Jozsef leylek, Mitchel Richmond, Jarrad Gollihue ve Andrea Sanchez yapıcı ve eleştirel tartışma için teşekkür ederiz. Dr. Sharyn Perry fenotipik fotoğraflar için. Bu malzeme Kooperatifi anlaşması No 1355438 altında Ulusal Bilim Vakfı tarafından desteklenen çalışma üzerine kuruludur.

1. seyreltme kitaplığı oluşturma
<ol><li>625 seyreltme Kütüphane plakaları el ile kimyasal kitaplığından kendi karşılık gelen plakasına eşleştikleri sağlanması etiketleyin. Ayrıca, bağlantı akışı ve akış hortumlar için çok kanallı ipucu yıkama otomatik aygıtlar Pozisyoner (ALP) dışarı 5 galon rezervuar konsol sürücü geçerek ( Tablo malzemelerigörmek).</li>
	<li>Bilgisayara erişmek ve çok kanallı ipucu yıkama ALP için aygıt denetleyicisi bağlantısı üzerinden yıkama pompası su geçirilip yorumlanması için dolaştırmak için açın. Bu protokol sonunda otomatik olarak kapanır.</li>
	<li>Yük, el, Stacker 10'ın Stacker Carousel, otel A - D (şekil 4, Stacker); aşağıdaki sırayla bağlı bir kutu AP96 P20 pipet ipuçları Oda 1, dört 96-şey V-Odalar 2-5 alt levha sipariş edilen Kütüphane ve iki alt plaka hisse senedi konsantrasyonları içeren iki üst plakalı boş (şekil 5, Stacker). Ayrıca, Oda 6 ve dört 96-şey V AP96 P20 pipet ipuçlarında, bir kutu yüklemek-Odalar 7-9 alt levha sipariş edilen Kütüphane ve iki alt plaka hisse senedi konsantrasyonları içeren iki üst plakalı boş (şekil 5, Stacker).</li>
	<li>P3, 300 mL % 70 alkol banyo P7 üzerinde üzerinde 300 mL su haznesi ile güverte, el ile ayarla ipucu yükleyici ALP (TL1) ve çok kanallı ipucu yıkama ALP (TW1) (şekil 4, güverte ve şekil 5, güverte).</li>
	<li>İşletim yazılımı kullanarak, Stacker 10'dan AP96 P20 pipet ipuçları sunmak ve ipucu yükleyici ALP taşıyın. Not: 1.5 1.12 tüm aracılığıyla robotun çalışma yazılım işleme sıvı ile yapılır; Tablo malzemelerigörmek.</li>
	<li>Mevcut Oda 2 otel a ve tüm dört 96-şey V ayrı-alt tabaklar güverte üzerinde alt yerleştirerek iki P4 ve P8 ve ilk iki P5 ve P9 (şekil 4).</li>
	<li>AP96 P20 pipet ipuçları ile uç yüklü ALP 96-kanal 200 µL baş üzerine yükleyin. 300 mL su rezervuar 90 µL Aspire edin ve 96-şey V dağıtmak-alt seyreltme plaka P4 üzerinde. P8 tabağa için bu adımı yineleyin.</li>
	<li>P5 kimyasal Kütüphane tabağa hkr alıyorum ve 15 µL üç kez dağıtımı ile karıştırın. Ayrıca, P5 kimyasal Kütüphane tabağa üzerinden 10 µL Aspire edin ve P4 seyreltme tabağın içine 10 µL dağıtmak.</li>
	<li>P4 tabağa çözümleri sürekli alıyorum ve 50 µL dağıtımı tarafından toplam üç kez karıştırın. Temiz karışınca alıyorum ve % 70 70 µL dağıtımı tarafından AP96 P20 pipet ipuçları sonra onları yıkama çok kanallı ipucu yıkama ALP alıyorum ve su dört bir %110 hacim dağıtımı tarafından alkol P7, üzerinden zaman.</li>
	<li>P8 ve P9 Tabaklarda ikinci çifti için 1,8 1,9 adımları yineleyin. İkinci 96-şey V oluşturma üzerine-alt seyreltme plaka, plaka ile aşağıdaki sırada alt üst yığını: P9, P5, P8 ve P4. Sonra yığının üzerinde bir boş statik ALP yer; P1, P2, P6, P10, P11, P12 veya P13.</li>
	<li>Oda 5 otel a boş 1.6-1,10 tamamlayana dek. Oda 6 ulaşan, yeni AP96 P20 pipet ipuçları için ipucu yükleyici ALP hareketli ve kullanılan AP96 P20 pipet ipuçları üzerinde boş bir statik ALP yerleştirerek üzerine 1.5 adımı yineleyin.</li>
	<li>Oda otel 9 A boş 1.6-1,10 tamamlayana dek. Ancak, otel B için devam etmek için tabakalar ve ipuçları güvertede otel A. yeniden yüklenmesi gerekir</li>
