Yazarların laboratuvarındaki çalışmalar Deutsche Forschungsgemeinschaft (http://www.dfg.de tarafından desteklendi; CO 1139/1-1), Fonds der Chemischen Industrie (http://www.vci.de/fonds) ve Göttingen Moleküler Biyoloji Merkezi (GZMB). Yazarlar Jörg Stülke'yi yararlı yorumlar ve makalenin eleştirel okuması için kabul etmek istiyor.

1. Agar Tabaklarının, Kültür Medyasının, Kriyostockların ve Ön Kültürlerin Hazırlanması
<ol><li>Büyüme ortamı ve gerekli reaktifleri hazırlayın (bkz. malzeme ve reaktifler tablosu).</li>
	<li>B. subtilis suşlarını çizgile (örneğin. BP40 (rocG+ gudBCR amyE::PgudB-yfp) ve BP52 (rocG+ gudB+ amyE::PgudB-cfp) sırasıyla bir ve iki aktif GDH ifade eden)2 tek koloniler elde etmek için SP orta agar plakaları üzerinde yapılacak yarışma deneyinde kullanılacaktır. Plakaları gece boyunca 37 °C'de kuluçkaya yatırın.</li>
	<li>Tek kolonileri alın ve 4 ml LB sıvı ortam içeren steril kültür tüplerini aşılayın. Bakterileri gece boyunca 28 °C ve 220 rpm'de yetiştirin.</li>
	<li>Hücrelerin 28 °C'de geceleme veya sporulate olmasını önlemek için gece kültürlerini büyütün.</li>
	<li>Standart bir spektrofotometre kullanarak kültürlerin optik yoğunluğunu 600 nm (OD600)dalga boyunda ölçün.</li>
	<li>Kültürler2.0'ın 600'üne ulaştıysa, 0.75 ml'lik kültürleri 1.5 ml reaksiyon tüplerinde 0.75 ml steril% 50 gliserol çözeltisi ile karıştırın. Yaklaşık10 8 hücre/ml içeren kriyostocklar elde etmek için son OD 600 1.0 olmalıdır.</li>
	<li>Tüpleri -80 °C'de saklayın.</li>
	<li>Cfp ve yfp kodlama florofor genleri ile etiketlenmiş her bir suşun üç ön kültürünü yapın. -80 °C kriyostocklardan 1 μl hücre ile ön ürünleri (4 ml LB sıvı ortam içeren steril kültür tüpleri) aşılayın. Kültürleri bir gecede 28 °C ve 220 rpm'de kuluçkaya yatırın.</li>
</ol>2. Bakterilerin BirlikteLiği, Numune Toplama ve Numune Depolama
<ol><li>100 ml C-Glc ve CE-Glc minimal ortamı yeni hazırlayın (reaktifler ve malzemeler tablosuna bakın) ve her ortamın 20 ml'lik kısmını steril 100 ml sallama şişelerine aktarın.</li>
	<li>Bir gecede yetiştirilen 0,1 ml'lik ön ürünleri alın, 1,5 ml'lik bir cuvette 0,9 ml LB orta ile seyreltin ve OD600'übelirleyin.</li>
	<li>Yarışma deneyi için,1.0-1.5 arasında benzer bir OD 600'e sahip farklı suşların ön kültürlerini alın.</li>
	<li>Karışık hücre popülasyonları elde etmek için, uygun OD600'e sahip olan ön kültürlerin hücrelerini 20 ml C-Glc'de 0,05'in 600'ünE ve 100 ml sallama şişelerinde desteklenmiş CE-Glc minimal ortamına seyreltin. Yarışma deneyi için iki suş 1:1 oranında karıştırılmalıdır.</li>
	<li>Şişelerden 10 ml numune alın, 15 ml plastik tüplere aktarın, hücreleri standart bir masa üstü santrifüjde 4.000 x g'da 10 dakika santrifüjleme ile hasat edin ve süpernatantı atın.</li>
	<li>Hücreleri 0,5 ml taze LB ortamında yeniden biriktirin ve hücreleri steril 1,5 ml reaksiyon kabına aktarın. 0,5 ml% 50 steril gliserol ekleyin, süspansiyonu titiz bir girdapla karıştırın ve numuneleri bir sonraki tedaviye kadar -80 ° C dondurucuda saklayın.</li>
	<li>Sallama şişelerinde kalan hücreleri 37 °C ve 220 rpm'de 24 saate kadar kuluçkaya yatırın. Floroforların fotoğraf ağartmasını önlemek için sallama şişelerini karanlıkta tutun.</li>
	<li>7 saat ve 24 saat büyümeden sonra 0,1 ml'lik daha fazla örnek alın. Numunelerin1:10 seyreltmelerinin (C-Glc veya CE-Glc minimal ortamında) OD 600'lerini ölçün ve not edin.</li>
	<li>OD600/1.0 olan kriyostocklar yapmak için her kültürden uygun miktarda hücre alın. Numuneleri bir sonraki tedaviye kadar -80 °C'de saklayın.</li>
</ol>3. Nicel Analizler için Numune Tedavisi, Kaplama ve Kuluçka
<ol><li>Tüm örnekleri topladıktan sonra, kriyostockları çözün ve hücreleri% 0,9 tuzlu su çözeltisinde seyreltin (reaktifler ve malzemeler tablosuna bakın) 10-3'ekadar.</li>
	<li>SP orta agar plakaları üzerinde 10 -3 seyreltmenin0.1 ml'si plaka ve steril cam pipetler kullanarak hücreleri dağıtın.</li>
	<li>Plakaları tek koloniler ortaya çıkana kadar karanlıkta 37 °C'de bir gecede kuluçkaya yatırın.</li>
</ol>4. Kantitatif Analiz için Stereo Floresan Mikroskopisi ile Hayatta Kalanları Saymak
<ol><li>Stereo floresan mikroskobu altında hayatta kalanları sayarken daha iyi bir yönlendirme için agar plakasının altını siyah bir kalemle dört parçaya bölün.</li>
	<li>Plakayı mikroskobun altına baş aşağı yerleştirin ve soğuk ışık kaynağını kullanarak kolonileri odak noktasına getirin.</li>
	<li>Koloniler odakta olduğunda, CFP etiketli gerinimde kalan hücreleri görselleştirmek için CFPiçin uygun filtre kümesine geçin. Kolonileri bir kalemle etiketleyerek bu gerginlikte hayatta kalanları sayın ve sayıyı not edin.</li>
	<li>Etiketleri etanol ile çıkarın ve yfp etiketli gerinimde kalan hücreleri görselleştirmek için YFPfiltre kümesine geçin. Kolonileri bir kalemle etiketleyarak hayatta kalanları tekrar sayın ve sayıyı not edin.</li>
</ol>5. Yarı Saydam Analizler için Örnek Tedavi ve Mikroskopi
<ol><li>Açıklayıcı rakamlar için, SP agar plakasındaki 3.1 adımından10 -4 seyreltmenin (yaklaşık 100 hücre) 10 μl'sini tespit edin (bkz. Şekil 3).</li>
	<li>Plakaları tek koloniler ortaya çıkana kadar karanlıkta 37 °C'de bir gecede kuluçkaya yatırın.</li>
	<li>Petri kabını kapaksız olarak mikroskop altına yerleştirin ve soğuk ışık kaynağını kullanarak noktayı odak noktasına getirin.</li>
	<li>Açıklayıcı figürler için lekelerin fotoğraflarını çekin. Soğuk ışık kaynağı ile kolonilerin fotoğraflarını çekmek için uygun bir pozlama süresi seçin.</li>
	<li>Plakayı hareket ettirmeden CFP filtre kümesine değiştirin, pozlama süresini ayarlayın ve fotoğraf çekin. Aynı şeyi YFP filtre kümesiyle de yapın ve daha fazla analiz için resimleri kaydedin.</li>
</ol>6. Veri Analizi
<ol><li>Nicel veri analizleri için Excel gibi yazılımları kullanın. Sayılan kolonilere dayanarak, sarı ve mavi kolonilerin yüzdesini% 100 olarak ayarlanmış tüm koloni sayısına göre hesaplayın.</li>
	<li>Yığılmış çubuk diyagramı oluşturmak için hesaplanan sayıları kullanın (bkz. Şekil 4 ve Şekil 5). Farklı noktalardan çekilen resimlerin birleştirilmiş resimlerini oluşturmak için Adobe Photoshop gibi bir görüntü işleme programı kullanın. Alternatif olarak, <a href="http://rsbweb.nih.gov/ij/">http://rsbweb.nih.gov/ij/</a> indirilebilen serbestçe kullanılabilen ImageJ yazılımı görüntü işleme için kullanılabilir.</li>
	<li>CfP filtre kümesi ve YFP filtre kümesiyle çekilen resimleri tek bir noktadan açın. Medyadan gelen arka plan floresanlarını azaltmak için kontrastı ve parlaklığı optimize edin.</li>
	<li>Resimlerden birine gidin ve tümlerini seçin. Resmi kopyalayın ve diğer resme yapıştırın.</li>
	<li>Resimleri birleştirmek için "renk atlatma" işlevini kullanın veya kanallar sekmesini kullanarak floresan resimlerin üzerine bindirin. Farklı ortamlardan ve farklı zaman noktalarından kolonilerin birleştirilmiş resimleri, sıvı kültürü içindeki farklı suşların büyümesini temsil eder.</li>
</ol>7. Özel İpuçları: Cfp ve yfp ile Etiketlenmiş İzojenik Suşların Boya Anahtarı Deneyi ve Cocultivation
B. subtilis'teki iki florofor kodlayan genden birinin ifadesi zindeliği ve dolayısıyla bakterilerin büyüme hızını etkileyebilir. Bu nedenle, ekim sırasında bir rakip suşunun hücre popülasyonundan elimine edilmesi sadece floroforun olumsuz etkisinden kaynaklandığını dışlamak için aşağıdaki deneylerin yapılması önerilir:
<ol><li>Tüm deneyi tekrarlayın ve ters etiketli suşları birlikte kullanın(örneğin, önceki cfpetiketli suş şimdi yfp geni ile etiketlenmiştir ve bunun tersi de vardır). Ters olmasına rağmen, elde edilen sonuçlar, bir suşun diğer suşa göre seçici bir büyüme avantajına sahip olduğu önceki gözlemle karşılaştırılabilir olmalıdır.</li>
	<li>Tüm deneyi tekrarlayın ve yfp ve cfp ile etiketlenmiş izojenik suşları koordine edin (örneğin BP40 (rocG+ gudBCR amyE::PgudB-yfp) ve BP41 (rocG+ gudBCR amyE::PgudB-cfp)). Zaman içinde hücre popülasyonunun bileşimini takip ederek iki florofordan birinin bakterilerin zindeliği üzerindeki olumsuz etkisini tahmin edin.</li>
</ol>

