This study was financially supported by the Korean Ministry of Science, ICT and Future Planning (Intelligent Synthetic Biology Center program 2012M3A6A8054887). P. Kim was supported by a fellowship from the Catholic University of Korea (2015).

1.機器の準備<ol><li>入口ポートと出口ポートを含むケモスタットジャー（150〜250 ml）または三角フラスコ（250ml）に取得します。シリコンチューブ10〜100ミリリットル/ hrの流量を可能にするとポートを接続します。任意に、エアベント、吹出口、および温度制御された水の入口および出口ポートを使用します。 </li><li>攪拌および温度制御を提供するケモスタットジャーに適した装置を得る（または回転振とう培養器を使用します）。 </li><li>新鮮な培地を提供し、文化を収集するために、2つの蠕動ポンプを取得します。 </li><li>媒体排出口と吸気口を含む貯蔵瓶（10〜20 L）を得ます。 </li><li>希釈率（; L / S 13チューブすなわち 、ID 0.8ミリメートル、流量範囲0.06〜36ミリリットル/分）に適したシリコンチューブを入手します。 </li></ol> 2.ミディアム調製と滅菌<ol><li>初期ミディアム<ol><li> 0.3グラムのグルコース0.08 gでNH 4を溶解Cl、0.05グラムのNaCl、0.75グラムのNa 2 HPO 4・2H 2 O、および0.3グラムのKH 2 PO 4 90 mlの蒸留水ケモスタットジャー中（DW）。 </li><li>クランプを使用してチューブと一緒にケモスタットジャーを封印。通気口を密閉しないでください。 </li><li> 121℃で15分間オートクレーブ中でケモスタットの瓶を滅菌します。滅菌後、室温でケモスタットjarファイルを格納します。 </li><li> 0.02 gでのMgSO 4・7H 2 O 0.01グラムのCaCl 2、及び10mlのDW（溶液A）中の0.1mgのチアミンを溶解します。 </li><li>シリンジおよび予め滅菌シリンジフィルター（0.45μmの細孔のフィルター）を使用してフィルタソリューションA。 </li><li> Aは、ケモスタットジャーにろ液溶液を加えます。 </li></ol></li><li>ストレスミディアム<ol><li> 30gのグルコース8 gでNH 4 Clで5 gでのNaCl 75 G 2のNa HPO 4・を溶解2H 2 O、30gのKH 2 PO 4、300gをコハク酸二ナトリウム六水和物（ 硫酸ナトリウム・コハク・6H &lt;サブ&gt; 2 O;貯留瓶で9.9 L DWで、この実験で使用したスト​​レッサー）。 </li><li>クランプを使用してチューブと一緒に貯留瓶を密封します。通気口を密閉しないでください。 </li><li> 121℃で15分間オートクレーブ内の貯留瓶を滅菌します。滅菌後、室温でjarファイルを格納します。 </li><li> 2 gでのMgSO 4・7H 2 O、1gのCaCl 2を、100mlのDW（溶液A）中の10 mgのチアミンを溶解します。 </li><li>シリンジおよび予め滅菌シリンジフィルター（0.45μmの孔径フィルタ）のフィルタ溶液A。 </li><li> Aが貯留瓶に濾液溶液を加えます。 </li><li>無菌的に貯留瓶に滅菌シリコンチューブを接続し、蠕動ポンプを取り付けます。 </li></ol></li><li>高ストレスミディアム<ol><li>セクション2.2のようにメディアを準備したが、ストレッサーの高い濃度で（ すなわち 、コハク酸適応3-5 G / L以上）。 注：このプロトコルは、ストレス番目への適応のためのものですATは、媒体を介して配信することができます。例えば、温度、攪拌、または照明などの物理的ストレス因子の場合には、栽培はそれに応じて設計する必要があります。 </li></ol></li></ol> 3.初期栽培<ol><li>野生型E.の単一コロニーを接種します初期の培地4mlを含有する15mLの試験管中で大腸菌 。 </li><li> 37℃で12時間および220 rpmで振盪インキュベーター中で試験管をインキュベートします。 </li><li>無菌的に転送ケモスタット瓶に前培養液の1ミリリットル。 </li><li> 6時間37℃で、通気（空気50ml /分）、撹拌（200rpm）しを提供する、ケモスタットの瓶をインキュベートします。 </li></ol> 4.ストレス適応<ol><li>無菌的ケモスタットジャーにポンプからシリコンチューブの端を接続します。 </li><li>出口ポンプ（10ミリリットル/時以上）を起動し、文化を集めます。 注意：文化は、通常、4-8時間の初期培養後、対数期にあるべきです。 </li><li> Chの出口管から培養物の光学密度（600 nm）をECK。 </li><li> （0.1時間-1の希釈率に対応した10ミリリットル/時）入口ポンプを起動します。 </li><li> 24時間毎にチューブコンセントから600nmでの培養物の光学密度を確認してください。 </li><li> 96時間（9.6倍の売上高）以上のためのケモスタットを操作します。光学濃度が安定している場合、高負荷培地を含有するリザーバーを交換します。光学濃度が0.2未満である場合には、6時間供給入口ポンプを停止させます。入口ポンプを再起動し、光学濃度が0.2以上であることを確認してください。 </li><li>徐々に高いストレッサー濃度を含有するリザーバーに変更することにより、ストレッサーの濃度を高めます。 </li><li>それはマイルストーンに到達するたびに適応文化のサンプルを取る（ 例えば 、100グラム/ Lのコハク酸ストレスに適応株）さらなるゲノム解析のために、と店。 </li><li>サンプル保存のために、滅菌した80％グリセロールsolutiで培養試料（0.5ミリリットル）を混ぜます（0.5ミリリットル）に、-80℃で保管してください。 注：微生物がALE処理中にストレッサーを分解する能力を取得した場合、発酵ジャーの中ストレッサー濃度は、新鮮な貯水池のものと同じではありません。 </li></ol>ストレスに適応した株の5シングルコロニーの分離<ol><li>同じストレッサーを含む寒天平板培地（1.6％寒天）を準備し、媒体の同一濃度で。 </li><li>ケモスタットからの出口文化（0.1 ml）をプレートし、16時間37℃でインキュベートします。 </li><li>滅菌爪楊枝を用いてプレートから単一コロニーを選択し、同じストレッサーを含む15ミリリットル試験管内およびケモスタットと同じ中濃度でそれらを接種し、6時間インキュベートします。 </li><li>転写培地50 mlを含む250 mlの三角フラスコに培養液1 mlです。収穫培養液0.5ミリリットル毎に1時間、および600nmでのODを測定します。適応straiの成長率を比較ストレッサー与えられた野生型株のものとn個。 </li></ol>