	<li>, El ile 300 mL su haznesi yeniden doldurun. Bu adım çok önemlidir ve bilgisayar programı 'devam, sonraki adımı gerçekleştirmeden önce' vurmak kullanıcı gerektiren bu mesajı ayrıntılı bir duraklama dahil edebilirsiniz.</li>
	<li>Sonraki Hotel'e devam etmeden önce her zaman bir tam 300 mL su haznesi sağlanması kalan otel 1.5-1,13 arasındaki adımları yineleyin.</li>
</ol>2. medya-tohum karışımı plakaları eleme için ekleme
<ol><li>½ Murashige ve Skoog (MS) medya ile % 0,1 Agar 4.3 g MS Salts, 0.50 g MES, 1.0 g Agar 1 L DI H2O. eklenmesiyle ayarlamak pH 5,7 için yine de 5 M potasyum hidroksit ilavesi ile pH probe izleme sırasında.</li>
	<li>Onları % 1 çamaşır suyu ve SDS 15 ve 30 dk arasında sallayarak tohum sterilize ve sonra 4 kez Santrifüjü tarafından eşit bir su hacmi ile durulayın. Tohum steril olduğunda, onları vernilization için 7 gün 24 saat üzerinden 4 ° C yerleştirin. Arabidopsis biyolojik kaynak merkezi sterilizasyon, vernilization ve büyüme22ek yöntemlerini açıklar.</li>
	<li>Tohum medyaya bir yoğunluk 0,1 g/100 mL el ile ekleyin. Bu yoğunluğu 3-10 tohum iyi bir 96-şey plaka başına ortalama sonuçlanır.</li>
	<li>Yer, elle, dört 96-şey düz-Oda sayısı 1 ve 2 otel a (6 rakam, otel A) alt levha. İpucu yükleyici ALP AP96 P250 pipet ipuçları bir kutu yerleştirin, 2.1-2,3 üzerinde P3 adımlarda oluşturduğunuz medya-tohum karışımı ile 300 mL su deposu dolu ve 300 mL su deposu dolu ile % 70 alkol P7 üzerinde (şekil 4, güverte ve şekil 6 Güverte). Not: 2,5 ile 2,8 işletim yazılımı ile yapılır.</li>
	<li>Oda sayısı 1 ve 2 otel a mevcut ve dört tabak yığınları ayırın. Bir tabak her boş statik Alpler (P4, P5, P6, P8, P9, P10, P11 ve P12) üzerinde yerleştirin. AP96 P250 pipet ipuçları 96-kanal 200 µL kafasına yük.</li>
	<li>P3 üzerinde 300 mL medya-tohum rezervuar 90 µL Aspire edin ve ilk 96-şey düz dağıtmak-alt plaka. Tüm sekiz plakaları medya-tohum karışımı içeren kadar bu işlemi yineleyin.</li>
	<li>AP96 P250 pipet keyif alıyorum ve 70 µL % 70 ile dolu 300 mL rezervuar dağıtımı temizleyin alkol P7 üzerinde. İpuçları çok kanallı ipucu yıkama ALP içinde alıyorum ve su bir %110 hacim dört kez dağıtımı tarafından yıkama ipuçları TL1, kaldırma ve tabakları el ile toplamak.</li>
</ol>3. küçük moleküller plakaları eleme için ekleme
<ol><li>Yük, elle, bir kutu AP96 P250 pipet ipuçları Oda 1, otel haline A, iki 96-şey V-2, 4, 6 ve 8 oda ve iki 96 de düz-alt eleme kalıplara Odalar 3, 5, 7 ve 9 (şekil 4 alt seyreltme Kütüphane kalıplara Stacker ve Şekil 7, otel A). Ayrıca, hortumlar ve çok kanallı ipucu yıkama ALP 5 galon su deposu üzerinden bağlayın. Not: 3.10 üzerinden 3.2 işletim yazılımı ile yapılır.</li>
	<li>Güverte P7 bir 300 mL % 70 alkol yıkama rezervuar içeren yapılandırma; medya-tohum rezervuar P3, güvertede yaptı (şekil 4, güverte ve Şekil 7, güverte). Buna ek olarak, çok kanallı ipucu yıkama ALP suda dolaşmaya aygıt denetleyicisi bağlantısı üzerinden konsol sürücü açın. Bu protokol sonunda otomatik olarak kapanır.</li>
	<li>AP96 P250 pipet İpucu kutusunu Hotel A'dan sunmak ve ipucu yükleyici ALP taşıyın.</li>
	<li>96-şey V mevcut-alt seyreltme Kütüphane plakaları otel Oda 2 A güverte ve statik ALP P4 ve P8 üstünde bir yer. 96-şey düz sunmak-alt tarama plakalar üzerinden Oda 3 otel a güverte ve bir statik ALP P5 ve P9 üstünde bir yer.</li>
	<li>AP96 P250 pipet ipuçları ile uç yüklü ALP 96-kanal 200 µL baş üzerine yükleyin.</li>
	<li>96-şey V mix-alt seyreltme plaka alıyorum ve üç kez 50 µL dağıtımı P4 üzerinde. Bu, bu plaka 10 µL Aspire edin ve 96-şey düz dağıtmak-alt tarama plaka üzerinde P5.</li>