<ol>
	<li>Buescher, F. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+network+reorganization+during+dynamic+adaptations+of+Bacillus+subtilis+metabolism.">Global network reorganization during dynamic adaptations of Bacillus subtilis metabolism.</a> Science. 335, 1099-1103 (2012).</li><li>Gunka, K., Stannek, L., Care, R. A., Commichau, F. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selection-driven+accumulation+of+suppressor+mutants+in+Bacillus+subtilis:+the+apparent+high+mutation+frequency+of+the+cryptic+gudB+gene+and+the+rapid+clonal+expansion+of+gudB++suppressors+are+due+to+growth+under+selection.">Selection-driven accumulation of suppressor mutants in Bacillus subtilis: the apparent high mutation frequency of the cryptic gudB gene and the rapid clonal expansion of gudB+ suppressors are due to growth under selection.</a> PLoS One. 8, (2013).</li><li>Al Mamum, A. A. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identity+and+function+of+a+large+gene+network+underlying+mutagenic+repair+of+DNA+breaks.">Identity and function of a large gene network underlying mutagenic repair of DNA breaks.</a> Science. 338, 1344-1348 (2012).</li><li>Koeppel, A. F., Wertheim, J. O., Barone, L., Gentile, N., Krizanc, D., Cohan, F. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Speedy+speciation+in+a+bacterial+microcosm:+new+species+can+arise+as+frequently+as+adaptations+within+a+species.">Speedy speciation in a bacterial microcosm: new species can arise as frequently as adaptations within a species.</a> ISME J. 7, 1080-1091 (2013).</li><li>Maughan, H., Nicholson, W. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+fitness+and+alteration+of+metabolic+pathways+during+Bacillus+subtilis+evolution+in+the+laboratory.">Increased fitness and alteration of metabolic pathways during Bacillus subtilis evolution in the laboratory.</a> Appl. Environ. Microbiol. 77, 4105-4118 (2011).</li><li>Burkholder, P. R., Giles, N. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induced+biochemical+mutations+in+Bacillus+subtilis.">Induced biochemical mutations in Bacillus subtilis.</a> Am. J. Bot. 34, 345-348 (1947).</li><li>McLoon, A. L., Guttenplan, S. B., Kearns, D. B., Kolter, R., Losick, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tracing+the+domestication+of+a+biofilm-forming+bacterium.">Tracing the domestication of a biofilm-forming bacterium.</a> J. Bacteriol. 193, 2027-2034 (2011).</li><li>Zeigler, D. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+origins+of+168,+W23,+and+other+Bacillus+subtilis+legacy+strains.">The origins of 168, W23, and other Bacillus subtilis legacy strains.</a> J. Bacteriol. 190, 6983-6995 (2008).</li><li>Gunka, K., Tholen, S., Gerwig, J., Herzberg, C., St&#252;lke, J., Commichau, F. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+high-frequency+mutation+in+Bacillus+subtilis:+requirements+for+the+decryptification+of+the+gudB+glutamate+dehydrogenase.">A high-frequency mutation in Bacillus subtilis: requirements for the decryptification of the gudB glutamate dehydrogenase.</a> 194, 1036-1044 (2012).</li><li>Commichau, F. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+Bacillus+subtilis+mutants+with+carbon+source-independent+glutamate+biosynthesis.">Characterization of Bacillus subtilis mutants with carbon source-independent glutamate biosynthesis.</a> J. Mol. Microbiol. Biotechnol. 12, 106-113 (2007).</li><li>Beckwith, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+suppressors+and+recovery+of+repressed+biochemical+memory.">Genetic suppressors and recovery of repressed biochemical memory.</a> J. Biol. Chem. 284, 12585-12592 (2009).</li><li>Gunka, K., Commichau, F. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+glutamate+homeostasis+in+Bacillus+subtilis:+a+complex+interplay+between+ammonium+assimilation,+glutamate+biosynthesis+and+degradation.">Control of glutamate homeostasis in Bacillus subtilis: a complex interplay between ammonium assimilation, glutamate biosynthesis and degradation.</a> Mol. Microbiol. 85, 213-224 (2012).</li><li>Yan, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protection+of+the+glutamate+pool+concentrations+in+enteric+bacteria.">Protection of the glutamate pool concentrations in enteric bacteria.</a> Proc. Natl. Acad. Sci. U.S.A. 104, 9475-9480 (2007).</li><li>Belitsky, B. R., Sonenshein, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+and+regulation+of+Bacillus+subtilis+glutamate+dehydrogenase+genes.">Role and regulation of Bacillus subtilis glutamate dehydrogenase genes.</a> J. Bacteriol. 180, 6298-6305 (1998).</li><li>Commichau, F. M., Herzberg, C., Tripal, P., Valerius, O., St&#252;lke, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+regulatory+protein-protein+interaction+governs+glutamate+biosynthesis+in+Bacillus+subtilis:+the+glutamate+dehydrogenase+RocG+moonlights+in+controlling+the+transcription+factor+GltC.">A regulatory protein-protein interaction governs glutamate biosynthesis in Bacillus subtilis: the glutamate dehydrogenase RocG moonlights in controlling the transcription factor GltC.</a> Mol. Microbiol. 65, 642-654 (2007).</li><li>Gordo, I., Perfeito, L., Sousa, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fitness+effects+of+mutations+in+bacteria.">Fitness effects of mutations in bacteria.</a> J. Mol. Microbiol. Biotechnol. 21, 20-35 (2011).</li><li>Rabatinov&#225;, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+&#948;+subunit+of+RNA+polymerase+is+required+for+rapid+changes+in+gene+expression+and+competitive+fitness+of+the+cell.">The &#948; subunit of RNA polymerase is required for rapid changes in gene expression and competitive fitness of the cell.</a> J. Bacteriol. 195, 2603-2611 (2013).</li><li>Capra, E. J., Perchuk, B. S., Skerker, J. M., Laub, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adaptive+mutations+that+prevent+crosstalk+enable+the+expansion+of+paralogous+signalling+protein+families.">Adaptive mutations that prevent crosstalk enable the expansion of paralogous signalling protein families.</a> Cell. 150, 222-232 (2012).</li><li>Garc&#237;a-Betancur, J. C., Yepes, A., Schneider, J., Lopez, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+analysis+of+Bacillus+subtilis+biofilms+using+fluorescence+microscopy+and+flow+cytometry.">Single-cell analysis of Bacillus subtilis biofilms using fluorescence microscopy and flow cytometry.</a> J. Vis. Exp. (15), (2012).</li></ol>