<ol><li>Rando, O. J., Verstrepen, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Timescales+of+genetic+and+epigenetic+inheritance%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Timescales of genetic and epigenetic inheritance.</a> Cell. 128, 655-668 (2007).</li>
<li>Kim, H. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Short-term+differential+adaptation+to+anaerobic+stress+via+genomic+mutations+by+Escherichia+coli+strains+K-12+and+B+lacking+alcohol+dehydrogenase%5BTitle%5D)+AND+%22Front+Microbiol%22%5BJournal%5D)">Short-term differential adaptation to anaerobic stress via genomic mutations by Escherichia coli strains K-12 and B lacking alcohol dehydrogenase.</a> Front Microbiol. 5, 476(2014).</li>
<li>Mendizabal, I., Keller, T. E., Zeng, J., Yi, S. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Epigenetics+and+evolution%5BTitle%5D)+AND+%22Integr+Comp+Biol%22%5BJournal%5D)">Epigenetics and evolution.</a> Integr Comp Biol. 54, 31-42 (2014).</li>
<li>Lee, J. Y., Seo, J., Kim, E. S., Lee, H. S., Kim, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Adaptive+evolution+of+Corynebacterium+glutamicum+resistant+to+oxidative+stress+and+its+global+gene+expression+profiling%5BTitle%5D)+AND+%22Biotechnol+Lett%22%5BJournal%5D)">Adaptive evolution of Corynebacterium glutamicum resistant to oxidative stress and its global gene expression profiling.</a> Biotechnol Lett. 35, 709-717 (2013).</li>
<li>Lee, J. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Artificial+oxidative+stress-tolerant+Corynebacterium+glutamicum%5BTitle%5D)+AND+%22AMB+Express%22%5BJournal%5D)">Artificial oxidative stress-tolerant Corynebacterium glutamicum.</a> AMB Express. 4, 15(2014).</li>
<li>Narang, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+steady+states+of+microbial+growth+on+mixtures+of+substitutable+substrates+in+a+chemostat%5BTitle%5D)+AND+%22J+Theor+Biol%22%5BJournal%5D)">The steady states of microbial growth on mixtures of substitutable substrates in a chemostat.</a> J Theor Biol. 190, 241-261 (1998).</li>
<li>Kwon, Y. D., Kim, S., Lee, S. Y., Kim, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Long-term+continuous+adaptation+of+Escherichia+coli+to+high+succinate+stress+and+transcriptome+analysis+of+the+tolerant+strain%5BTitle%5D)+AND+%22J+Biosci+Bioeng%22%5BJournal%5D)">Long-term continuous adaptation of Escherichia coli to high succinate stress and transcriptome analysis of the tolerant strain.</a> J Biosci Bioeng. 111, 26-30 (2011).</li>
<li>Barrick, J. E., Lenski, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Genome+dynamics+during+experimental+evolution%5BTitle%5D)+AND+%22Nat+Rev+Genet%22%5BJournal%5D)">Genome dynamics during experimental evolution.</a> Nat Rev Genet. 14, 827-839 (2013).</li>
<li>Li, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+Sequence+Alignment%2FMap+format+and+SAMtools%5BTitle%5D)+AND+%22Bioinformatics%22%5BJournal%5D)">The Sequence Alignment/Map format and SAMtools.</a> Bioinformatics. 25, 2078-2079 (2009).</li>
<li>McKenna, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+Genome+Analysis+Toolkit%3A+a+MapReduce+framework+for+analyzing+next-generation+DNA+sequencing+data%5BTitle%5D)+AND+%22Genome+Res%22%5BJournal%5D)">The Genome Analysis Toolkit: a MapReduce framework for analyzing next-generation DNA sequencing data.</a> Genome Res. 20, 1297-1303 (2010).</li>
<li>Deatherage, D. E., Barrick, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Identification+of+mutations+in+laboratory-evolved+microbes+from+next-generation+sequencing+data+using+breseq%5BTitle%5D)+AND+%22Methods+Mol+Biol%22%5BJournal%5D)">Identification of mutations in laboratory-evolved microbes from next-generation sequencing data using breseq.</a> Methods Mol Biol. 1151, 165-188 (2014).</li>
</ol>

