Name: design and use of a low cost, automated morbidostat for adaptive evolution of bacteria under antibiotic drug selection
Uploaded: 2016-09-27T15:00:00.000+00:00
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The authors would like to thank Prof. Sze-Bi Hsu and Ms. Zhenzhen for useful discussions and help in the theoretical analysis and numerical simulation. Y. T. Y. would like to acknowledge funding support from the Ministry of Science and Technology under grant numbers MOST 103-2220-E-007-026 and MOST 104-2220-E-007-011, and from the National Tsing Hua University under grant numbers 103N2042E1, 104N2042E1, and 105N518CE1.

Morbidostat Cihaz 1. Montaj ve Ön test <ol><li> Morbidostat Meclisi <ol><li> 18 G şırınga iğne ile kültür flakon kapağı 3 delikler. ~ Uzunluğunda 7 cm polietilen boru üç parça kesin. kap polietilen boru bu üç parça yerleştirin. </li><li> polidimetilsiloksan (PDMS) karışım için döküm olarak hizmet kapağın kenarını sarmak için bant kullanın. bir kürdan ile elle karıştırma ile bir bileşenin 5 g ve 150 ml&#39;lik plastik bir kap içinde PDMS B bileşeninin 0.5 g karıştırın. 10 ml şırınga içine karışımı yerleştirin. <ol><li> şırınga ile kap PDMS karışımı dökün. 70 ° C&#39;de bir fırında 8 saat PDMS tedavi etmek için, tüm kapak fırında. </li></ol></li><li> Bir havya ile karbon direnci ile 24 ayar tel ve LED katot LED anot lehimleyin. 24 G teli ile bir 680 Ω karbon direnç lehim. Elektrik bandı kullanarak tel bağlantısı izole. </li><li> Yani24 G tel ve 100 kÊ karbon direnci ile photodetector verici ile photodetector kollektör lder. Elektrik bandı kullanarak tel bağlantısı izole. </li><li> kültür flakon tutucu ışık yayan diyot yerleştirin. Ek Şekil 2&#39;de devre şeması takip ederek, LED IR (örneğin, bir cep telefonu kamera) tespit edebilen bir dijital fotoğraf makinesi kullanılarak çalıştığından emin olun 5 V güç kaynağı ile diyot ve güç onu yayan ışık bağlayın. </li><li> Kültür şişe tutucu fotodetektör yerleştirin. Ek Şekil 2&#39;de devre şeması takip ederek, elektronik kontrol yönüyle voltajı ölçmek için foto detektör dirençlerin her iki tarafına kabloyu 5 V güç kaynağı ile fotodetektör ve güç bağlayın. </li><li> Manyetik karıştırıcı birim olarak görev soğutma fanı, şaftı üzerinde bir mıknatıs Tutkal. alignm Notkültür flakon fan ve kültür şişe ve mıknatıs arasındaki boşluk milin ent manyetik karıştırma ünitesinin düzgün fonksiyonu için çok önemlidir. </li><li> Mikro-pompalar, orta şişe ve atık şişe silikon tüp karşılık gelen parça içine kültür flakonun polietilen boru her parça bağlayın. 1 saat pompa açın ve toplam hacim pompa hızını belirlemek için kültür şişesine orta şişeden pompalanan ölçün. Tipik pompalama oranı seyreltme oranı D = 0.33 sa -1 kültür flakon hacmini çalışan bir 12 ml karşılık ~ 4 ml / saat, olması gerektiğini unutmayın. </li><li> Bir orta ölçekli (34 cm x 34 cm x 43.5 cm) çalkalama inkübatör bütün aksamını yerleştirin. </li></ol></li><li> Morbidostat ön test <ol><li> 5 V serin fan set güç kaynağı voltajı manyetik karıştırma çubuğu test etmek onun motoru başlatmak için. karıştırma işlemi görsel olarak kontrol edilebilir unutmayın. sürücü gerilimisoğutma fanı motoru tam darbe genişlik modülasyonu (PWM) tarafından kontrol edilir s. </li><li> Yabani tip E. bir dizi hazırlanması 600 optik yoğunlukları ile coli örnekleri genellikle 0.07 den kalibrasyonu 0.3 arasında değişen nm. Bir testi E. yerleştirin Kültür şişede coli numune ve fotodetektör