	<li>P5 plaka çözümlerinde alıyorum ve üç kez 50 µL dağıtımı ile karıştırın. AP96 P250 pipet ipuçları etanol ile temiz alıyorum ve % 70 70 µL dağıtımı tarafından alkol P7 ve sonra yıkama ipuçları çok kanallı ipucu yıkama alıyorum ve % 110 hacmi dağıtımı ALP, dört kez su rezervuar.</li>
	<li>İkinci 96-şey V için 3.5 ve 3.6 adımları yineleyin-alt seyreltme Kütüphane plaka (P8) ve 96-şey düz-alt tarama plaka (P9).</li>
	<li>Yığın iki 96-şey V-alt seyreltme Kütüphane tabak birlikte ve iki 96-şey düz alt tarama tabak birlikte. Tabakları statik ALPs P1, P2, P6, P10, P11, P12 veya P13 taşıyın.</li>
	<li>3.4-3,9 üç kez adımları yineleyin, tarama için seyreltilmiş kimyasallar ekleyerek toplam sekiz kez tabaklar. Son olarak, her iyi görsel uyum aracılığıyla tarama plakaların tohumların numarayı denetleyin ve bu wells ile ek steril ve vernalized tohum tarafından üçten tohum ek.</li>
</ol>4. kuluçka ve görselleştirme plakaları tarama
<ol><li>96-şey düz-alt eleme plakaları dört gün boyunca 22 ° c çevre bir odasında bir 16/8 açık/koyu döngüsünde bir kuruma geçirmez konteyner kuluçkaya. 96-şey düz-alt eleme plakaları diseksiyon mikroskop altında görselleştirin. Daha fazla araştırma yapılması için tüm anormal fenotipleri kaydedin.</li>
</ol>

<ol>
	<li>Blackwell, H. E., Zhao, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+genetic+approaches+to+plant+biology.">Chemical genetic approaches to plant biology.</a> Plant Physiol. 133 (2), 448-455 (2003).</li><li>Dejonghe, W., Russinova, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plant+chemical+genetics:+From+phenotype-based+screens+to+synthetic+biology.">Plant chemical genetics: From phenotype-based screens to synthetic biology.</a> Plant Physiol. 174 (1), 5-20 (2017).</li><li>McCourt, P., Desveaux, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plant+chemical+genetics.">Plant chemical genetics.</a> New Phytol. 185 (1), 15-26 (2010).</li><li>Lumba, S., Cutler, S., McCourt, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plant+nuclear+hormone+receptors:+A+role+for+small+molecules+in+protein-protein+interactions.">Plant nuclear hormone receptors: A role for small molecules in protein-protein interactions.</a> Annu Rev Cell Dev Biol. 26, 445-469 (2010).</li><li>Hicks, G. R., Raikhel, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Opportunities+and+challenges+in+plant+chemical+biology.">Opportunities and challenges in plant chemical biology.</a> Nat Chem Biol. 5 (5), 268-272 (2009).</li><li>De Rybel, B., 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=A+role+for+the+root+cap+in+root+branching+revealed+by+the+non-auxin+probe+naxillin.">A role for the root cap in root branching revealed by the non-auxin probe naxillin.</a> Nat Chem Biol. 8 (9), 798-805 (2012).</li><li>Koornneef, M., Meinke, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+development+of+Arabidopsis+as+a+model+plant.">The development of Arabidopsis as a model plant.</a> Plant J. 61 (6), 909-921 (2010).</li><li>Serrano, M., Kombrink, E., Meesters, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Considerations+for+designing+chemical+screening+strategies+in+plant+biology.">Considerations for designing chemical screening strategies in plant biology.</a> Front Plant Sci. 6, 131 (2015).