The authors have nothing to disclose.

微生物は、それらの急速な成長率と遺伝的多様性のほとんどすべての環境に適応することが可能です。適応研究室の進化は、与えられた条件の下で有益な自然突然変異を保有する生物個体を選択する方法を提供している、設計条件の下で進化した微生物を可能にします。 ケモスタット法は、以下の理由により転送技術より人工的に駆動される進化を達成するために、よ?...

微生物が生存し、多様な環境に適応することができます。重度のストレスの下では、適応はランダムなゲノム変異とそれに続く正の選択1-3により有益な表現型の買収を経て発生する可能性があります。したがって、微生物細胞は、「適応進化」と呼ばれる最適な成長のために代謝または調節ネットワークを変更することにより、適合させることができます。このようなスーパーバグや堅牢な微生物株の発生の発生などの最近の重要な微生物の傾向は、非常に密接にストレスの多い条件下での適応進化に関連しています。定義された実験室条件の下で、我々は分子進化のメカニズムを研究、さらには様々なアプリケーションのための微生物の進化の方向を制御することができます。多細胞生物とは異なり、単細胞生物は、次のような理由から、適応実験室進化（ALE）に適しています：彼らは多くの人口を維持するため、すぐに再生成し、それはホーンを作成し、維持するのは簡単ですogeneous環境。 DNA配列決定技術と高スループット技術の最近の進歩と組み合わせることで、ALEは、全身規制の変更につながるゲノム変化を直接観察することができます。変異動態​​と人口の多様性も観測可能です。遺伝子工学戦略はALE株4,5の分析から決定することができます。 ケモスタット培養は定常状態の細胞を取得し、発酵工程6の生産性を増加させるために使用される方法です。新鮮な培地を加​​え、培養液は、工程（後者は、媒体とバイオマスを含む）中に回収されます。長期ケモスタット培養は、しかし、文化の定常状態の生産性を変化させ、培養物（ 図1a）の間に自然突然変異と選択の蓄積をもたらします。様々な選択圧（ストレス）下で、突然変異の蓄積が促進されます。長期的にストレスの緩やかな増加<html>ケモスタットは、コロニーの固体培地からの転送と、液体媒体からシリアル転送（繰り返しなど 、温度、pH、浸透圧、栄養飢餓、酸化、有毒な最終製品として、与えられたストレス因子に対して機能変異の連続選択を提供しますバッチ培養）は、研究者が進化した微生物（ 図1Bおよび1C）を取得することを可能にします。ケモスタット培養は、複雑な方法が必要ですが、多様性のプール（複製と集団サイズの数）は、コロニー転送とシリアル転送技術によって得られるものよりも高くなっています。個々の細胞への安定的なストレス暴露とケモスタット培養物（定常状態）の間、細胞状態の変化を減少させたバッチ培養ベースの技術と比較して、ALEの他の利点です。高コハク酸条件にさらされた大腸菌のストレス誘発ALEは、この記事で紹介されています。 ジル/ ftp_upload / 54446 / 54446fig1.jpg &quot;/&gt; 図1：適応実験室進化の方法 （A）ケモスタット;。 （B）シリアル転送。 （C）コロニー転送。トップ数字はALEのための方法の概念を示し、下部の図は、ALEの間に成長した細胞の数を示している。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54446/54446fig1large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a>

