli by P1 Transduction with Excisable Antibiotic Resistance Cassettes

Here we present a protocol for the use of pre-existing antibiotic resistance-cassette deletion constructs as a basis for making deletion mutants in other E. coli strains. Such deletion mutations can be mobilized and inserted into the corresponding locus of a recipient strain using P1 bacteriophage transduction.

物品税課税対象の抗生物質耐性カセット P1 伝達による大腸菌の遺伝子の削除を生産

A first approach to study the function of an unknown gene in bacteria is to create a knock-out of this gene. Here, we describe a robust and fast protocol ...

In Situ Measurement and Correlation of Cell Density and Light Emission of Bioluminescent Bacteria

There is a considerable number of bacterial species capable of emitting light. All of them share the same gene cluster, namely the lux operon. Despite this similarity, these bacteria show extreme variations in characteristics like growth behavior, intensity of light emission or regulation of bioluminescence. The method presented here is a newly developed assay that combines recording of cell growth and bioluminescent light emission intensity over time utilizing a plate reader. The resulting growth and light emission characteristics can be linked to important features of the respective bacterial strain, such as quorum sensing regulation. The cultivation of a range of bioluminescent bacteria requires a specific medium (e.g., artificial sea water medium) and defined temperatures. The easy to handle, non-bioluminescent standard-research bacterium Escherichia coli (E. coli), on the other hand, can be cultivated inexpensively in high quantities in laboratory scale. Exploiting E. coli by introducing a plasmid containing the whole lux operon can simplify experimental conditions and additionally opens up many possibilities for future applications. The expression of all lux genes utilizing an E. coli expression strain was achieved by construction of an expression plasmid via Gibson cloning and insertion of four fragments containing seven lux genes and three rib genes of the lux-rib operon into a pET28a vector. E. coli based lux gene expression can be induced and controlled via Isopropyl-&#946;-D-thiogalactopyranosid (IPTG) addition resulting in bioluminescent E. coli cells. The advantages of this system are to avoid quorum sensing regulation restrictions and complex medium compositions along with non-standard growth conditions, such as defined temperatures. This system enables analysis of lux genes and their interplay, by the exclusion of the respective gene from the lux operon, or even addition of novel genes, exchanging the luxAB genes from one bacterial strain by another, or analyzing protein complexes, such as luxCDE.

In Situ Measurement and Correlation of Cell Density and Light Emission of Bioluminescent Bacteria

Bioluminescent bacteria regulate light production through a variety of mechanisms, such as quorum sensing. This novel method allows the in situ investigation of bioluminescence and the correlation of light emission to cell density. An artificial bioluminescent Escherichia coli system allows the characterization of lux operons, Lux proteins, and their interplay.

その場で測定と細胞密度の相関、発光細菌の発光

There is a considerable number of bacterial species capable of emitting light. All of them share the same gene cluster, namely the lux operon. Despite ...

生化学

A Fluorescence-based Method to Study Bacterial Gene Regulation in Infected Tissues

Bacterial virulence genes are often regulated at the transcriptional level by multiple factors that respond to different environmental signals. Some factors act directly on virulence genes; others control pathogenesis by adjusting the expression of downstream regulators or the accumulation of signals that affect regulator activity. While regulation has been studied extensively during in vitro growth, relatively little is known about how gene expression is adjusted during infection. Such information is important when a particular gene product is a candidate for therapeutic intervention. Transcriptional approaches like quantitative, real-time RT-PCR and RNA-Seq are powerful ways to examine gene expression on a global level but suffer from many technical challenges including low abundance of bacterial RNA compared to host RNA, and sample degradation by RNases. Evaluating regulation using fluorescent reporters is relatively easy and can be multiplexed with fluorescent proteins with unique spectral properties. The method allows for single-cell, spatiotemporal analysis of gene expression in tissues that exhibit complex three-dimensional architecture and physiochemical gradients that affect bacterial regulatory networks. Such information is lost when data are averaged over the bulk population. Herein, we describe a method for quantifying gene expression in bacterial pathogens in situ. The method is based on simple tissue processing and direct observation of fluorescence from reporter proteins. We demonstrate the utility of this system by examining the expression of Staphylococcus aureus thermonuclease (nuc), whose gene product is required for immune evasion and full virulence ex vivo and in vivo. We show that nuc-gfp is strongly expressed in renal abscesses and reveal heterogeneous gene expression due in part to apparent spatial regulation of nuc promoter activity in abscesses fully engaged with the immune response. The method can be applied to any bacterium with a manipulatable genetic system and any infection model, providing valuable information for preclinical studies and drug development.