</li><li>Yoshitani, N., 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=A+structure-based+strategy+for+discovery+of+small+ligands+binding+to+functionally+unknown+proteins:+Combination+of+in+silico+screening+and+surface+plasmon+resonance+measurements.">A structure-based strategy for discovery of small ligands binding to functionally unknown proteins: Combination of in silico screening and surface plasmon resonance measurements.</a> Proteomics. 5 (6), 1472-1480 (2005).</li><li>Macarron, 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=Impact+of+high-throughput+screening+in+biomedical+research.">Impact of high-throughput screening in biomedical research.</a> Nat Rev Drug Discov. 10 (3), 188-195 (2011).</li><li>DeBolt, S., 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=Morlin,+an+inhibitor+of+cortical+microtubule+dynamics+and+cellulose+synthase+movement.">Morlin, an inhibitor of cortical microtubule dynamics and cellulose synthase movement.</a> Proc Natl Acad Sci U S A. 104 (14), 5854-5859 (2007).</li><li>Christian, M., Hannah, W. B., Luthen, H., Jones, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+auxins+by+a+chemical+genomics+approach.">Identification of auxins by a chemical genomics approach.</a> J Exp Bot. 59 (10), 2757-2767 (2008).</li><li>Drakakaki, G., 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=Clusters+of+bioactive+compounds+target+dynamic+endomembrane+networks+in+vivo.">Clusters of bioactive compounds target dynamic endomembrane networks in vivo.</a> PNAS. 108 (43), 17850-17855 (2011).</li><li>Armstrong, J. I., Yuan, S., Dale, J. M., Tanner, V. N., Theologis, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+inhibitors+of+auxin+transcriptional+activation+by+means+of+chemical+genetics+in+Arabidopsis.">Identification of inhibitors of auxin transcriptional activation by means of chemical genetics in Arabidopsis.</a> Proc Natl Acad Sci U S A. 101 (41), 14978-14983 (2004).</li><li>Brown, L. 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=A+small+molecule+with+differential+effects+on+the+PTS1+and+PTS2+peroxisome+matrix+import+pathways.">A small molecule with differential effects on the PTS1 and PTS2 peroxisome matrix import pathways.</a> Plant J. 65 (6), 980-990 (2011).</li><li>De Rybel, B., 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=Chemical+inhibition+of+a+subset+of+Arabidopsis+thaliana+GSK3-like+kinases+activates+brassinosteroid+signaling.">Chemical inhibition of a subset of Arabidopsis thaliana GSK3-like kinases activates brassinosteroid signaling.</a> Chem Biol. 16 (6), 594-604 (2009).</li><li>Arkin, M. R., Tang, Y., Wells, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small-molecule+inhibitors+of+protein-protein+interactions:+progressing+toward+the+reality.">Small-molecule inhibitors of protein-protein interactions: progressing toward the reality.</a> Chem Biol. 21 (9), 1102-1114 (2014).</li><li>St Onge, R., Schlecht, U., Scharfe, C., Evangelista, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Forward+chemical+genetics+in+yeast+for+discovery+of+chemical+probes+targeting+metabolism.">Forward chemical genetics in yeast for discovery of chemical probes targeting metabolism.</a> Molecules. 17 (11), 13098-13115 (2012).</li><li>Vassilev, L. T., 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=In+vivo+activation+of+the+p53+pathway+by+small-molecule+antagonists+of+MDM2.">In vivo activation of the p53 pathway by small-molecule antagonists of MDM2.</a> Science. 303 (5659), 844-848 (2004).</li><li>Zhao, Y., 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=Chemical+genetic+interrogation+of+natural+variation+uncovers+a+molecule+that+is+glycoactivated.">Chemical genetic interrogation of natural variation uncovers a molecule that is glycoactivated.