<table><tbody><tr><td>Mini-chemostat fermentor</td><td>Biotron Inc.</td><td>-</td><td>manufactured by special order</td></tr><tr><td>silicon tubing</td><td>Cole-Parmer</td><td>Masterflex L/S 13</td><td>tubing size can be varied depending on the dilution rate and the size of fermentor jar.</td></tr><tr><td>reservoir jar</td><td>Bellco</td><td>Media storage bottle</td><td>20 L</td></tr><tr><td>chemicals</td><td>Sigma-Aldrich</td><td>-</td><td>reagent grade</td></tr><tr><td>glucose</td><td>Sigma-Aldrich</td><td>G5767</td><td>ACS reagent</td></tr><tr><td>NH&lt;sub&gt;4&lt;/sub&gt;Cl</td><td>Sigma-Aldrich</td><td>A9434</td><td>for molecular biology, suitable for cell culture, &amp;ge;99.5%</td></tr><tr><td>NaCl</td><td>Sigma-Aldrich</td><td>746398</td><td>ACS reagent, &amp;ge;99%</td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;&amp;middot;2H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma-Aldrich</td><td>4272</td><td>98.5-101%</td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>795488</td><td>ACS reagent, &amp;ge;99%</td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt;&amp;middot;7H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma-Aldrich</td><td>230391</td><td>ACS reagent, &amp;ge;98%</td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>793639</td><td>ACS reagent, &amp;ge;96%</td></tr><tr><td>thiamine&amp;middot;HCl</td><td>Sigma-Aldrich</td><td>T4625</td><td>reagent grade, &amp;ge;99%</td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;&amp;middot;succinate&amp;middot;6H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma-Aldrich</td><td>S2378</td><td>ReagentPlus, &amp;ge;99%</td></tr></tbody></table>

procedure for adaptive laboratory evolution of microorganisms using a chemostat

Natural evolution involves genetic diversity such as environmental change and a selection between small populations. Adaptive laboratory evolution (ALE) refers to the experimental situation in which evolution is observed using living organisms under controlled conditions and stressors; organisms are thereby artificially forced to make evolutionary changes. Microorganisms are subject to a variety of stressors in the environment and are capable of regulating certain stress-inducible proteins to increase their chances of survival. Naturally occurring spontaneous mutations bring about changes in a microorganism's genome that affect its chances of survival. Long-term exposure to chemostat culture provokes an accumulation of spontaneous mutations and renders the most adaptable strain dominant. Compared to the colony transfer and serial transfer methods, chemostat culture entails the highest number of cell divisions and, therefore, the highest number of diverse populations. Although chemostat culture for ALE requires more complicated culture devices, it is less labor intensive once the operation begins. Comparative genomic and transcriptome analyses of the adapted strain provide evolutionary clues as to how the stressors contribute to mutations that overcome the stress. The goal of the current paper is to bring about accelerated evolution of microorganisms under controlled laboratory conditions.

高コハク酸のストレス適応のため、野生型E.大腸菌 W3110株は、D = 0.1時間-1 270日間（ 図2）でケモスタットで培養しました。 <img alt="図2" src="/files/ftp_upload/54446/54446fig2.jpg" /> 図2：Eの高コハク酸ストレス適応ケモスタット培養を使用して大腸菌 W3110。細い矢印は、ストレッサーの濃度が増加し?...

Watch this Scientific Journal Video about Procedure for Adaptive Laboratory Evolution of Microorganisms Using a Chemostat at JoVE.com

ケモスタットを使用した微生物の適応的実験室進化の手順 |遺伝学 |ビデオ

Procedure for Adaptive Laboratory Evolution of Microorganisms Using a Chemostat

ここでは、ケモスタット培養を使用した条件下での微生物の適応実験室進化を取得するためのプロトコルを提示します。また、進化した株のゲノム解析について説明します。

ケモスタットを用いた微生物の適応研究所進化のための手順

Natural evolution involves genetic diversity such as environmental change and a selection between small populations. Adaptive laboratory evolution (ALE) ...