Yazarlar rakip finansal çıkarları olmadığını beyan ederler.

Bakterilerin rekabetçi uygunluğunu analiz etmek için çeşitli yöntemler geliştirilmiştir16. Çoğu durumda bakteriler farklı antibiyotik direnç kasetleri ile etiketlenmiştir17. Yaklaşımımıza benzer şekilde, hücrelerin antibiyotik direnç kasetleri ile etiketlanması, tanımlanmış büyüme koşulları altında kokültivasyon sırasında bakterilerin rekabetçi zindeliğinin değerlendirilmesini sağlar. Ayrıca, bu yöntem kromozom17'dekibelirli bir çekirgede birbirinden...

Toprak bakterileri genellikle esnek düzenleyici ağlar ve geniş metabolik kapasitelerle donatılmıştır. Her iki özellik de hücrelerin katabolik ve anabolik yollarını, belirli bir ekolojik nişte bulunan besinler için arkadaşları ve diğer mikroorganizmalarla rekabet edecek şekilde ayarlamalarını sağlar1. Bununla birlikte, bakteriler çevrelerine uyum sağlayamazsa, diğer mekanizmalar bir türün hayatta kalmasını açıklayabilir. Gerçekten de, birçok bakteri hızlı çoğaldığı ve popülasyonlar yüksek hücre yoğunluklarına ulaşabildiğinden, alt popülasyonlar hücrelere seçici bir büyüme avantajı sağlayan ve bu nedenle zindeliklerini artıran kendiliğinden birikmiş faydalı mutasyonlara sahip olabilir. Ayrıca, mutasyonel sıcak noktalar ve strese bağlı adaptif mutajensis, maladapted bir bakterinin evrimini kolaylaştırabilir2,3. Bu nedenle, sürekli seçim altında mutasyonların ve büyümenin birikmesi, aynı cins içinde bile muazzam mikrobiyal çeşitliliğin kökenidir4,5. Doğada olduğu gibi, bakteri genomlarının şekillendirilmesi de seçim altında sürekli ekim nedeniyle laboratuvarda meydana gelir. Bu, temel araştırmalarda ve endüstride dünya çapında kullanılan Gram-pozitif bakteri B. subtilis'in evcilleştirilmesi ile örneklenmiştir. 1940'larda B. subtilis DNA'ya zarar veren X ışınları ile tedavi edildi ve ardından belirli bir büyüme koşulu altında ekimyapıldı 6. Evcilleştikleri sırada bakterilerde biriken mutasyonlar, birçok büyüme özelliğinin kaybını, yani B. subtilis laboratuvar suşu 168 karmaşık koloniler oluşturma yeteneğini kaybetti7,8.
Günümüzde, en iyi çalışılan model bakteriler Escherichia coli ve B. subtilisiçin, belirli bilimsel soruları ele almak için genomlarını genetik olarak manipüle etmek için çeşitli güçlü araçlar mevcuttur. Bazen bir ilgi geninin inaktivasyonu, daha sonra standart büyüme ortamında açıkça görülebilen ciddi bir büyüme kusuruna neden olur9. Bunun aksine, zayıf bir büyüme kusuru neden olan ve böylece suşun kondisyonunu sadece biraz etkileyen mutasyonlar genellikle göz ardı edilir. Bununla birlikte, her iki durumda da mutant suşlarının birkaç nesil boyunca uzun süreli inkübasyonu ve geçişi genellikle ana suşun fenotipini geri kazandıran baskılayıcı mutantların birikmesine neden2,9. Baskılayıcı mutantların karakterizasyonu ve ebeveyn mutant suşunun büyüme kusurunu geri kazandıran mutasyonların tanımlanması, önemli ve genellikle yeni hücresel süreçlerin aydınlatılmasına izin veren çok yararlı bir yaklaşımdır10,11.
Biz B. subtilis12glutamat homeostaz kontrolü ile ilgileniyoruz. E. coli'yebenzer şekilde, B. subtilis glutamat homeostazının(yaniglutamat bozulmasında blok2)bastırıcı mutantların birikmesiyle pertürbasyonuna yanıt verir. Spontan mutasyonla elde edilen bu baskılayıcı mutantlardaki genomik değişikliklerin glutamat homeostazını hızla geri getirdiği gösterilmiştir9,13. Bu nedenle, B. subtilis'in bakterinin evcilleştirmesi sırasında belirli bir büyüme durumuna adaptasyonunun enzim sentezinde ve glutamat metabolizmasında yer alan evrimleşmiş enzimatik aktivitelerde yansıtılması şaşırtıcı değildir12. Evcilleştirme işlemi sırasında büyüme ortamında eksojen glutamat eksikliğinin, laboratuvar suşu 1682,14'tekriyotik glutamat dehidrogenaz (GDH) gudBCR geninin ortaya çıkması ve sabitlenmesi için itici güç olduğu ileri sürülmüştür. Bu hipotez, laboratuvar suşundaki GDH aktivitesinin azalmasının, eksojen glutamat az olduğunda bakterilere seçici bir büyüme avantajı sağladığı gözlemimiz ile desteklenmektedir2. Ayrıca, bir B. subtilis suşunun yetiştirilmesi, GDH GudB sentezlenmesi, eksojen glutamat yokluğunda gudB genini inaktive eden baskılayıcı mutantların birikmesi ile sonuçlanır2. Açıkçası, katabolik olarak aktif bir GDH'nin varlığı hücre için dezavantajlıdır, çünkü anabolizma için kullanılabilecek endojen olarak üretilen glutamat amonyum ve 2-oksioglutarat olarak bozulur (Şekil 1). Bunun aksine, glutamat ortam tarafından sağlandığında, üst düzey GDH aktivitesi ile donatılmış bir B. subtilis suşu, sadece bir fonksiyonel GDH sentezleyen bir suş üzerinde seçici bir büyüme avantajına sahiptir. Üst düzey GDH aktivitesinin, bakterilerin orta2 tarafından sağlanan diğer karbon kaynaklarına ek olarak ikinci bir karbon kaynağı olarak glutamat kullanmasına izin verdiğini varsaymak mantıklıdır (bkz. Şekil 1). Bu nedenle, GDH aktivitesi, eksojen glutamanın mevcudiyetine bağlı olarak bakterilerin zindeliğini güçlü bir şekilde etkiler.
Burada, kromozom üzerinde tek bir lokusta farklılık gösteren iki B. subtilis suşu arasındaki tür içi rekabeti izlemek ve görselleştirmek için çok açıklayıcı bir yöntem sunuyoruz (Şekil 2). İki suş, floroforlar YFP ve CFP'yi kodlayan yfp ve cfp genleri ile etiketlendi ve farklı beslenme koşulları altında birlikte çalıştı. Zamanla örnekleme yaparak ve agar plakalarına uygun seyreltmeler yaparak, kültürlerin her birinde hayatta kalanlar ortak bir stereo floresan mikroskobu kullanılarak kolayca izlenebilir. Bu makalede açıklanan prosedürün gerçekleştirilmesi kolaydır ve zaman içinde bir hücre popülasyonunda sırasıyla yararlı ve zararlı mutasyonların hızlı klonal genişlemesini ve ortadan kaldırılmasını görselleştirmek için uygundur.