voltajı kaydedin. <ol><li> Yeri ~ 100 plaka okuyucu aynı numunenin ul ve optik yoğunluk kaydedin. kalibrasyon eğrisini çizmek için optik yoğunluk verilerinin karşı fotodetektör voltajını kullanın. tipik optik yoğunluk bir birim değişikliği burada kullanılan önyargı düzeni ile fotodetektör olarak ~ 7.6 V değişiklik karşılık unutmayın. </li></ol></li></ol></li></ol> 2. Morbidostat Koşu <ol><li> Günlük Deneme Başlamadan Önce Hazırlık <ol><li> girişleri bölümünde olduğu gibi cihaz oluşturmak için iç çapı 1/32 inç ve dış çapı 3/32 inç üç ultra-kimyasala dayanıklı Tygon boru donanımları ile kültür flakon hazırlayın1.1 ve Ek Şekil 1&#39;de. </li><li> M9 minimal ortam (% 0.2 glukoz) ve trimetoprim (TMP) ilaç içeren bir ortam hazırlar. 121 ° C&#39;de bir otoklav içinde orta, orta şişe, ilaç orta şişe, atık şişe ve kültür flakon sterilize edin. Not: TMP, daha sonra Tablo 1 &#39;de konsantrasyonları ve ortam içeren TMP ilaç otoklava değildir elde etmek için kullanılan bir hazır bir çözümdür. </li></ol></li><li> Morbidostat Koşu <ol><li> Deneyin ilk gününde, donmuş vahşi tip 1 ml E. çözülme Oda sıcaklığında ve transfer ihtiva eden kültür şişesine hücreler 120 ul, 5 dakika boyunca coli MG1655 hücreler, M9 büyüme ortamı 12 ml. İlk günden sonra, dondurulmuş E. 1 ml çözülme coli 5 dakika ve transfer içeren yeni bir kültür şişesine hücrelerin 120 ul için önceki gün hücreleri ~ M9 büyüme ortamı 12 ml. </li><li> çalkalayıcı inkübatör açın ve tempe ayarlamak<html> Resim çalkalanarak 30 ° C&#39;ye kadar süreyle. </li><li> mikro-pompa, manyetik bir karıştırma birimi, ve optik yoğunluk ölçümü açarak morbidostat işlemi başlatır. NOT: Çalışma sırasında, bir geri besleme eşik algoritması ilaç enjeksiyonu kontrol etmek için kullanılır. ölçülen optik yoğunluk önceden belirlenmiş bir eşiği aştığında, ilaç enjeksiyon pompası açık olacak ve orta pompa kapalı olacaktır. Aksi takdirde, ilaç enjeksiyon pompası devre dışı kalır ve orta pompa açıktır. </li><li> mikrobiyal büyüme olmadığından emin olmak için zaman zaman gerilim izleyin. Not: Çünkü donma ve çözülme döngüsü, E. coli hücreleri, her gün büyüme sırasında yaklaşık 4 saatlik bir gecikme sergiler. </li><li> ~ 23 saat sonra, onun besleme gerilimini kapatarak mikro pompayı durdurun ve kültür şişesini çıkarmak. Kültür şişeye% 15 gliserol ekleyin ve depolama için -80 ° C&#39;de örnek dondurma. </li><li> Tekrarlayın 2.2.5 2.2.1 adımları. </li></ol></li></ol>le &quot;&gt; 3. Antibiyotik Duyarlılık Ölçümü 96 Kuyu Formatında NOT: antibiyotik direnci seviyesini belirlemek için 96 kuyu biçiminde bir plaka okuyucu büyüme hızı ölçümü yapın. <ol><li> 5 dakika boyunca oda sıcaklığında, her gün, dondurulmuş numune çözülme ve • 15 mi M9 ortam ihtiva eden kültür şişesine hücre 15 ul transfer. Onları ~ 0.1 ulaştığı 600 nm&#39;de optik yoğunluk kadar büyümeye izin verin. 1 ml&#39;lik şişeler içine 15 ml ayırın ve Tablo 1 &#39;de gösterildiği gibi, istenen ilaç konsantrasyonları elde etmek için bu adımda TMP ilaç ekleyin. </li><li> Plaka okuyucu içinde her kuyuya adım 3.1 ve yerden numune 200 ul almak ve onları 12 saat 5 için büyümeye olanak sağlar. Dalga boyu 600 nm olduğunda optik yoğunluk ölçümü sırasında otomatik olarak kaydedilir. NOT: Her bir veri noktası için, üçlü ölçümler kaydedilir ve veri tutarlılığı sağlamak için ortalaması alınır. zamana karşı optik yoğunluk veri olabilir be büyüme oranı elde etmek için kullanılır. </li><li> IC 50 elde etmek için ilaç