procedure-for-adaptive-laboratory-evolution-microorganisms-using

Research

Education

JoVE Journal

JoVE Core

Biology

Genetics

Evolution

Evolutionary History

SCIENTISTS IN ACTION

14.3K ビュー.  Korea Research Institute of Bioscience and Biotechnology (KRIBB). ここでは、ケモスタット培養を使用した条件下での微生物の適応実験室進化を取得するためのプロトコルを提示します。また、進化した株のゲノム解析について説明します。 

ビデオ: Procedure for Adaptive Laboratory Evolution of Microorganisms Using a Chemostat

Design and Use of a Low Cost, Automated Morbidostat for Adaptive Evolution of Bacteria Under Antibiotic Drug Selection

We describe a low cost, configurable morbidostat for characterizing the evolutionary pathway of antibiotic resistance. The morbidostat is a bacterial culture device that continuously monitors bacterial growth and dynamically adjusts the drug concentration to constantly challenge the bacteria as they evolve to acquire drug resistance. The device features a working volume of ~10 ml and is fully automated and equipped with optical density measurement and micro-pumps for medium and drug delivery. To validate the platform, we measured the stepwise acquisition of trimethoprim resistance in Escherichia coli MG 1655, and integrated the device with a multiplexed microfluidic platform to investigate cell morphology and antibiotic susceptibility. The approach can be up-scaled to laboratory studies of antibiotic drug resistance, and is extendible to adaptive evolution for strain improvements in metabolic engineering and other bacterial culture experiments.

We describe a low cost, configurable morbidostat that enables the characterization of antibiotic drug resistance by dynamically adjusting the drug concentration. The device can be integrated with a multiplexed microfluidic platform. The approach can be scaled up for laboratory antibiotic drug resistance studies.

設計と低コストの使用、自動Morbidostat細菌の適応進化のための抗生物質選択下

We describe a low cost, configurable morbidostat for characterizing the evolutionary pathway of antibiotic resistance. The morbidostat is a bacterial ...

遺伝学

CRISPR/Cas9-mediated Targeted Integration In Vivo Using a Homology-mediated End Joining-based Strategy

As a promising genome editing platform, the CRISPR/Cas9 system has great potential for efficient genetic manipulation, especially for targeted integration of transgenes. However, due to the low efficiency of homologous recombination (HR) and various indel mutations of non-homologous end joining (NHEJ)-based strategies in non-dividing cells, in vivo genome editing remains a great challenge. Here, we describe a homology-mediated end joining (HMEJ)-based CRISPR/Cas9 system for efficient in vivo precise targeted integration. In this system, the targeted genome and the donor vector containing homology arms (~800 bp) flanked by single guide RNA (sgRNA) target sequences are cleaved by CRISPR/Cas9. This HMEJ-based strategy achieves efficient transgene integration in mouse zygotes, as well as in hepatocytes in vivo. Moreover, a HMEJ-based strategy offers an efficient approach for correction of fumarylacetoacetate hydrolase (Fah) mutation in the hepatocytes and rescues Fah-deficiency induced liver failure mice. Taken together, focusing on targeted integration, this HMEJ-based strategy provides a promising tool for a variety of applications, including generation of genetically modified animal models and targeted gene therapies.

CRISPR/Cas9-mediated Targeted Integration In Vivo Using a Homology-mediated End Joining-based Strategy

The clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9 (CRISPR/Cas9) system provides a promising tool for genetic engineering, and opens up the possibility of targeted integration of transgenes. We describe a homology-mediated end joining (HMEJ)-based strategy for efficient DNA targeted integration in vivo and targeted gene therapies using CRISPR/Cas9.

CRISPR/Cas9 仲介の対象となる統合生体内で相同性を介した終わり参加ベースの戦略を使用して

As a promising genome editing platform, the CRISPR/Cas9 system has great potential for efficient genetic manipulation, especially for targeted integration ...

Genome-wide Surveillance of Transcription Errors in Eukaryotic Organisms

Accurate transcription is required for the faithful expression of genetic information. Surprisingly though, little is known about the mechanisms that control the fidelity of transcription. To fill this gap in scientific knowledge, we recently optimized the circle-sequencing assay to detect transcription errors throughout the transcriptome of Saccharomyces cerevisiae, Drosophila melanogaster, and Caenorhabditis elegans. This protocol will provide researchers with a powerful new tool to map the landscape of transcription errors in eukaryotic cells so that the mechanisms that control the fidelity of transcription can be elucidated in unprecedented detail.

This protocol provides researchers with a new tool to monitor the fidelity of transcription in multiple model organisms.

真核生物の転写エラーのゲノム全体の監視

Accurate transcription is required for the faithful expression of genetic information. Surprisingly though, little is known about the mechanisms that ...