<table><tbody><tr><td>(NH&lt;sub&gt;4&lt;/sub&gt;)&lt;sub&gt;2&lt;/sub&gt;SO&lt;sub&gt;4&lt;/sub&gt;&amp;nbsp;</td><td>Roth, Germany</td><td>3746</td><td></td></tr><tr><td>Agar</td><td>Difco, USA</td><td>214010</td><td></td></tr><tr><td>Ammonium ferric citrate (CAF)</td><td>Sigma-Aldrich, Germany</td><td>9714</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&amp;nbsp;&lt;/sub&gt;</td><td>Roth, Germany</td><td>5239</td><td></td></tr><tr><td>Glucose</td><td>Applichem, Germany</td><td>A3617</td><td></td></tr><tr><td>Glycerol</td><td>Roth, Germany</td><td>4043</td><td></td></tr><tr><td>K&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;&amp;bull;3H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Roth, Germany</td><td>6878</td><td></td></tr><tr><td>KCl</td><td>Applichem, Germany</td><td>A3582</td><td></td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Roth, Germany</td><td>3904</td><td></td></tr><tr><td>KOH</td><td>Roth, Germany</td><td>6751</td><td></td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt;&amp;bull;7H&lt;sub&gt;2&lt;/sub&gt;O&amp;nbsp;</td><td>Roth, Germany</td><td>P027</td><td></td></tr><tr><td>MnCl&lt;sub&gt;2&lt;/sub&gt;&amp;nbsp;</td><td>Roth, Germany</td><td>T881</td><td></td></tr><tr><td>MnSO&lt;sub&gt;4&lt;/sub&gt;&amp;bull;4H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Merck Millipore, Germany</td><td>102786</td><td></td></tr><tr><td>NaCl</td><td>Roth, Germany</td><td>9265</td><td></td></tr><tr><td>Nutrient broth</td><td>Roth, Germany</td><td>X929</td><td></td></tr><tr><td>Potassium glutamate</td><td>Applichem, Germany</td><td>A3712</td><td></td></tr><tr><td>Tryptone</td><td>Roth, Germany</td><td>8952</td><td></td></tr><tr><td>Tryptophan</td><td>Applichem, Germany</td><td>A3445</td><td></td></tr><tr><td>Yeast extract</td><td>Roth, Germany</td><td>2363</td><td></td></tr><tr><td>1.5 ml Reaction tubes</td><td>Sarstedt, Germany</td><td>72,690,001</td><td></td></tr><tr><td>2.0 ml Reaction tubes</td><td>Sarstedt, Germany</td><td>72,691</td><td></td></tr><tr><td>15 ml Plastic tubes with screw cap</td><td>Sarstedt, Germany</td><td>62,554,001</td><td></td></tr><tr><td>Petri dishes</td><td>Sarstedt, Germany</td><td>82.1473</td><td></td></tr><tr><td>1.5 ml Polystyrene cuvettes</td><td>Sarstedt, Germany</td><td>67,742</td><td></td></tr><tr><td>15 ml Glass culture tubes&amp;nbsp;</td><td>Brand, Germany</td><td>7790 22</td><td></td></tr><tr><td>100 ml Shake flasks with aluminium caps</td><td>Brand, Germany</td><td>928 24</td><td></td></tr><tr><td>Sterile 10 ml glass pipettes</td><td>Brand, Germany</td><td>278 23</td><td></td></tr><tr><td>Incubator (28 and 37 &amp;deg;C)</td><td>New Brunswick</td><td>M1282-0012</td><td></td></tr><tr><td>Standard pipette set (2-20 &amp;mu;l, 10-100 &amp;mu;l, 100-1,000 &amp;mu;l)</td><td>Eppendorf, Germany</td><td>4910 000.034, 4910 000.042,</td><td></td></tr><tr><td>Table top centrifuge for 1.5 and 2 ml reaction tubes</td><td>Thermo Scientific, Heraeus Fresco 21, Germany</td><td>75002425</td><td></td></tr><tr><td>Table top centrifuge for 15 ml plastic tubes</td><td>Heraeus Biofuge Primo R, Germany</td><td>75005440</td><td></td></tr><tr><td>Standard spectrophotometer</td><td>Amersham Biosciences Ultrospec 2100 pro, Germany</td><td>80-2112-21</td><td></td></tr><tr><td>Stereofluorescence microscope&amp;nbsp;</td><td>Zeiss SteREO Lumar V12, Germany</td><td>495008-0009-000</td><td></td></tr><tr><td>Freezer (-20 and -80 &amp;deg;C)</td><td>-</td><td>-</td><td></td></tr><tr><td>Fridge (4 &amp;deg;C)</td><td>-</td><td>-</td><td></td></tr><tr><td>Autoclave</td><td>Zirbus, LTA 2x3x4, Germany</td><td>-</td><td></td></tr><tr><td>pH meter</td><td>pH-meter 766, Calimatic, Knick, Germany</td><td>766</td><td></td></tr><tr><td>Vortex</td><td>Vortex 3, IKA, Germany</td><td>3340000</td><td></td></tr><tr><td>Balance</td><td>CP2202S, Sartorius, Germany</td><td>replaced by</td><td></td></tr><tr><td>Black pen (permanent marker)</td><td>Staedler, Germany</td><td>317-9</td><td></td></tr><tr><td>Powerpoint program</td><td>Microsoft, USA</td><td>-</td><td></td></tr><tr><td>Office Excel program</td><td>Microsoft, USA</td><td>-</td><td>Program for data processing</td></tr><tr><td>Adobe Photoshop CS5</td><td>Adobe, USA</td><td>replaced by CS6, download</td><td>Computer program for image processing</td></tr><tr><td>Computer</td><td>PC or Mac</td><td>-</td><td></td></tr><tr><td>ZEN pro 2011 software for the stereofluorescence microscope</td><td>Zeiss, Germany</td><td>410135 1002 110</td><td>AxioCam MRc Rev. Obtained through Zeiss</td></tr><tr><td colspan="4">&lt;strong&gt;Specific solution recipes&lt;/strong&gt;</td></tr><tr><td colspan="4">&lt;strong&gt;SP Medium&lt;/strong&gt;</td></tr><tr><td>8 g Nutrient broth</td><td></td><td></td><td></td></tr><tr><td>0.25 mg MgSO&lt;sub&gt;4&lt;/sub&gt;&amp;bull;7H&lt;sub&gt;2&lt;/sub&gt;O</td><td></td><td></td><td></td></tr><tr><td>1 g KCl</td><td></td><td></td><td></td></tr><tr><td>15 g agar for solid SP medium</td><td></td><td></td><td>if required</td></tr><tr><td>add 1 L with H&lt;sub&gt;2&lt;/sub&gt;O</td><td></td><td></td><td>autoclave for 20 min at 121 &amp;deg;C</td></tr><tr><td>1 ml CaCl&lt;sub&gt;2&lt;/sub&gt; (0.5 M), sterilized by filtration</td><td></td><td></td><td></td></tr><tr><td>1 ml MnCl&lt;sub&gt;2&lt;/sub&gt; (10 mM), sterilized by filtration</td><td></td><td></td><td></td></tr><tr><td>2 ml ammonium ferric citrate (CAF, 2.2 mg/ml), sterilized by filtration</td><td></td><td></td><td></td></tr><tr><td colspan="4">&lt;strong&gt;LB Medium&lt;/strong&gt;</td></tr><tr><td>10 g Tryptone</td><td></td><td></td><td></td></tr><tr><td>5 g Yeast extract</td><td></td><td></td><td></td></tr><tr><td>10 g NaCl</td><td></td><td></td><td></td></tr><tr><td>15 g agar for solid LB medium</td><td></td><td></td><td>if required</td></tr><tr><td>add 1 L with H&lt;sub&gt;2&lt;/sub&gt;O</td><td></td><td></td><td>autoclave for 20 min at 121 &amp;deg;C</td></tr><tr><td colspan="4">&lt;strong&gt;C-Glc Minimal Medium&lt;/strong&gt;</td></tr><tr><td>200 ml 5 x C salts</td><td></td><td></td><td></td></tr><tr><td>10 ml L-Tryptophan (5 mg/ml), sterilized by filtration</td><td></td><td></td><td></td></tr><tr><td>10 ml ammonium ferric citrate (CAF, 2.2 mg/ml), sterilized by filtration</td><td></td><td></td><td></td></tr><tr><td>10 ml III&amp;rsquo; salts</td><td></td><td></td><td></td></tr><tr><td>25 ml Glucose (20%)</td><td></td><td></td><td>autoclaved for 20 min at 121 &amp;deg;C</td></tr><tr><td>add 1 L with sterile H&lt;sub&gt;2&lt;/sub&gt;O</td><td></td><td></td><td></td></tr><tr><td colspan="4">&lt;strong&gt;CE-Glc Minimal Medium&lt;/strong&gt;</td></tr><tr><td>200 ml 5 x C salts</td><td></td><td></td><td></td></tr><tr><td>10 ml L-Tryptophan (5 mg/ml), sterilized by filtration</td><td></td><td></td><td></td></tr><tr><td>10 ml ammonium ferric citrate (CAF, 2.2 mg/ml), sterilized by filtration</td><td></td><td></td><td></td></tr><tr><td>10 ml III&amp;rsquo; salts</td><td></td><td></td><td></td></tr><tr><td>20 ml Glutamate (40%)</td><td></td><td></td><td></td></tr><tr><td>25 ml Glucose (20%), autoclaved for 20 min at 121 &amp;deg;C</td><td></td><td></td><td></td></tr><tr><td>add 1 L with sterile H&lt;sub&gt;2&lt;/sub&gt;O</td><td></td><td></td><td></td></tr><tr><td>5 x C salts&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>20 g KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td></td><td></td><td></td></tr><tr><td>80 g K&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;&amp;bull;3H&lt;sub&gt;2&lt;/sub&gt;O</td><td></td><td></td><td></td></tr><tr><td>16.5 g (NH&lt;sub&gt;4&lt;/sub&gt;)&lt;sub&gt;2&lt;/sub&gt;SO&lt;sub&gt;4&lt;/sub&gt;</td><td></td><td></td><td></td></tr><tr><td>add 1 L with sterile H&lt;sub&gt;2&lt;/sub&gt;O</td><td></td><td></td><td>autoclave for 20 min at 121 &amp;deg;C</td></tr><tr><td>III&amp;rsquo; salts</td><td></td><td></td><td></td></tr><tr><td>0.232 g MnSO&lt;sub&gt;4&lt;/sub&gt;&amp;bull;4H&lt;sub&gt;2&lt;/sub&gt;O</td><td></td><td></td><td></td></tr><tr><td>12.3 g MgSO&lt;sub&gt;4&lt;/sub&gt;&amp;bull;7H&lt;sub&gt;2&lt;/sub&gt;O</td><td></td><td></td><td></td></tr><tr><td>add 1 L with sterile H&lt;sub&gt;2&lt;/sub&gt;O, autoclave for 20 min at 121 &amp;deg;C</td><td></td><td></td><td></td></tr><tr><td>40% Glutamate solution</td><td></td><td></td><td></td></tr><tr><td>200 g L-Glutamic acid</td><td></td><td></td><td></td></tr><tr><td>adjust the pH to 7.0 by adding approximately 80 g KOH</td><td></td><td></td><td></td></tr><tr><td>add 0.5 L with sterile H&lt;sub&gt;2&lt;/sub&gt;O</td><td></td><td></td><td>autoclave for 20 min at 121 &amp;deg;C</td></tr><tr><td colspan="4">&lt;strong&gt;0.9% Saline (NaCl) Solution&lt;/strong&gt;</td></tr><tr><td>add 1 L with sterile H&lt;sub&gt;2&lt;/sub&gt;O</td><td></td><td></td><td>autoclave for 20 min at 121 &amp;deg;C</td></tr><tr><td>50% Glycerol solution</td><td></td><td></td><td></td></tr><tr><td>295 ml Glycerol (87%)</td><td></td><td></td><td></td></tr><tr><td>add 0.5 L with sterile H&lt;sub&gt;2&lt;/sub&gt;O</td><td></td><td></td><td>autoclave for 20 min at 121 &amp;deg;C</td></tr><tr><td colspan="4">Bacteria (All strains are based on the &lt;em&gt;Bacillus subtilis&lt;/em&gt; strain 168)</td></tr><tr><td>&lt;em&gt;Bacillus subtilis&lt;/em&gt; BP40 (rocG&lt;sup&gt;+&lt;/sup&gt; gudB&lt;sup&gt;CR&lt;/sup&gt; amyE::PgudB-yfp)&amp;nbsp;</td><td>Laboratory strain collection</td><td></td><td></td></tr><tr><td>&lt;em&gt;Bacillus subtilis&lt;/em&gt; BP41 (rocG&lt;sup&gt;+&lt;/sup&gt; gudB&lt;sup&gt;CR&lt;/sup&gt; amyE::PgudB-cfp)&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>&lt;em&gt;Bacillus subtilis&lt;/em&gt; BP52 (rocG&lt;sup&gt;+&lt;/sup&gt; gudB&lt;sup&gt;+&lt;/sup&gt; amyE::PgudB-cfp)</td><td></td><td></td><td></td></tr><tr><td>&lt;em&gt;Bacillus subtilis&lt;/em&gt; BP156 (rocG&lt;sup&gt;+&lt;/sup&gt; gudB&lt;sup&gt;+&lt;/sup&gt; amyE::PgudB-yfp)</td><td></td><td></td><td></td></tr></tbody></table>