konsantrasyonuna karşı büyüme oranı verilerini çizilir. IC50 büyüme maksimal büyüme oranı 5 yüzde elli olan ilaç konsantrasyonu olarak tanımlanır unutmayın. </li></ol> Mikroakışkan Cihazlar 4. Tek Hücre Morfolojisi ve Antibiyotik Duyarlılık Ölçme <ol><li> Başka yerde 15 açıklandığı gibi PDMS katmanlı yumuşak litografi yoluyla mikroakışkan cihazların imalatı gerçekleştirin. <ol><li> Kontrol ve akış tabakaları için fotorezist kalıp yapmak fotolitografi kullanın. Kontrol katmanı için, bir çıplak silikon bir kalıp üretmek için negatif ışığa (~ 15 mikron yükseklik) ve pozitif ışığa (~ 8 mm yükseklik) kullandığını unutmayın. </li><li> sırasıyla, kontrol tabakası ve akış tabakası, 1: 1 ve 20: 5 çapraz bağlantı oranları PDMS karışımları hazırlayın. kabarcıkları uzaklaştırmak için vakum altında bir desikatör içinde karışımlarını koyun1 saat için. </li><li> (TMSC) buhar trimetilklorosilan silikon gofret kontrol katmanı fotorezist kalıpları Açığa. PDMS karışımı döküm ~ 6 mm kalınlığındaki kontrol katman olun (çapraz bağlama oranı 5: 1) denetim katmanı fotorezist kalıp üzerine silikon gofret. Fırında, 1 saat boyunca 80 ° C&#39;de 40 dakika boyunca kontrol katmanı fırında. </li><li> (: 1, sıkma devir 60 saniye boyunca 3.000 rpm çapraz bağlama oranı 20 ile) bir akış tabakası fotorezist kalıpta bir PDMS karışımı kaplama Spin akış katman olun. Fırında, 1 saat boyunca 80 ° C&#39;de 30 dakika boyunca akış tabakası fırında. </li><li> İstenilen yonga boyutu kontrol tabakası (genellikle 3 cm x 2 cm) kesilir ve elle kontrol tabakası kalkmasına bir jilet kullanın. Akış tabakasının üstüne denetim katmanı yerleştirin ve bir stereomikroskop altında bunları hizalayın. Fırın gece boyunca 80 ° C &#39;de hizalanmış montaj Cure. Daha sonra, bakiye TMCS çözmek için 5 saat için plastik bir kap içinde deiyonize su, 250 ml montaj emmek. </li><li> Daha sonra, 20 G iğne ile giriş delikler ve (PDMS çapraz Oranı 5 bağlama: sıkma devir 2.000 rpm sn 30, 1) boş bir PDMS tabaka ile kaplı bir cam slayt için tüm elastomer bağ 40 sn için hava plazma muamelesi ile 1.2 Torr hava basıncı. </li><li> PDMS gelen sitotoksik etkilerini azaltmak için 80 ° C&#39;de 36 saat boyunca yoğun pişirme yürütmek. Bu pişirme adımı bakteriyel hücrelere toksisite en aza indirmek için kritik olduğunu not edin. </li></ol></li><li> Chip Büyüme Deney üzerinde <ol><li> mikroakışkan cihazın içine yerleştirmeden önce, 1 saat süre ile, 1 saat, M9 ortam için deiyonize su ile yıkayın. </li><li> 5 dakika boyunca oda sıcaklığında, her gün, dondurulmuş numune çözülme ve taze M9 ortam ile yeni bir test tüpü inoküle. gece boyunca 37 ° C&#39;de çalkalamalı bir inkübatör içinde örnek yerleştirin. </li><li> M9 orta mikrobiyal örnek 10 kat sulandırmak ve 5 psi mikrobiyal örnek kabı baskı ile mikroakışkan cihazın içine yükleyin. Daha sonra, TMP ilaç yükü5 psi ilaç ortamı kap baskı çip halinde araç. 15 dakika mikroakışkan yonga üzerinde iki oda arasında Mikromekanik valfi harekete geçirerek mikrop ve ilaç arasındaki karıştırma yürütün. </li><li> ters mikroskop üzerine monte mikroskop inkübatör mikroakışkan çip yerleştirin. CCD kamera ile 8 saat boyunca saat ters mikroskop üzerine monte edilmiş bir büyüme odasında hücre görüntü elde. </li><li> Hücre görüntü verileri 18 bir eşik algoritma ile hücre sayıları hesaplamak için özel program komut dosyasını kullanın. hücre sayısı verileri genellikle üzerinde üç mikroakışkan odaları ortalama unutmayın. </li></ol></li></ol>