Designing Automated, High-throughput, Continuous Cell Growth Experiments Using eVOLVER

Continuous culture methods enable cells to be grown under quantitatively controlled environmental conditions, and are thus broadly useful for measuring fitness phenotypes and improving our understanding of how genotypes are shaped by selection. Extensive recent efforts to develop and apply niche continuous culture devices have revealed the benefits of conducting new forms of cell culture control. This includes defining custom selection pressures and increasing throughput for studies ranging from long-term experimental evolution to genome-wide library selections and synthetic gene circuit characterization. The eVOLVER platform was recently developed to meet this growing demand: a continuous culture platform with a high degree of scalability, flexibility, and automation. eVOLVER provides a single standardizing platform that can be (re)-configured and scaled with minimal effort to perform many different types of high-throughput or multi-dimensional growth selection experiments. Here, a protocol is presented to provide users of the eVOLVER framework a description for configuring the system to conduct a custom, large-scale continuous growth experiment. Specifically, the protocol guides users on how to program the system to multiplex two selection pressures &#8212; temperature and osmolarity &#8212; across many eVOLVER vials in order to quantify fitness landscapes of Saccharomyces cerevisiae mutants at fine resolution. We show how the device can be configured both programmatically, through its open-source web-based software, and physically, by arranging fluidic and hardware layouts. The process of physically setting up the device, programming the culture routine, monitoring and interacting with the experiment in real-time over the internet, sampling vials for subsequent offline analysis, and post experiment data analysis are detailed. This should serve as a starting point for researchers across diverse disciplines to apply eVOLVER in the design of their own complex and high-throughput cell growth experiments to study and manipulate biological systems.

The eVOLVER framework enables high-throughput continuous microbial culture with high resolution and dynamic control over experimental parameters. This protocol demonstrates how to apply the system to conduct a complex fitness experiment, guiding users on programming automated control over many individual cultures, measuring, collecting, and interacting with experimental data in real-time.

EVOLVER を用いた自動、高スループット、連続細胞増殖実験の設計

Continuous culture methods enable cells to be grown under quantitatively controlled environmental conditions, and are thus broadly useful for measuring ...

Daily Transfers, Archiving Populations, and Measuring Fitness in the Long-Term Evolution Experiment with Escherichia coli

The Long-Term Evolution Experiment (LTEE) has followed twelve populations of Escherichia coli as they have adapted to a simple laboratory environment for more than 35 years and 77,000 bacterial generations. The setup and procedures used in the LTEE epitomize reliable and reproducible methods for studying microbial evolution. In this protocol, we first describe how the LTEE populations are transferred to fresh medium and cultured each day. Then, we describe how the LTEE populations are regularly checked for possible signs of contamination and archived to provide a permanent frozen "fossil record" for later study. Multiple safeguards included in these procedures are designed to prevent contamination, detect various problems when they occur, and recover from disruptions without appreciably setting back the progress of the experiment. One way that the overall tempo and character of evolutionary changes are monitored in the LTEE is by measuring the competitive fitness of populations and strains from the experiment. We describe how co-culture competition assays are conducted and provide both a spreadsheet and an R package (fitnessR) for calculating relative fitness from the results. Over the course of the LTEE, the behaviors of some populations have changed in interesting ways, and new technologies like whole-genome sequencing have provided additional avenues for investigating how the populations have evolved. We end by discussing how the original LTEE procedures have been updated to accommodate or take advantage of these changes. This protocol will be useful for researchers who use the LTEE as a model system for studying connections between evolution and genetics, molecular biology, systems biology, and ecology. More broadly, the LTEE provides a tried-and-true template for those who are beginning their own evolution experiments with new microbes, environments, and questions.

Daily Transfers, Archiving Populations, and Measuring Fitness in the Long-Term Evolution Experiment with Escherichia coli

This protocol describes how to maintain the Escherichia coli Long-Term Evolution Experiment (LTEE) by performing its daily transfers and periodic freeze-downs and how to conduct competition assays to measure fitness improvements in evolved bacteria. These procedures can serve as a template for researchers starting their own microbial evolution experiments.

大腸菌を用いた長期進化実験における毎日の移動、集団のアーカイブ、および適応度の測定

The Long-Term Evolution Experiment (LTEE) has followed twelve populations of Escherichia coli as they have adapted to a simple laboratory environment for ...