monitoring intraspecies competition in a bacterial cell population by cocultivation of fluorescently labelled strains

Bakteri gibi birçok mikroorganizma son derece hızlı çoğalır ve popülasyonlar yüksek hücre yoğunluklarına ulaşabilir. Bir popülasyondaki hücrelerin küçük fraksiyonları her zaman hücre için zararlı veya faydalı olan birikmiş mutasyonlara sahiptir. Bir mutasyonun fitness etkisi, alt nüfusa güçlü bir seçici büyüme avantajı sağlarsa, bu alt nüfusa sahip bireyler hızla rakip olabilir ve hatta yakın arkadaşlarını tamamen ortadan kaldırabilir. Bu nedenle, yararlı mutasyonlar elde eden hücrelerin küçük genetik değişiklikleri ve seçim odaklı birikimi, bir hücre popülasyonunun genotipinin tamamen kaymasına neden olabilir. Burada, gram-pozitif model bakteri bacillus subtilisfloresan etiketli bireylerinin birlikte çalıştırıldığı zaman içinde bakteri hücre popülasyonunda, sırasıyla yararlı ve zararlı mutasyonların hızlı klonal genişlemesini ve ortadan kaldırılmasını izlemek için bir prosedür sunuyoruz. Yöntemin gerçekleştirilmesi kolaydır ve bakteri hücre popülasyonundaki bireyler arasında tür içi rekabeti göstermek çok açıklayıcıdır.

Burada açıklanan yöntem, sırasıyla florofor cfp ve YFP'yi kodlayan cfp ve yfp genleri ile etiketlenmiş B. subtilis suşlarından oluşan bir hücre popülasyonunda tür içi rekabeti görselleştirmek için başarıyla uygulanmıştır. Şekil 3'tegösterildiği gibi, yöntem tür içi rekabeti çok açıklayıcı bir şekilde görselleştirmek için kullanılabilir. Örneklerin küçük alanlarda tespit haline getirerek, hücre popülasyonunun klonal bileşimi bir bakı?...