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C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tools+for+developing+tolerance+to+toxic+chemicals+in+microbial+systems+and+perspectives+on+moving+the+field+forward+and+into+the+industrial+setting.">Tools for developing tolerance to toxic chemicals in microbial systems and perspectives on moving the field forward and into the industrial setting.</a> Curr Opin in Chem Eng. 6, 9-17 (2014).</li></ol>

The authors have nothing to disclose.

düşük maliyetli bileşenleri Düşük ayak izi morbidostat cihaz gösterilmiştir. Cihaz tarafından kayıtlı ilaç direnç seviyesi artar önceki raporlarda 5 ile tutarlıdır. ilaç direnci evrimsel çalışmalar için tasarlanan cihaz, diğer birçok deneylere potansiyel uygulanabilir. İlk olarak, ilaç kaynaklı mutasyon Ayrıntılı bir klinik açıdan önemli antibiyotik büyük bir set için kurulabilir. Örneğin, çoklu ilaç direncinin evrimsel yolun sadece deneyde kullanılan ilaç sayısını...

İlk antibiyotik ilaç penisilin tanıtılmasından bu yana, mikrobiyal antibiyotik direnci küresel bir sağlık sorunu 1 dönüşmüştür. Antibiyotik direnci elde edilmesi geriye in vivo olarak incelenebilir karşın, bu deneylerin koşulları genellikle bütün evrim 2 boyunca kontrol edilmez. Alternatif olarak, adaptif laboratuvar evrim bir antibiyotik ilaç 3&#39;ten çevresel streslere veya seçim baskısı altında bir mikrobiyal türlerin moleküler evrim ortaya çıkarabilir. Son zamanlarda, antibiyotik ilaç direncinin çok iyi kontrol evrimsel deneyler antibiyotik ilaç direnci ortaya çıkmasını aydınlatmıştır. Örneğin, Austin&#39;in grup düzgün tasarlanmış mikroakışkan bölmeli bir ortamda 4 hızlı çıkmasını gösterdi. Son zamanlarda geliştirilen morbidostat ilaç seçim baskısı 5,6 altında sistematik mutasyonlar neden olur. morbidostat, mikrobiyal SendaiSürekli neredeyse sabit nüfusu korumak için antibiyotik konsantrasyonunu ayarlar yon cihazı, mikrobiyoloji 7,8 kullanılan dalgalanma testi önemli bir ilerlemedir. dalgalanma testinde, bir antibiyotik ilaç yüksek bir konsantrasyonda enjekte edilir ve kalan mutantlar elenir ve sayılır. Bunun yerine, bir morbidostat içinde mikropların sürekli meydan ve çoklu mutasyonları elde edilir. Morbidostat kemostat, mikrobiyal nüfusu 9 sulandırarak sırasında sürekli olarak besinleri sağlayarak sabit bir nüfus tutar 1950 yılında Novick&#39;le ve Szliard tarafından icat bir kültür cihazına benzer çalışır. piyasaya sunulmasından bu yana, kemostat gelişmiş ve geliştirilmiştir. Güncel mikroakışkan chemostats nanoliter ve tek hücreli kapasiteleri ulaşmıştır. Bununla birlikte, bu cihazlar, bir çok mutasyon olayları 10,11 ile büyük hücre popülasyonunu gerektiren adaptif evrimi deneyler için uygun değildir. Son zamanlarda, mini~ 10 ml çalışma hacmine sahip chemostats da litre ölçek Biyoreaktörlerde ve mikroakışkan kemostat 12,13 arasındaki boşluğu doldurmak için geliştirilmiştir. Burada bir antibiyotik ilaç direnci çalışma için tasarım ve düşük maliyet, otomatikleştirilmiş morbidostat sunuyoruz. Önerilen modül minimum donanım gereksinimi ile bir mikrobiyoloji laboratuvarında bir çalkalama inkübatör kullanılabilir. Açık kaynak yazılım aynı zamanda kolayca metabolik mühendislik 3 olarak adaptif evrim özel uygulamalar, için özel olarak tasarlanmıştır. Son olarak, morbidostat antibiyotik duyarlılık testi 14 için multiplekslenmiş mikroakışkan platforma entegre edilmiştir.