Generating Controlled, Dynamic Chemical Landscapes to Study Microbial Behavior

We demonstrate a method for the generation of controlled, dynamic chemical pulses&#8213;where localized chemoattractant becomes suddenly available at the microscale&#8213;to create micro-environments for microbial chemotaxis experiments. To create chemical pulses, we developed a system to introduce amino acid sources near-instantaneously by photolysis of caged amino acids within a polydimethylsiloxane (PDMS) microfluidic chamber containing a bacterial suspension. We applied this method to the chemotactic bacterium, Vibrio ordalii, which can actively climb these dynamic chemical gradients while being tracked by video microscopy. Amino acids, rendered biologically inert (&#39;caged&#39;) by chemical modification with a photoremovable protecting group, are uniformly present in the suspension but not available for consumption until their sudden release, which occurs at user-defined points in time and space by means of a near-UV-A focused LED beam. The number of molecules released in the pulse can be determined by a calibration relationship between exposure time and uncaging fraction, where the absorption spectrum after photolysis is characterized by using UV-Vis spectroscopy. A nanoporous polycarbonate (PCTE) membrane can be integrated into the microfluidic device to allow the continuous removal by flow of the uncaged compounds and the spent media. A strong, irreversible bond between the PCTE membrane and the PDMS microfluidic structure is achieved by coating the membrane with a solution of 3-aminopropyltriethoxysilane (APTES) followed by plasma activation of the surfaces to be bonded. A computer-controlled system can generate user-defined sequences of pulses at different locations and with different intensities, so as to create resource landscapes with prescribed spatial and temporal variability. In each chemical landscape, the dynamics of bacterial movement at the individual scale and their accumulation at the population level can be obtained, thereby allowing the quantification of chemotactic performance and its effects on bacterial aggregations in ecologically relevant environments.

A protocol for the generation of dynamic chemical landscapes by photolysis within microfluidic and millifluidic setups is presented. This methodology is suitable to study diverse biological processes, including the motile behavior, nutrient uptake, or adaptation to chemicals of microorganisms, both at the single cell and population level.

微生物の挙動を研究するための制御された動的化学景観の生成

We demonstrate a method for the generation of controlled, dynamic chemical pulses&#8213;where localized chemoattractant becomes suddenly available at the ...

生物工学

Automated Microbial Cultivation and Adaptive Evolution using Microbial Microdroplet Culture System (MMC)

Conventional microbial cultivation methods usually have cumbersome operations, low throughput, low efficiency, and large consumption of labor and reagents. Moreover, microplate-based high-throughput cultivation methods developed in recent years have poor microbial growth status and experiment parallelization because of their low dissolved oxygen, poor mixture, and severe evaporation and thermal effect. Due to many advantages of micro-droplets, such as small volume, high throughput, and strong controllability, the droplet-based microfluidic technology can overcome these problems, which has been used in many kinds of research of high-throughput microbial cultivation, screening, and evolution. However, most prior studies remain&#160;at the stage of laboratory construction and application. Some key issues, such as high operational requirements, high construction difficulty, and lack of automated integration technology, restrict the wide application of droplet microfluidic technology in microbial research. Here, an automated Microbial Microdroplet Culture system (MMC) was successfully developed based on droplet microfluidic technology, achieving the integration of functions such as inoculation, cultivation, online monitoring, sub-cultivation, sorting, and sampling required by the process of microbial droplet cultivation. In this protocol, wild-type Escherichia coli (E. coli) MG1655 and a methanol-essential E. coli strain (MeSV2.2) were taken as examples to introduce how to use the MMC to conduct automated and relatively high-throughput microbial cultivation and adaptive evolution in detail. This method is easy to operate, consumes less labor and reagents, and has high experimental throughput and good data parallelity, which has great advantages compared with conventional cultivation methods. It provides a low-cost, operation-friendly, and result-reliable experimental platform for scientific researchers to conduct related microbial research.

Automated Microbial Cultivation and Adaptive Evolution using Microbial Microdroplet Culture System MMC

This protocol describes how to use the Microbial Microdroplet Culture system (MMC) to conduct automated microbial cultivation and adaptive evolution. MMC can cultivate and sub-cultivate microorganisms automatically and continuously and monitor online their growth with relatively high throughput and good parallelization, reducing labor and reagent consumption.

微生物微小液滴培養システム(MMC)を用いた微生物の自動培養と適応進化

Conventional microbial cultivation methods usually have cumbersome operations, low throughput, low efficiency, and large consumption of labor and ...

The Use of Chemostats in Microbial Systems Biology

Cells regulate their rate of growth in response to signals from the external world. As the cell grows, diverse cellular processes must be coordinated including macromolecular synthesis, metabolism and ultimately, commitment to the cell division cycle. The chemostat, a method of experimentally controlling cell growth rate, provides a powerful means of systematically studying how growth rate impacts cellular processes - including gene expression and metabolism - and the regulatory networks that control the rate of cell growth. When maintained for hundreds of generations chemostats can be used to study adaptive evolution of microbes in environmental conditions that limit cell growth. We describe the principle of chemostat cultures, demonstrate their operation and provide examples of their various applications. Following a period of disuse after their introduction in the middle of the twentieth century, the convergence of genome-scale methodologies with a renewed interest in the regulation of cell growth and the molecular basis of adaptive evolution is stimulating a renaissance in the use of chemostats in biological research.