Watch this Scientific Journal Video about Monitoring Intraspecies Competition in a Bacterial Cell Population by Cocultivation of Fluorescently Labelled Strains at JoVE.com

Monitoring Intraspecies Competition in a Bacterial Cell Population by Cocultivation of Fluorescently Labelled Strains

Bakteriler yaşamları boyunca zararlı veya faydalı mutasyonlar biriktirebilir. Bir hücre popülasyonunda, yararlı mutasyonlar biriktirmiş bireyler, arkadaşlarını hızla geride kalabilir. Burada, floresan etiketli bireyleri kullanarak zaman içinde bakteri hücresi popülasyonunda tür içi rekabeti görselleştirmek için basit bir prosedür sunuyoruz.

Floresan Etiketli Suşların Cocultivation'ı ile Bakteri Hücre Popülasyonunda İntraspecies Rekabetini İzleme

Many microorganisms such as bacteria proliferate extremely fast and the populations may reach high cell densities. Small fractions of cells in a ...

monitoring-intraspecies-competition-bacterial-cell-population

Research

JoVE Journal

Biology

8.5K Görüntüleme.  Georg-August University. Bakteriler yaşamları boyunca zararlı veya faydalı mutasyonlar biriktirebilir. Bir hücre popülasyonunda, yararlı mutasyonlar biriktirmiş bireyler, arkadaşlarını hızla geride kalabilir. Burada, floresan etiketli bireyleri kullanarak zaman içinde bakteri hücresi popülasyonunda tür içi rekabeti görselleştirmek için basit bir prosedür sunuyoruz.

Video: Monitoring Intraspecies Competition in a Bacterial Cell Population by Cocultivation of Fluorescently Labelled Strains

Monitoring Spatial Segregation in Surface Colonizing Microbial Populations

Microbes provide an intriguing system to study social interaction among individuals within a population. The short generation times and relatively simple genetic modification procedures of microbes facilitate the development of the sociomicrobiology field. To assess the fitness of certain microbial species, selected strains or their genetically modified derivatives within one population, can be fluorescently labelled and tracked using microscopy adapted with appropriate fluorescence filters. Expanding colonies of diverse microbial species on agar media can be used to monitor the spatial distribution of cells producing distinctive fluorescent proteins.
Here, we present a detailed protocol for the use of green- and red-fluorescent protein producing bacterial strains to follow spatial arrangement during surface colonization, including flagellum-driven community movement (swarming), exopolysaccharide- and hydrophobin-dependent growth mediated spreading (sliding), and complex colony biofilm formation. Non-domesticated isolates of the Gram-positive bacterium, Bacillus subtilis can be utilized to scrutinize certain surface spreading traits and their effect on two-dimensional distribution on the agar-solidified medium. By altering the number of cells used to initiate colony biofilms, the assortment levels can be varied on a continuous scale. Time-lapse fluorescent microscopy can be used to witness the interaction between different phenotypes and genotypes at a certain assortment level and to determine the relative success of either.

The role of assortment (spatial segregation) in evolutionary scenarios can be examined using simple microbial systems in the laboratory that allow controlled adjustment of spatial distribution. By modifying the founder cell density, various assortment levels can be visualized using fluorescently labelled bacterial strains in the colony biofilms of Bacillus subtilis.

Yüzey koloniler Mikrobiyal Populasyonlarında Mekansal Ayrışma İzleme

Microbes provide an intriguing system to study social interaction among individuals within a population. The short generation times and relatively simple ...

Genetik

Title Cell Encapsulation by Droplets

My name is Hang Moon. I'm working with the professor of condoms. He and me work with the XYJ stage to make a 3D structure of cell.Hi, I am PE and Lynn and I'm working with the cell printing project with cell encapsulation and media and gels. This is solenoid be for ejection of a cell and cell mixture of aros over cell media. The so valve is activated by two signal line.The signal line is came from a dry board here. Signal came from here. That line through the that line, that one is the function first generator.So first is generated by our programmed way. So if you want, you want to change the purse, you can change the program with the that switches line or you can control that process generator to computer. The cellular valve is open and closed by the electric signal, but the, the cell is supplied through that line.The cell and the media or agro gel is supplied through the syringe syringes. The syringes inside of the water, inside of syringe is the press light by the air. The pressurized air is supplied from a tank like here the line.So the little line and the ized air is fly through the air filter. The air filter is discriminate the dust and other kind of the dirt. That is pressure generator we call it, is generating the signal used for open the solar air bell or closed the solar air valve computer is the connected through P-I-B-U-S-V connector.So we can use the USB port by directly our computer, the laptop computer. So that one is connected to that side here. And then we can control through the control laptop here.So if we want to synchronize of the movement of the stage and opening the bell is can be controlled by the same controller as a laptop. So we can control the timing when it open, when it close, and when it moves and when it stops. And we can synchronize the motion and opening bell.That stage is the motorized stage. It is automatically automatically moved by through the death signal line and motor. So motorization is 0.1 micrometers.So it can ideally it can draw the line up to 100 millimeter line. So jet axis is moving, going up and down like that way. So it control the cap between the substrate and the solar belt.So it can control the height of ejection. That substrate is the connected to the XY stages here, X here and Y there. So it's moving around a plan XY plan.So it draws some feature demo right stages is three Xs, X, Y, and G.So the jet con jet axis or XY axis is controlled by signal here. So signal line is connected to the motion controller here. That motion controller doesn't have any manual switch at all because it cannot be controlled manually.It's controlled through the laptop computer. So laptop computer is connected. The backside of the controller, we have three xs for a printing cell XY jet.So it make three different kind of signal independently. And then motion controller is communicate through the rf 2, 3, 2 connectors is directly connected to the USV. And then USV is connected to laptop computer.So laptop controlled borrower. We use the AutoCAD program, we make a gen A path like that way bam. And then it change to the machine language like that.So machine language is transferred to the like that the simple character and numbers like that. There is each position related to the X, Y, Z stages here I choose the file, contain that kind of program and then open downloading program like that way. And then I choose from file like does exact the same, the text file.Just the text file is downloaded, choose text file and automatically download from laptop to com motion controller executive and then it going the BAM character as I mentioned before, BAM. In this flask are N IH 3G three mouse fiberblast cells. And I'm going to first aspirate the old media.This process is to passage the cells and the cells are adhered to the bottom of the flask. So after aspirating the media, I'm going to put in six RYS in which is an enzyme.Okay? So after the trypsin has been put into the flask, we need to wait three to five minutes for the cells to become detached from the bottom of the flask.Now we're ready to put in the media and passage the cells into a centrifuge tube. It's a one-to-one dilution. So I'm putting in six mil of this media right here and I'm gonna wash out the saws a little bit to make sure all of them are passaged.And now the cells are ready to be centrifuge in this tube. Now we're going to centrifuge these cells at a thousand RPM three to five minutes. Now that the cells have been spun down into a pellet, I'm going to aspirate the supernatant.Now I'm going to wash out the cells with PBS. And this is to, this is for staining the cells. The stains that we are using are esterase substrates.Okay, now we we're going to centrifuge again and spin down the cells into a palate. And I'm going to aspirate the PBS and I'm going to resin the cells. I'm in media to be counted.I'm mixing the cells so that when we take the a sample of it, the concentration will be even throughout the tube. Now I'm going to take a hundred microliters of the cells to measure the cell density. Now I'm putting in a hundred microliters of type and blue, which is a stain.We're going to wait three to five minutes in order for the stain to permeate dead cell membranes. When we count the cells, the blue ones we don't count. The cells are ready to be counted with the hemo cytometer, I'm going to transfer 50 microliters of the cell mixture to each side of the hemo cytometer.Okay, I'm going to cover it with a glass slide side. There's two sides to the hemo cytometer. I'm gonna count the first side and after counting both sides, I'm going to take the average and then multiply by two because we had a one-to-one dilution with the type in blue.So basically our cell density will be 135 times 10 to the four cells per mil. For our experiment, we will be using a cell solution with a concentration of half a million cells per mil. And so since we have 13.5 million cells, I will be resus suspending the cells in 27 mil of media.We are using two different types of stains. This is a live dead assay from molecular probes. Our first stain is called calcium am.Our second stain is called AUM homodimer. It stains dead cells. We use blue excitation to get green fluorescence and green excitation to get red fluorescence For these two stains, when we're imaging our dice solution will be prepared in PBS.So first I'm going to transfer five mil of PBS into a tube phosphate buffered saline. That's a saline solution. Now I'm going to add half a microliter of calcium am per mil.Now I'm adding 2.5 microliters of calcium am to the tube and two microliters of athe homodimer per mil of PBS. Here is the btex machine. It makes the cell and media well distributed through the whole volume.Now we load the cell 200 microliter onto the seven here. And then we put on the cap, this is a tight tightly sealed through like that way here I open the valve and then it's the pressure light air comes in and then I adjust the belt like that. And then it change the pressure.So I adjust. The pressure is five PSI. The inside of tubing pressure is five PSI.From here to here, it connected through the this inside the hole. So just we open the bell here and then the cell injected with the five PSI pressure and then starting open. And then Sona valve starting operate.And then we roll the substrate. It substrate is just a simple slide glass. And then we load the proper portion and I put some sketch tape to fix the substrate like that, right?And simply I just fix it. So we are setting whole thing the cell and ary valve and the substrate during the moving the ary working, start working. So we are set up whole thing and then open ary valve start.And then we move the stage like that, right? And then we make some pattern onto the substrate pattern onto the substrate and bam character. I'm going to immerse these droplets that we printed in this dye solution that we made earlier.And now the droplets will be incubated for 10 minutes and then imaged. First I'm going to adjust the image through the eyepiece. Our first image is just a bright field image.I'm gonna turn off the light and change the filter so that we have blue excitation, which will show green fluorescence. And the third image we'll be taking is the dead cells, which is the Thm Homodimer stain. And with green light, we will get red fluorescence.