<table><tbody><tr><td>Environmental Shaker Incubator</td><td>BioSan</td><td>ES-20</td><td></td></tr><tr><td>Arduino Leonardo board</td><td>Arduino</td><td>Leonardo</td><td></td></tr><tr><td>680 Ohm Carbon Resistor</td><td>Digikey</td><td></td><td>Bias resistor for LED</td></tr><tr><td>100k Ohm Carbon resistor</td><td>Digikey</td><td></td><td>Bias resistor for phototransistor</td></tr><tr><td>940 nm light emitting diode</td><td>Bright LED Electronic</td><td>BIR-BM13E4G-2</td><td>Optical density measurement</td></tr><tr><td>940 nm phototransistor</td><td>Kodenshi&amp;nbsp;</td><td>ST-2L2B</td><td>Optical density measurement</td></tr><tr><td>Darlington pair IC Toshiba</td><td>Mouser</td><td>ULN2803APG</td><td>this IC drives micropumps and magnetic stirring unit</td></tr><tr><td>5 V DC brushless fan</td><td>ADDA</td><td>AD0405LX-G70</td><td>spec: 5 V supply voltage and 80&amp;nbsp;mA; available www.jameco.com</td></tr><tr><td>Piezoelectric micropump</td><td>CurieJet</td><td>PS15I-FT-5L</td><td>Pressure &gt; 3&amp;nbsp;kPa; Flow rate &gt;5 ml/min</td></tr><tr><td>Tygon 3350 Tuning</td><td>Saint Gobain</td><td>ABW00001</td><td>ID: 1/32&quot; OD: 3/32&quot; L:50&#039;&amp;nbsp;</td></tr><tr><td>Magnetic Stir bar</td><td>COWIE</td><td></td><td>tapered shape dim: 10&amp;nbsp;mm x 4 mm</td></tr><tr><td>Glass scintillation 20 ml vial</td><td>DGS</td><td></td><td>Pyrex glass 28 mm (diameter) x 61 mm (height)</td></tr><tr><td>Culture vial holder</td><td></td><td></td><td>Custom made from Polyformaldehyde&amp;nbsp;</td></tr><tr><td>Silicone</td><td>Dow Corning</td><td>Sylgald 184</td><td>used to seal the glass vial</td></tr><tr><td>Medium bottle</td><td>VWR</td><td>66022-065</td><td></td></tr><tr><td>Difco M9 minimal salt 5x</td><td>BD</td><td></td><td>Medium</td></tr><tr><td>Cadamino Acid</td><td>BD</td><td></td><td>Medium</td></tr><tr><td>glucose</td><td>Sigma</td><td></td><td></td></tr><tr><td>Agar Bateriological</td><td>Oxoid</td><td></td><td>for agar plate</td></tr><tr><td>Luria Bertani medium</td><td></td><td></td><td></td></tr><tr><td>Inverted microscope</td><td>Leica Microsystems</td><td>Leica DMI-LED</td><td>used for microfluidic measurement Use 40X objective NA = 0.55</td></tr><tr><td>Microscope Incubator</td><td>Live Cell Instrument</td><td>CU-109</td><td>used for microfluidic measurement</td></tr><tr><td>Solenoidal