Cell growth rate is a regulated process and a primary determinant of cell physiology. Continuous culturing using chemostats enables extrinsic control of cell growth rate by nutrient limitation facilitating the study of molecular networks that control cell growth and how those networks evolve to optimize cell growth.

微生物システム生物学のケモスタットの使用

Cells regulate their rate of growth in response to signals from the external world. As the cell grows, diverse cellular processes must be coordinated ...

生物学

Design and Use of Multiplexed Chemostat Arrays

Chemostats are continuous culture systems in which cells are grown in a tightly controlled, chemically constant environment where culture density is constrained by limiting specific nutrients.1,2 Data from chemostats are highly reproducible for the measurement of quantitative phenotypes as they provide a constant growth rate and environment at steady state. For these reasons, chemostats have become useful tools for fine-scale characterization of physiology through analysis of gene expression3-6 and other characteristics of cultures at steady-state equilibrium.7 Long-term experiments in chemostats can highlight specific trajectories that microbial populations adopt during adaptive evolution in a controlled environment. In fact, chemostats have been used for experimental evolution since their invention.8 A common result in evolution experiments is for each biological replicate to acquire a unique repertoire of mutations.9-13 This diversity suggests that there is much left to be discovered by performing evolution experiments with far greater throughput. 
We present here the design and operation of a relatively simple, low cost array of miniature chemostats&mdash;or ministats&mdash;and validate their use in determination of physiology and in evolution experiments with yeast. This approach entails growth of tens of chemostats run off a single multiplexed peristaltic pump. The cultures are maintained at a 20 ml working volume, which is practical for a variety of applications. It is our hope that increasing throughput, decreasing expense, and providing detailed building and operation instructions may also motivate research and industrial application of this design as a general platform for functionally characterizing large numbers of strains, species, and growth parameters, as well as genetic or drug libraries.

We developed and validated a small-footprint array of miniature chemostats built from readily available parts for low cost. Physiological and experimental evolution results were similar to larger volume chemostats. The ministat array provides a compact, inexpensive, and accessible platform for traditional chemostat experiments, functional genomics, and chemical screening applications.

多重ケモスタットアレイの設計および使用

Chemostats are continuous culture systems in which cells are grown in a tightly controlled, chemically constant environment where culture density is ...

<meta charset="utf8"></head><body>低コストのサーモミキサーを用いた好熱剤適応のための実験的進化プロトコル

Adaptation at the Extremes of Life: Experimental Evolution with the Extremophile Archaeon Sulfolobus acidocaldarius

The archaeon Sulfolobus acidocaldarius has emerged as a promising thermophilic model system. Investigating how thermophiles adapt to changing temperatures is a key requirement, not only for understanding fundamental evolutionary processes but also for developing S. acidocaldarius as a chassis for bioengineering. One major obstacle to conducting experimental evolution with thermophiles is the expense of equipment maintenance and energy usage of traditional incubators for high-temperature growth. To address this challenge, a comprehensive experimental protocol for conducting experimental evolution in S. acidocaldarius is presented, utilizing low-cost and energy-efficient bench-top thermomixers. The protocol involves a batch culture technique with relatively small volumes (1.5 mL), enabling tracking of adaptation in multiple independent lineages. This method is easily scalable through the use of additional thermomixers. Such an approach increases the accessibility of S. acidocaldarius as a model system by reducing both initial investment and ongoing costs associated with experimental investigations. Moreover, the technique is transferable to other microbial systems for exploring adaptation to diverse environmental conditions.

<meta charset="utf8"></head><body>著者スポットライト:好熱性進化を探求するための革新的な技術を使用した微生物の適応の理解

Here, we present an experimental evolution protocol for adaptation in thermophiles utilizing low-cost, energy-efficient bench-top thermomixers as incubators. The technique is demonstrated through the characterization of temperature adaptation in Sulfolobus acidocaldarius, an archaeon with an optimal growth temperature of 75 &#176;C.

生命の極限状態での適応:極限環境微生物アーキオンSulfolobus acidocaldariusによる実験的進化

The archaeon Sulfolobus acidocaldarius has emerged as a promising thermophilic model system. Investigating how thermophiles adapt to changing temperatures ...