Droplets tarafından Başlığı Hücre Kapsülleme

Biyoloji

Derivation of Human Embryonic Stem Cells by Immunosurgery

The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both medical applications and as a research 
tool for addressing fundamental questions in development and disease. Here, we provide a 
concise, step-by-step protocol for the derivation of human embryonic stem cells from embryos 
by immunosurgical isolation of the inner cell mass. 


The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both medical applications and as a research 
tool for addressing fundamental questions in development and disease. Here, we provide a 
concise, step-by-step protocol for the derivation of human embryonic stem cells from embryos 
by immunosurgical isolation of the inner cell mass.

Immunosurgery İnsan Embriyonik Kök Hücreleri türetilmesi

The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both ...

A Visual Assay to Monitor T6SS-mediated Bacterial Competition

Type VI secretion systems (T6SSs) are molecular nanomachines allowing Gram-negative bacteria to transport and inject proteins into a wide variety of target cells1,2. The T6SS is composed of 13 core components and displays structural similarities with the tail-tube of bacteriophages3. The phage uses a tube and a puncturing device to penetrate the cell envelope of target bacteria and inject DNA. It is proposed that the T6SS is an inverted bacteriophage device creating a specific path in the bacterial cell envelope to drive effectors and toxins to the surface. The process could be taken further and the T6SS device could perforate other cells with which the bacterium is in contact, thus injecting the effectors into these targets. The tail tube and puncturing device parts of the T6SS are made with Hcp and VgrG proteins, respectively4,5.
The versatility of the T6SS has been demonstrated through studies using various bacterial pathogens. The Vibrio cholerae T6SS can remodel the cytoskeleton of eukaryotic host cells by injecting an "evolved" VgrG carrying a C-terminal actin cross-linking domain6,7. Another striking example was recently documented using Pseudomonas aeruginosa which is able to target and kill bacteria in a T6SS-dependent manner, therefore promoting the establishment of bacteria in specific microbial niches and competitive environment8,9,10.
In the latter case, three T6SS-secreted proteins, namely Tse1, Tse2 and Tse3 have been identified as the toxins injected in the target bacteria (Figure 1). The donor cell is protected from the deleterious effect of these effectors via an anti-toxin mechanism, mediated by the Tsi1, Tsi2 and Tsi3 immunity proteins8,9,10. This antimicrobial activity can be monitored when T6SS-proficient bacteria are co-cultivated on solid surfaces in competition with other bacterial species or with T6SS-inactive bacteria of the same species8,11,12,13.
The data available emphasized a numerical approach to the bacterial competition assay, including time-consuming CFU counting that depends greatly on antibiotic makers. In the case of antibiotic resistant strains like P. aeruginosa, these methods can be inappropriate. Moreover, with the identification of about 200 different T6SS loci in more than 100 bacterial genomes14, a convenient screening tool is highly desirable. We developed an assay that is easy to use and requires standard laboratory material and reagents. The method offers a rapid and qualitative technique to monitor the T6SS-dependent bactericidal/bacteriostasis activity by using a reporter strain as a prey (in this case Escherichia coli DH5&alpha;) allowing a-complementation of the lacZ gene. Overall, this method is graphic and allows rapid identification of T6SS-related phenotypes on agar plates. This experimental protocol may be adapted to other strains or bacterial species taking into account specific conditions such as growth media, temperature or time of contact.

We describe a qualitative assay to monitor bacterial competition mediated by the Pseudomonas aeruginosa type VI secretion system (T6SS). The assay relies on the survival/killing of Escherichia coli target cells carrying a lacZ-reporter. This technique is adjustable to assess the bactericidal/bacteriostasis activity of T6SS-proficient microorganisms.

T6SS aracılı Bakteriyel Rekabet Monitör A Görsel Assay

Type VI secretion systems (T6SSs) are molecular  nanomachines allowing Gram-negative bacteria to transport and inject proteins  into a wide variety of ...

İmmünoloji ve Enfeksiyon

Monitoring Dynamic Changes In Mitochondrial Calcium Levels During Apoptosis Using A Genetically Encoded Calcium Sensor

Dynamic changes in intracellular calcium concentration in response to various stimuli regulates many cellular processes such as proliferation, differentiation, and apoptosis1. During apoptosis, calcium accumulation in mitochondria promotes the release of pro-apoptotic factors from the mitochondria into the cytosol2. It is therefore of interest to directly measure mitochondrial calcium in living cells in situ during apoptosis. High-resolution fluorescent imaging of cells loaded with dual-excitation ratiometric and non-ratiometric synthetic calcium indicator dyes has been proven to be a reliable and versatile tool to study various aspects of intracellular calcium signaling. Measuring cytosolic calcium fluxes using these techniques is relatively straightforward. However, measuring intramitochondrial calcium levels in intact cells using synthetic calcium indicators such as rhod-2 and rhod-FF is more challenging. Synthetic indicators targeted to mitochondria have blunted responses to repetitive increases in mitochondrial calcium, and disrupt mitochondrial morphology3. Additionally, synthetic indicators tend to leak out of mitochondria over several hours which makes them unsuitable for long-term experiments. Thus, genetically encoded calcium indicators based upon green fluorescent protein (GFP)4 or aequorin5 targeted to mitochondria have greatly facilitated measurement of mitochondrial calcium dynamics. Here, we describe a simple method for real-time measurement of mitochondrial calcium fluxes in response to different stimuli. The method is based on fluorescence microscopy of 'ratiometric-pericam' which is selectively targeted to mitochondria. Ratiometric pericam is a calcium indicator based on a fusion of circularly permuted yellow fluorescent protein and calmodulin4. Binding of calcium to ratiometric pericam causes a shift of its excitation peak from 415 nm to 494 nm, while the emission spectrum, which peaks around 515 nm, remains unchanged. Ratiometric pericam binds a single calcium ion with a dissociation constant in vitro of ~1.7 &mu;M4. These properties of ratiometric pericam allow the quantification of rapid and long-term changes in mitochondrial calcium concentration. Furthermore, we describe adaptation of this methodology to a standard wide-field calcium imaging microscope with commonly available filter sets. Using two distinct agonists, the purinergic agonist ATP and apoptosis-inducing drug staurosporine, we demonstrate that this method is appropriate for monitoring changes in mitochondrial calcium concentration with a temporal resolution of seconds to hours. Furthermore, we also demonstrate that ratiometric pericam is also useful for measuring mitochondrial fission/fragmentation during apoptosis. Thus, ratiometric pericam is particularly well suited for continuous long-term measurement of mitochondrial calcium dynamics during apoptosis.