</a> Nat Chem Biol. 3 (11), 716-721 (2007).</li><li>Walsh, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+emerging+field+of+chemical+genetics:+Potential+applications+for+pesticide+discovery.">The emerging field of chemical genetics: Potential applications for pesticide discovery.</a> Pest Manag Sci. 63 (12), 1165-1171 (2007).</li><li>. Seed Handling Available from: <a target="_blank" href="https://abrc.osu.edu/seed-handling">https://abrc.osu.edu/seed-handling</a> (2013)</li><li>Knoth, C., Salus, M. S., Girke, T., Eulgem, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+synthetic+elicitor+3,5-dichloroanthranilic+acid+induces+NPR1-dependent+and+NPR1-independent+mechanisms+of+disease+resistance+in+Arabidopsis.">The synthetic elicitor 3,5-dichloroanthranilic acid induces NPR1-dependent and NPR1-independent mechanisms of disease resistance in Arabidopsis.</a> Plant Physiol. 150 (1), 333-347 (2009).</li><li>Conway, M. K., 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=Scalable+96-well+Plate+based+iPSC+culture+and+production+using+a+robotic+liquid+handling+system.">Scalable 96-well Plate based iPSC culture and production using a robotic liquid handling system.</a> J Vis Exp. , (2015).</li><li>Daniszewski, 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=Automated+cell+culture+systems+and+their+applications+to+human+pluripotent+stem+cell+studies.">Automated cell culture systems and their applications to human pluripotent stem cell studies.</a> SLAS Technol. , (2017).</li><li>Popa-Burke, I., Russell, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Compound+precipitation+in+high-concentration+DMSO+solutions.">Compound precipitation in high-concentration DMSO solutions.</a> J Biomol Screen. 19 (9), 1302-1308 (2014).</li><li>Partridge, F. 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=An+automated+high-throughput+system+for+phenotypic+screening+of+chemical+libraries+on+C.+elegans+and+parasitic+nematodes.">An automated high-throughput system for phenotypic screening of chemical libraries on C. elegans and parasitic nematodes.</a> Cold Spring Harb Protoc. , (2017).</li></ol>

Yazarlar onlar rakip hiçbir mali çıkarları var bildirin.

Bu iletişim kuralı araştırmacılar Arabidopsis ileri kimyasal genetik ekranda yerine getirmeye yardımcı olmak için tasarlanmıştır. Biz temsilcisi sonuçları sağlamak 50.000 bileşikleri (Şekil 2 ve şekil 3) ekranında, en büyük ileri kimyasal genetik ekranlar birini9,13,23bugüne kadar Arabidopsis üzerinde gerçekleştirilen. Sıvı işleme robot kullan...

Son 20 yılda bitki Biyoloji alanındaki araştırmacılar hücre duvarı sentezi, sitoiskeleti, hormon sentezi anlayışımızı geliştirmek ve sinyal kimyasal genetik yaklaşımları, ileriye ve geriye doğru kullanarak büyük atılımlar yapmış, gravitropism, Patogenez, pürin biyosentezi ve1,2,3,4,5kaçakçılığı endomembrane. İleri kimyasal genetik teknikler kullanan fenotipleri ilgi kimliği sağlar ve belirli işlemleri genotypic temellerini anlamak araştırmacılar sağlar. Diğer taraftan, ters kimyasal genetik bir önceden belirlenmiş protein hedef6ile etkileşim kimyasallar arar. Eşlenen ve açıklamalı genomunu küçük olduğu için Arabidopsis bu keşifler bitki Biyoloji ön planda olmuştur. Bu kısa oluşturma süresi ve anormal hücre altı makine7tanımlaması kolaylaştırmak kullanılabilir birden çok mutant/muhabir hatları vardır.