Described here is a method for analyzing bacterial gene expression in animal tissues at a cellular level. This method provides a resource for studying the phenotypic diversity occurring within a bacterial population in response to the tissue environment during an infection.

感染した組織の細菌の遺伝子の規則を勉強する蛍光ベースのメソッド

Bacterial virulence genes are often regulated at the transcriptional level by multiple factors that respond to different environmental signals. Some ...

Phage-mediated Delivery of Targeted sRNA Constructs to Knock Down Gene Expression in E. coli

RNA-mediated knockdowns are widely used to control gene expression. This versatile family of techniques makes use of short RNA (sRNA) that can be synthesized with any sequence and designed to complement any gene targeted for silencing. Because sRNA constructs can be introduced to many cell types directly or using a variety of vectors, gene expression can be repressed in living cells without laborious genetic modification. The most common RNA knockdown technology, RNA interference (RNAi), makes use of the endogenous RNA-induced silencing complex (RISC) to mediate sequence recognition and cleavage of the target mRNA. Applications of this technique are therefore limited to RISC-expressing organisms, primarily eukaryotes. Recently, a new generation of RNA biotechnologists have developed alternative mechanisms for controlling gene expression through RNA, and so made possible RNA-mediated gene knockdowns in bacteria. Here we describe a method for silencing gene expression in E. coli that functionally resembles RNAi. In this system a synthetic phagemid is designed to express sRNA, which may designed to target any sequence. The expression construct is delivered to a population of E. coli cells with non-lytic M13 phage, after which it is able to stably replicate as a plasmid. Antisense recognition and silencing of the target mRNA is mediated by the Hfq protein, endogenous to E. coli. This protocol includes methods for designing the antisense sRNA, constructing the phagemid vector, packaging the phagemid into M13 bacteriophage, preparing a live cell population for infection, and performing the infection itself. The fluorescent protein mKate2 and the antibiotic resistance gene chloramphenicol acetyltransferase (CAT) are targeted to generate representative data and to quantify knockdown effectiveness.

Phage-mediated Delivery of Targeted sRNA Constructs to Knock Down Gene Expression in E. coli

We describe a method to knock down gene expression in a growing population of E. coli cells using sequence-targeted sRNA expression cassettes delivered by an M13 phagemid vector.

ターゲティングされたのsRNAのファージ媒介送達はにおける遺伝子発現をノックダウンするために構築します<em&gt; E。大腸菌</em

RNA-mediated knockdowns are widely used to control gene expression. This versatile family of techniques makes use of short RNA (sRNA) that can be ...

生物学

Generation of Marked and Markerless Mutants in Model Cyanobacterial Species

Cyanobacteria are ecologically important organisms and potential platforms for production of biofuels and useful industrial products. Genetic manipulation of cyanobacteria, especially model organisms such as Synechocystis sp. PCC6803 and Synechococcus sp. PCC7002, is a key tool for both basic and applied research. Generation of unmarked mutants, whereby chromosomal alterations are introduced into a strain via insertion of an antibiotic resistance cassette (a manipulatable fragment of DNA containing one or more genes), followed by subsequent removal of this cassette using a negative selectable marker, is a particularly powerful technique. Unmarked mutants can be repeatedly genetically manipulated, allowing as many alterations to be introduced into a strain as desired. In addition, the absence of genes encoding antibiotic resistance proteins in the mutated strain is desirable, as it avoids the possibility of 'escape' of antibiotic resistant organisms into the environment. However, detailed methods for repeated rounds of genetic manipulation of cyanobacteria are not well described in the scientific literature. Here we provide a comprehensive description of this technique, which we have successfully used to generate mutants with multiple deletions, single point mutations within a gene of interest and insertion of novel gene cassettes.

Introducing multiple genomic alterations into cyanobacteria is an essential tool in the development of strains for industrial and basic research purposes. We describe a system for generating unmarked mutants in the model cyanobacterial species Synechocystis sp. PCC6803 and marked mutants in Synechococcus sp. PCC7002.

モデルシアノバクテリア種のマークとマーカーレス変異体の生成

Cyanobacteria are ecologically important organisms and potential platforms for production of biofuels and useful industrial products. Genetic manipulation ...