This protocol describes a method for real-time measurement of mitochondrial calcium fluxes by fluorescent imaging. The method takes advantage of a circularly permutated YFP-based dual-excitation ratiometric calcium sensor (ratiometric pericam-mt) selectively expressed in mitochondria.

Apoptozis sırasında Mitokondrial Kalsiyum Düzeyleri Dinamik değişiklikler izlenmesi Genetiği Kodlanmış Kalsiyum Sensor Kullanımı

Dynamic changes in intracellular calcium concentration in response to various stimuli regulates many cellular processes such as proliferation, ...

Coincubation Assay for Quantifying Competitive Interactions between Vibrio fischeri Isolates

This manuscript describes a culture-based, coincubation assay for detecting and characterizing competitive interactions between two bacterial populations. This method employs stable plasmids that allow each population to be differentially tagged with distinct antibiotic resistance capabilities and fluorescent proteins for selection and visual discrimination of each population, respectively. Here, we describe the preparation and coincubation of competing Vibrio fischeri strains, fluorescence microscopy imaging, and quantitative data analysis. This approach is simple, yields quick results, and can be used to determine whether one population kills or inhibits the growth of another population, and whether competition is mediated through a diffusible molecule or requires direct cell-cell contact. Because each bacterial population expresses a different fluorescent protein, the assay permits the spatial discrimination of competing populations within a mixed colony. Although the described methods are performed with the symbiotic bacterium V. fischeri using conditions optimized for this species, the protocol can be adapted for most culturable bacterial isolates.

Coincubation Assay for Quantifying Competitive Interactions between Vibrio fischeri Isolates

Bacteria encode diverse mechanisms for engaging in interbacterial competition. Here, we present a culture-based protocol for characterizing competitive interactions between bacterial isolates and how they impact the spatial structure of a mixed population.

Vibrio fischeri Izolatlar arasında rekabetçi etkileşimleri ölçmek için kokuluçak assay

This manuscript describes a culture-based, coincubation assay for detecting and characterizing competitive interactions between two bacterial populations. ...

Kinetic Visualization of Single-Cell Interspecies Bacterial Interactions

Polymicrobial communities are ubiquitous in nature, yet studying their interactions at the single-cell level is difficult. Thus, a microscopy-based method has been developed for observing interspecies interactions between two bacterial pathogens. The use of this method to interrogate interactions between a motile Gram-negative pathogen, Pseudomonas aeruginosa and a non-motile Gram-positive pathogen, Staphylococcus aureus is demonstrated here. This protocol consists of co-inoculating each species between a coverslip and an agarose pad, which maintains the cells in a single plane and allows for visualization of bacterial behaviors in both space and time.
Furthermore, the time-lapse microscopy demonstrated here is ideal for visualizing the early interactions that take place between two or more bacterial species, including changes in bacterial species motility in monoculture and in coculture with other species. Due to the nature of the limited sample space in the microscopy setup, this protocol is less applicable for studying later interactions between bacterial species once cell populations are too high. However, there are several different applications of the protocol which include the use of staining for imaging live and dead bacterial cells, quantification of gene or protein expression through fluorescent reporters, and tracking bacterial cell movement in both single species and multispecies experiments.

This live-bacterial cell imaging protocol allows for visualization of interactions between multiple bacterial species at the single-cell level over time. Time-lapse imaging allows for observation of each bacterial species in monoculture or coculture to interrogate interspecies interactions in multispecies bacterial communities, including individual cell motility and viability.

Tek Hücreli Türler Arası Bakteri Etkileşimlerinin Kinetik Görselleştirilmesi

Polymicrobial communities are ubiquitous in nature, yet studying their interactions at the single-cell level is difficult. Thus, a microscopy-based method ...

Using Coculture to Detect Chemically Mediated Interspecies Interactions

In nature, bacteria rarely exist in isolation; they are instead surrounded by a diverse array of other microorganisms that alter the local environment by secreting metabolites. These metabolites have the potential to modulate the physiology and differentiation of their microbial neighbors and are likely important factors in the establishment and maintenance of complex microbial communities. We have developed a fluorescence-based coculture screen to identify such chemically mediated microbial interactions. The screen involves combining a fluorescent transcriptional reporter strain with environmental microbes on solid media and allowing the colonies to grow in coculture. The fluorescent transcriptional reporter is designed so that the chosen bacterial strain fluoresces when it is expressing a particular phenotype of interest (i.e. biofilm formation, sporulation, virulence factor production, etc.) Screening is performed under growth conditions where this phenotype is not expressed (and therefore the reporter strain is typically nonfluorescent). When an environmental microbe secretes a metabolite that activates this phenotype, it diffuses through the agar and activates the fluorescent reporter construct. This allows the inducing-metabolite-producing microbe to be detected: they are the nonfluorescent colonies most proximal to the fluorescent colonies. Thus, this screen allows the identification of environmental microbes that produce diffusible metabolites that activate a particular physiological response in a reporter strain. This publication discusses how to: a) select appropriate coculture screening conditions, b) prepare the reporter and environmental microbes for screening, c) perform the coculture screen, d) isolate putative inducing organisms, and e) confirm their activity in a secondary screen. We developed this method to screen for soil organisms that activate biofilm matrix-production in Bacillus subtilis; however, we also discuss considerations for applying this approach to other genetically tractable bacteria.

Bacteria produce secreted compounds that have the potential to affect the physiology of their microbial neighbors. Here we describe a coculture screen that allows detection of such chemically mediated interspecies interactions by mixing soil microbes with fluorescent transcriptional reporter strains of Bacillus subtilis on solid media.

Kimyasal Dolayımlı Türler arası etkileşimleri tespit etmek Coculture kullanma

In nature, bacteria rarely exist in isolation; they are instead surrounded by a diverse array of other microorganisms that alter the local environment by ...

Quantification of Interbacterial Competition using Single-Cell Fluorescence Imaging

Interbacterial competition can directly impact the structure and function of microbiomes. This work describes a fluorescence microscopy approach that can be used to visualize and quantify competitive interactions between different bacterial strains at the single-cell level. The protocol described here provides methods for advanced approaches in slide preparation on both upright and inverted epifluorescence microscopes, live-cell and time-lapse imaging techniques, and quantitative image analysis using the open-source software FIJI. The approach in this manuscript outlines the quantification of competitive interactions between symbiotic Vibrio fischeri populations by measuring the change in area over time for two coincubated strains that are expressing different fluorescent proteins from stable plasmids. Alternative methods are described for optimizing this protocol in bacterial model systems that require different growth conditions. Although the assay described here uses conditions optimized for V. fischeri, this approach is highly reproducible and can easily be adapted to study competition among culturable isolates from diverse microbiomes.

This manuscript describes a method for using single-cell fluorescence microscopy to visualize and quantify bacterial competition in coculture.

Tek Hücreli Floresan Görüntüleme Kullanılarak Interbakteriyel Rekabetin Nicelemesi

Interbacterial competition can directly impact the structure and function of microbiomes. This work describes a fluorescence microscopy approach that can ...

Bacterial Co-Incubation Assay: A Fluorescence Microscopy-Based Technique to Visualize Intraspecific Bacterial Competition at the Single-Cell Level

Source: Smith, S., et al., <a href="https://app.jove.com/v/62851">Quantification of Interbacterial Competition using Single-Cell Fluorescence Imaging.</a> J. Vis. Exp. (2021).
This video demonstrates a fluorescence microscopy-based method to study bacterial competition at the single-cell level. This method provides insights into the structure and function of bacterial communities.