İleri kimyasal genetik ekranlar ilerlemesini, ilk eleme süreci ve bileşik faiz8hedef belirleme yavaş iki önemli performans sorunları vardır. Küçük molekül seçimi hızını artırarak büyük bir yardım otomasyon ve otomatik ekipman9kullanmaktır. Sıvı işleme robotlar büyük kütüphaneler küçük moleküllerin işleme için mükemmel bir araçtır ve biyolojik bilimler10' ilerleme sürüş vesile olmuştur. Burada sunulan Protokolü biyoaktif küçük moleküller hızlı bir hızda. kimliği etkinleştirme tarama süreci ile ilgili performans sorunu hafifletmek için tasarlanmıştır Bu teknik emek ve zaman operatör adına da ilke araştırmacı için ekonomik maliyet azalan süre yükünü azaltır.
Şimdiye kadar en kimyasal kitaplıkları analiz 10.000 ve 20.000 bileşikleri, bazı kadar 150.000 ve bazı az 709,11,12,13,14, ile arasında düzenlenen 15 , 16. burada tanıttı protokolü 50.000 bileşikler (bkz. Tablo reçetesi) küçük molekül Kütüphanesi hayata geçirildi, bir büyük ileri kimyasal genetiğin ekranlar üzerinde yürütülen Arabidopsis bugüne. Özellikle ilgilidir gibi herbisit bulma, böcek ilacı bulma, mantar ilacı doğru artan verimlilik ve hız ile ilgili ileri kimyasal genetik, mevcut trendi ile bu iletişim kuralını uyar keşfetmek, ilaç keşif ve kanser Biyoloji17 ,18,19,20,21. Burada Arabidopsis ile uygulanan rağmen bu iletişim kuralı, kolayca olabilir hücre kültürleri, sporları ve potansiyel olarak bile böcekler için adapte 96, 384 veya 1536-şey tabak içinde sıvı ortamda. Kendi küçük boyutları nedeniyle, Arabidopsis mükellef taraması 96 iyi tabak içinde. Ancak, kuyular arasında eşit şekilde tohumu dağıtma bir mücadeledir. El tohum emek yoğun doğrudur ve tohum 96-şey kalıplara dağıtmak üzere tasarlanan aygıt olsa, onlar satın almak pahalıdır. İşte, nasıl bu adım küçük bir kayıp ile doğruluk atlatılabilmişlerdir göstermektedir.
Bu yöntem genel amacı doğruluğu, üzerinden bir sıvı işleme robot kullanımı ödün vermeden Arabidopsis daha yönetilebilir karşı büyük bir kimyasal Kütüphane eleme yapmaktı. İlk seyreltme serisi yönetimi ve örnekleri bir diseksiyon mikroskop altında ve hızlı hızlı görselleştirme izin sonraki fenotipik ekranlar tamamlamak için geçen süre azaltarak, bu yöntemin kullanılması araştırmacının daha etkili olmasını sağlar kimliği roman biyoaktif küçük moleküller. Şekil 1 4 adımda bu protokolün önemli sonuçlar gösterilmektedir.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57393/57393fig1.jpg"> Şekil 1: genel iş akışı ileri kimyasal genetik ekranın. Bazı detay ile her 4 önemli adımlar için açıklanan protokol genel bir bakış. 1: kimyasal Kütüphane, 2 alma: seyreltme Kütüphane, 3 yapım: yapım eleme plakaları ve 4: kuluçka ve eleme kaplamalar görselleşebilme. <a href="/files/ftp_upload/57393/57393fig1large.jpg" target="_blank">Bu rakam daha büyük bir versiyonunu görüntülemek için buraya tıklayınız.</a>

<table>
	<tbody>
		<tr>
			<td>Keyboard</td>
			<td>Local Provider</td>
			<td>N/A</td>
			<td>Used for protocol design and operating the Biomek FX</td>
		</tr>
		<tr>
			<td>Mouse</td>
			<td>Local Provider</td>
			<td>N/A</td>
			<td>Used for protocol design and operating the Biomek FX</td>
		</tr>
		<tr>
			<td>Computer Screen</td>
			<td>Local Provider</td>