Live-Cell Imaging of Transcriptional Activity at DNA Double-Strand Breaks

DNA double-strand breaks (DSB) are the most severe type of DNA damage. Despite the catastrophic consequences on genome integrity, it remains so far elusive how DSBs affect transcription. A reason for this was the lack of suitable tools to simultaneously monitor transcription and the induction of a genic DSB with sufficient temporal and spatial resolution. This work describes a set of new reporters that directly visualize transcription in live cells immediately after the induction of a DSB in the DNA template. Bacteriophage RNA stem-loops are employed to monitor the transcription with single-molecule sensitivity. For targetting the DSB to a specific gene region, the reporter genes are engineered to contain a single recognition sequence of the homing endonuclease I-SceI, otherwise absent from the human genome. A single copy of each reporter gene was integrated into the genome of human cell lines. This experimental system allows the detection of single RNA molecules generated by the canonical gene transcription or by DNA break-induced transcription initiation. These reporters provide an unprecedented opportunity for interpreting the reciprocal interactions between transcription and DNA damage and to disclose hitherto unappreciated aspects of DNA break-induced transcription.

This protocol presents a new reporter gene system and the experimental setup to detect transcription at DNA double-strand breaks with single-molecule sensitivity.

DNA二重鎖破断時の転写活性の生細胞イメージング

DNA double-strand breaks (DSB) are the most severe type of DNA damage. Despite the catastrophic consequences on genome integrity, it remains so far ...

CRISPR Interference-Based Gene Silencing: A Technique for Targeted Repression of Gene Function in Pathogenic Leptospira

Source: Fernandes, L. G. V., et al. <a href="https://jove.unicartagenaproxy.elogim.com/v/62631">Application of CRISPR Interference (CRISPRi) for Gene Silencing in Pathogenic Species of Leptospira.</a> J. Vis. Exp. (2021).
This video demonstrates a CRISPR interference-based gene silencing in pathogenic Leptospira. Targeted repression of gene expression using a CRISPR/Cas system that blocks mRNA formation helps identify candidate genes for pathological and pharmacological studies.

CRISPR干渉に基づく遺伝子サイレンシング:病原性レプトスピラにおける遺伝子機能の標的抑制技術

Source: Fernandes, L. G. V., et al. Application of CRISPR Interference (CRISPRi) for Gene Silencing in Pathogenic Species of Leptospira. J. Vis. Exp. ...

サイトを誘導する具体的なレプリケーション閉塞中

要約

さらに動画を探す

この動画の章

新規トランスポゾンを介したアプローチを使用してエシェリヒア属大腸菌DNA 要素の最適な染色体の場所の決定

抗生物質耐性の進化のマルチコピープラスミドのロールのテスト

単一の合成Curliオペロンを作成して付着細菌を工学

物品税課税対象の抗生物質耐性カセット P1 伝達による大腸菌の遺伝子の削除を生産

その場で測定と細胞密度の相関、発光細菌の発光

感染した組織の細菌の遺伝子の規則を勉強する蛍光ベースのメソッド

ターゲティングされたのsRNAのファージ媒介送達はにおける遺伝子発現をノックダウンするために構築します

モデルシアノバクテリア種のマークとマーカーレス変異体の生成

DNA二重鎖破断時の転写活性の生細胞イメージング

CRISPR干渉に基づく遺伝子サイレンシング:病原性レプトスピラにおける遺伝子機能の標的抑制技術

サイトを誘導する具体的なレプリケーション閉塞中

要約

さらに動画を探す

この動画の章

新規トランスポゾンを介したアプローチを使用してエシェリヒア属大腸菌DNA 要素の最適な染色体の場所の決定

抗生物質耐性の進化のマルチコピー プラスミドのロールのテスト

単一の合成Curliオペロンを作成して付着細菌を工学

物品税課税対象の抗生物質耐性カセット P1 伝達による大腸菌の遺伝子の削除を生産

その場で測定と細胞密度の相関、発光細菌の発光

感染した組織の細菌の遺伝子の規則を勉強する蛍光ベースのメソッド

ターゲティングされたのsRNAのファージ媒介送達はにおける遺伝子発現をノックダウンするために構築します

モデルシアノバクテリア種のマークとマーカーレス変異体の生成

DNA二重鎖破断時の転写活性の生細胞イメージング

CRISPR干渉に基づく遺伝子サイレンシング:病原性レプトスピラにおける遺伝子機能の標的抑制技術

抗生物質耐性の進化のマルチコピープラスミドのロールのテスト