valves</td><td>Pneumadyne</td><td>S10MM-31-12-3</td><td>Normally open 1.3 Watt 12 Vdc</td></tr><tr><td>USB interface card</td><td>Hobby Engineering</td><td>USBIO24-R Digital I/O Module&amp;nbsp;</td><td>for microfluidics measurement</td></tr><tr><td>Air compressor</td><td>Rocker Scientific</td><td>ROCKER 440</td><td>Pressure source for microfluidcs; Max. Pressure 80 psi</td></tr><tr><td>Male luer-lock fittings to 1/8&quot; barb</td><td>ValuePlastics.com</td><td>MTLL230-1</td><td>used for microfluidic control</td></tr><tr><td>1/8&quot; barb to 10-32 threaded port</td><td>ValuePlastics.com</td><td>B-1</td><td>used for microfluidic control</td></tr><tr><td>Female luer-lock fittings to 10-32 threaded port</td><td>ValuePlastics.com</td><td>KFTL-1</td><td>used for microfluidic control</td></tr><tr><td>NPN darlington transistor 500 mA, 40&amp;nbsp;V (2N6427)</td><td>DigiKey.com</td><td>2N6427GOS-ND</td><td>used for microfluidic control</td></tr><tr><td>10 kOhm, carbon film resistor, 0.25 W</td><td>DigiKey.com</td><td>P10KBACT-ND</td><td>used for microfluidic control</td></tr><tr><td>Tantalum capacitor, 10&amp;nbsp;&amp;mu;F, 25 V, 10%</td><td>DigiKey.com</td><td>478-1841-ND</td><td>used for microfluidic control</td></tr><tr><td>Andor CCD camera</td><td>Andor</td><td>Zyla 4.2 Plus SCMOS</td><td>used for microfluidic on chip imaging</td></tr><tr><td>ELISA plate reader</td><td></td><td></td><td></td></tr><tr><td>two component Silicone&amp;nbsp;</td><td>Momentive</td><td>RTV 615</td><td>used for microfluidic chip fabrication</td></tr><tr><td>SU-8 photoresist</td><td>Micrchem</td><td>SU8 2015</td><td>used for microfluidic chip fabrication</td></tr><tr><td>AZ4620 photoresist</td><td>Clariant</td><td>AZ 4620</td><td>used for microfluidic chip fabrication</td></tr><tr><td>Plasma cleaner</td><td>Harrick Plasma</td><td>PDC 32G</td><td>used for microfluidic chip fabrication</td></tr><tr><td>20 Gauge Syringe Needle</td><td>BD</td><td></td><td>used for microfluidic chip fabrication</td></tr><tr><td>Labcycler</td><td>Sensoquest</td><td>Labcycler</td><td>PCR</td></tr><tr><td>DNA polymerase</td><td>Toyobo</td><td>KDO Plus</td><td>PCR amplification</td></tr><tr><td>Trimethoprim</td><td>Sigma</td><td></td><td></td></tr><tr><td>Plate reader</td><td>Biotek</td><td>Synergy H1 hybrid&amp;nbsp;</td><td>antibiotic resistane measurement</td></tr></tbody></table>