			<td>N/A</td>
			<td>Used for protocol design and operating the Biomek FX</td>
		</tr>
		<tr>
			<td>Computer</td>
			<td>Local Provider</td>
			<td>N/A</td>
			<td>Used for protocol design and operating the Biomek FX</td>
		</tr>
		<tr>
			<td>DIVERSet Diverse Screening Library</td>
			<td>ChemBridge</td>
			<td>N/A</td>
			<td>Chemical library</td>
		</tr>
		<tr>
			<td>Biomek Software</td>
			<td>Beckman Coulter</td>
			<td>N/A</td>
			<td>Runs and designs the Biomek FX</td>
		</tr>
		<tr>
			<td>Device Controller</td>
			<td>Beckman Coulter</td>
			<td>719366</td>
			<td>Operates the water pump/tip washing station</td>
		</tr>
		<tr>
			<td>Stacker Carousel Pendent</td>
			<td>Beckman Coulter</td>
			<td>148240</td>
			<td>Manual operation of Biomek Stacker Carousel</td>
		</tr>
		<tr>
			<td>Biomek Stacker Carousel</td>
			<td>Beckman Coulter</td>
			<td>148520</td>
			<td>Rotary unit that houses all FX Stacker 10&#39;s</td>
		</tr>
		<tr>
			<td>FX Stacker 10</td>
			<td>Beckman Coulter</td>
			<td>148522</td>
			<td>Elevator unit that houses components for screen</td>
		</tr>
		<tr>
			<td>FX Stacker 10</td>
			<td>Beckman Coulter</td>
			<td>148522</td>
			<td>Elevator unit that houses components for screen</td>
		</tr>
		<tr>
			<td>FX Stacker 10</td>
			<td>Beckman Coulter</td>
			<td>148522</td>
			<td>Elevator unit that houses components for screen</td>
		</tr>
		<tr>
			<td>FX Stacker 10</td>
			<td>Beckman Coulter</td>
			<td>148522</td>
			<td>Elevator unit that houses components for screen</td>
		</tr>
		<tr>
			<td>Biomek FX</td>
			<td>Beckman Coulter</td>
			<td>https://www.beckman.com/liquid-handlers</td>
			<td>Robot that performs the desired operations</td>
		</tr>
		<tr>
			<td>Accuframe</td>
			<td>Artisan Technology Group</td>
			<td>76853-4</td>
			<td>Frames arm to place components corretly</td>
		</tr>
		<tr>
			<td>Framing Fixture</td>
			<td>Beckman Coulter</td>
			<td>719415</td>
			<td>Centers arm in the Accuframe</td>
		</tr>
		<tr>
			<td>Multichannel Tip Wash ALP</td>
			<td>Beckman Coulter</td>
			<td>719662</td>
			<td>Washes the tips after the ethanol bath</td>
		</tr>
		<tr>
			<td>Tip Loader ALP</td>
			<td>Beckman Coulter</td>
			<td>719356</td>
			<td>Pneumatically loads tips onto the arm</td>
		</tr>
		<tr>
			<td>Air Compressor</td>
			<td>Local Provider</td>
			<td>N/A</td>
			<td>Provides air for pneumatic tip loading</td>
		</tr>
		<tr>
			<td>MasterFlex Console Drive</td>
			<td>Cole-Parmer</td>
			<td>77200-65</td>
			<td>Pump used to circulate water through the Multichannel Tip Washer</td>
		</tr>
		<tr>
			<td>Air Hose</td>
			<td>Local Provider</td>
			<td>N/A</td>
			<td>Provides air from air compressor to Tip Loader</td>
		</tr>
		<tr>
			<td>Water Hose</td>
			<td>Local Provider</td>
			<td>N/A</td>
			<td>Provides water from 5 Gallon Reserviour to Tip Washer</td>
		</tr>
		<tr>
			<td>Static ALP&#39;s</td>
			<td>Beckman Coulter</td>
			<td>Comes with Biomek FX</td>
			<td>Supports equipment for the Screen</td>
		</tr>
		<tr>
			<td>5 Gallon Reserviour</td>
			<td>Local Provider</td>
			<td>N/A</td>
			<td>Recirculates the dirty water from cleaning the tips</td>
		</tr>