design and use of a low cost, automated morbidostat for adaptive evolution of bacteria under antibiotic drug selection

Biz antibiyotik direnci evrimsel yolu tanımlamak için düşük maliyetli, yapılandırılabilir morbidostat açıklar. morbidostat sürekli bakteri gelişimini izler ve dinamik onlar ilaç direnci elde etmek için geliştikçe sürekli bakteri meydan okumak için ilaç konsantrasyonunu ayarlayan bakteriyel kültür cihazdır. Cihaz ~ 10 ml çalışma hacmi bulunmaktadır ve tam otomatik ve optik yoğunluk ölçümü ve orta ve ilaç dağıtımı için mikro pompalar ile donatılmıştır. Platformu doğrulamak için, biz 1655 Escherichia coli MG trimetoprim direncinin aşamalı satın ölçüldü ve hücre morfolojisi ve antibiyotik duyarlılıklarının araştırmak için multiplekslenmiş mikroakışkan platformu cihazı entegre edilmiştir. yaklaşım antibiyotik ilaç direncinin laboratuvar çalışmalarına ölçekli yukarı ve metabolik mühendislik ve diğer bakteri kültürü deneylerinde gerilme geliştirmeleri için evrimi adaptif uzatılabilir olabilir.

Yukarıda açıklanan morbidostat Şekil 1&#39;de şematize edilmiştir. Deneysel evrim, antibiyotik duyarlılık testi ve hücre morfolojisi kontrolü dahil olmak üzere ortak morbidostat işlemleri, bir E. valide edildi (TMP) trimetoprimin maruz coli MG1655 kültürü, yaygın olarak kullanılan bir antibiyotik ilaç 5,6. TMP ilaç direnci çok belirgin adım adım artar uyarır ve mutasyonlar dihidrofolat redüktaz (DHFR) geni etrafında k...

Watch this Scientific Journal Video about Design and Use of a Low Cost, Automated Morbidostat for Adaptive Evolution of Bacteria Under Antibiotic Drug Selection at JoVE.com

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 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.

Tasarım ve Düşük Maliyetli kullanımı, Otomatik Morbidostat Bakterilerin Adaptif Evolution için antibiyotik ilaç Seçimi Altında

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

design-use-low-cost-automated-morbidostat-for-adaptive-evolution

Research

JoVE Journal

Genetics

9.7K Görüntüleme.  National Tsing Hua University. 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.

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

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.

Here, we present a protocol to obtain adaptive laboratory evolution of microorganisms under conditions using chemostat culture. Also, genomic analysis of the evolved strain is discussed.

Bir Kemostat kullanarak Mikroorganizmaların Adaptif Laboratuvarı Evolution Prosedürü

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

Genetik

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

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

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

Multicopy plazmid rolü antibiyotik direnci gelişimi test

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

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 kullanarak otomatik, yüksek verimlilik, sürekli hücre büyüme denemeleri tasarlama

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

Quantification of Plasmid-Mediated Antibiotic Resistance in an Experimental Evolution Approach

Plasmids play a major role in microbial ecology and evolution as vehicles of lateral gene transfer and reservoirs of accessory gene functions in microbial populations. This is especially the case under rapidly changing environments such as fluctuating antibiotics exposure. We recently showed that plasmids maintain antibiotic resistance genes in Escherichia coli without positive selection for the plasmid presence. Here we describe an experimental system that allows following both the plasmid genotype and phenotype in long-term evolution experiments. We use molecular techniques to design a model plasmid that is subsequently introduced to an experimental evolution batch system approach in an E. coli host. We follow the plasmid frequency over time by applying replica plating of the E. coli populations while quantifying the antibiotic resistance persistence. In addition, we monitor the conformation of plasmids in host cells by analyzing the extent of plasmid multimer formation by plasmid nicking and agarose gel electrophoresis. Such an approach allows us to visualize not only the genome size of evolving plasmids but also their topological conformation-a factor highly important for plasmid inheritance. Our system combines molecular strategies with traditional microbiology approaches and provides a set-up to follow plasmids in bacterial populations over a long time. The presented approach can be applied to study a wide range of mobile genetic elements in the future.

Our experimental approach provides a strategy to follow plasmid abundance and antibiotic resistance over time in bacterial populations.

Deneysel Evrim Yaklaşımında Plazmid Aracılı Antibiyotik Direncinin Ölçülmesi

Plasmids play a major role in microbial ecology and evolution as vehicles of lateral gene transfer and reservoirs of accessory gene functions in microbial ...

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.

Günlük Transferler, Popülasyonların Arşivlenmesi ve Escherichia coli ile Uzun Vadeli Evrim Deneyinde Uygunluğun Ölçülmesi

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

Mutagenesis and Functional Selection Protocols for Directed Evolution of Proteins in E. coli

The efficient generation of genetic diversity represents an invaluable molecular tool that can be used to label DNA synthesis, to create unique molecular signatures, or to evolve proteins in the laboratory. Here, we present a protocol that allows the generation of large (>1011) mutant libraries for a given target sequence. This method is based on replication of a ColE1 plasmid encoding the desired sequence by a low-fidelity variant of DNA polymerase I (LF-Pol I). The target plasmid is transformed into a mutator strain of E. coli and plated on solid media, yielding between 0.2 and 1 mutations/kb, depending on the location of the target gene. Higher mutation frequencies are achieved by iterating this process of mutagenesis. Compared to alternative methods of mutagenesis, our protocol stands out for its simplicity, as no cloning or PCR are involved. Thus, our method is ideal for mutational labeling of plasmids or other Pol I templates or to explore large sections of sequence space for the evolution of activities not present in the original target. The tight spatial control that PCR or randomized oligonucleotide-based methods offer can also be achieved through subsequent cloning of specific sections of the library. Here we provide protocols showing how to create a random mutant library and how to establish drug-based selections in E. coli to identify mutants exhibiting new biochemical activities.

Mutagenesis and Functional Selection Protocols for Directed Evolution of Proteins in E. coli

Here we demonstrate a simple protocol to create a random mutant library for a given target sequence. We show how this method, which is performed in vivo in Escherichia coli, can be coupled with functional selections to evolve new enzymatic activities.

Proteinlerin Yönetmen Evolution Mutagenez ve Fonksiyonel Seçim Protokolleri E. coli</em

The efficient generation of genetic diversity represents an invaluable molecular tool that can be used to label DNA synthesis, to create unique molecular ...

Biyoloji

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.

Mikrobiyal Systems Biyoloji Chemostats Kullanımı

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

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.

Multiplexed kemostat Diziler Tasarımı ve Kullanımı

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

An Automated Culture System for Use in Preclinical Testing of Host-Directed Therapies for Tuberculosis

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), was the most significant infectious disease killer globally until the advent of COVID-19. Mtb has evolved to persist in its intracellular environment, evade host defenses, and has developed resistance to many anti-tubercular drugs. One approach to solving resistance is identifying existing&#160;approved drugs that will boost the host immune response to Mtb. These drugs could then be repurposed as adjunctive host-directed therapies (HDT) to shorten treatment time and help overcome antibiotic resistance.
Quantification of intracellular Mtb growth in macrophages is a crucial aspect of assessing potential HDT. The gold standard for measuring Mtb growth is counting colony-forming units (CFU) on agar plates. This is a slow, labor-intensive assay that does not lend itself to rapid screening of drugs. In this protocol, an automated, broth-based culture system, which is more commonly used to detect Mtb in clinical specimens, has been adapted for preclinical screening of host-directed therapies. The capacity of the liquid culture assay system to investigate intracellular Mtb growth in macrophages treated with HDT was evaluated. The HDTs tested for their ability to inhibit Mtb growth were all-trans Retinoic acid (AtRA), both in solution and encapsulated in poly(lactic-co-glycolic acid) (PLGA) microparticles and the combination of interferon-gamma and linezolid. The advantages of this automated liquid culture-based technique over the CFU method include simplicity of setup, less labor-intensive preparation, and faster time to results (5-12 days compared to 21 days or more for agar plates).

Rapid and efficient quantification of intracellular M. tuberculosis growth is crucial for pursuing improved therapies against tuberculosis (TB). This protocol describes a broth-based colorimetric detection assay using an automated liquid culture system to quantify Mtb growth in macrophages treated with candidate host-directed therapies.

Tüberküloz için Ev Sahibi Tarafından Yönlendirilen Tedavilerin Preklinik Testlerinde Kullanılmak Üzere Otomatik Bir Kültür Sistemi

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), was the most significant infectious disease killer globally until the advent ...

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

Many microorganisms such as bacteria proliferate extremely fast and the populations may reach high cell densities. Small fractions of cells in a population always have accumulated mutations that are either detrimental or beneficial for the cell. If the fitness effect of a mutation provides the subpopulation with a strong selective growth advantage, the individuals of this subpopulation may rapidly outcompete and even completely eliminate their immediate fellows. Thus, small genetic changes and selection-driven accumulation of cells that have acquired beneficial mutations may lead to a complete shift of the genotype of a cell population. Here we present a procedure to monitor the rapid clonal expansion and elimination of beneficial and detrimental mutations, respectively, in a bacterial cell population over time by cocultivation of fluorescently labeled individuals of the Gram-positive model bacterium Bacillus subtilis. The method is easy to perform and very illustrative to display intraspecies competition among the individuals in a bacterial cell population.

Bacteria may accumulate either detrimental or beneficial mutations during their lifetime. In a population of cells individuals that have accumulated beneficial mutations may rapidly outcompete their fellows. Here we present a simple procedure to visualize intraspecies competition in a bacterial cell population over time using fluorescently labeled individuals.&#160;

Tasarım ve Düşük Maliyetli kullanımı, Otomatik Morbidostat Bakterilerin Adaptif Evolution için antibiyotik ilaç Seçimi Altında

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