		<tr>
			<td>Grippers</td>
			<td>Beckman Coulter</td>
			<td>Comes with Biomek FX</td>
			<td>Grabs and moves the equipment to the correct places</td>
		</tr>
		<tr>
			<td>96-Channel 200 &#181;L Head</td>
			<td>Beckman Coulter</td>
			<td>Comes with Biomek FX</td>
			<td>Holds the 96 tips used within the screen</td>
		</tr>
		<tr>
			<td>AP96 P200 Pipette Tips</td>
			<td>Beckman Coulter</td>
			<td>717251</td>
			<td>Used to make the screening library</td>
		</tr>
		<tr>
			<td>96 Well Flat Bottom Plate</td>
			<td>Costar</td>
			<td>9018</td>
			<td>Aids in visulization of screen</td>
		</tr>
		<tr>
			<td>96 Well V-Bottom Plate</td>
			<td>Costar</td>
			<td>3897</td>
			<td>Aids in storing of dilution library</td>
		</tr>
		<tr>
			<td>AlumaSeal 96 Sealing Film</td>
			<td>MedSci</td>
			<td>F-96-100</td>
			<td>Seals for storage both the chemicle library and dilution library</td>
		</tr>
		<tr>
			<td>Plastic ziplock sandwich bags</td>
			<td>Local Provider</td>
			<td>N/A</td>
			<td>Used to ensure a humid environment for screen</td>
		</tr>
		<tr>
			<td>AP96 P20 Pipette Tips</td>
			<td>Beckman Coulter</td>
			<td>717254</td>
			<td>Used in the dilution library creation</td>
		</tr>
		<tr>
			<td>Growth Chamber</td>
			<td>Percival</td>
			<td>AR36L3</td>
			<td>Germinates seeds for phenotypic visulization</td>
		</tr>
		<tr>
			<td>Spatula</td>
			<td>Local Provider</td>
			<td>N/A</td>
			<td>Holds seeds to add into wells where liquid seeding failed seed adequatly</td>
		</tr>
		<tr>
			<td>Toothpick</td>
			<td>Local Provider</td>
			<td>N/A</td>
			<td>Pushes seeds from spatula to wells</td>
		</tr>
		<tr>
			<td>Murashige and Skoog Basal Salt Mixture</td>
			<td>PhytoTechnology Laboratories</td>
			<td>M524</td>
			<td>Add to MS media mixture</td>
		</tr>
		<tr>
			<td>MES Free Acid Monohydrate</td>
			<td>Fisher Scientific</td>
			<td>ICN19483580</td>
			<td>Added to MS media to decrease pH</td>
		</tr>
		<tr>
			<td>Agar Powder</td>
			<td>Alfa Aesar</td>
			<td>9002-18-0</td>
			<td>Increases thickness of media to support seed suspension</td>
		</tr>
		<tr>
			<td>5M KOH</td>
			<td>Sigma-Aldrich</td>
			<td>484016</td>
			<td>Increases pH to adequate levels</td>
		</tr>
		<tr>
			<td>1L Media Storage Bottle</td>
			<td>Corning</td>
			<td>1395-1L</td>
			<td>Holds enough media for a screen</td>
		</tr>
		<tr>
			<td>Polypropylene Centrifuge Tubes</td>
			<td>Corning</td>
			<td>431470</td>
			<td>Sterilizes seeds prior to vernilization</td>
		</tr>
		<tr>
			<td>pH Probe</td>
			<td>Davis Instruments</td>
			<td>YX-58825-26</td>
			<td>Used for making media</td>
		</tr>
		<tr>
			<td>ALPs (Automated Labware Positioners) Users Manual</td>
			<td>Beckman Coulter</td>
			<td>PN 987836</td>
			<td>Aids in setting up the accompaning equipment for the Biomek FX</td>
		</tr>
		<tr>
			<td>Biomek 2000 Stacker Carousel Users Guide</td>
			<td>Beckman Coulter</td>
			<td>609862-AA</td>
			<td>Aids in setting up the Stacker Carousel</td>
		</tr>
		<tr>
			<td>Biomek FX and FXP Laboratory Automation Workstations Users Manual</td>
			<td>Beckman Coulter</td>
			<td>PN 987834</td>
			<td>Used to frame the Multichannel Pod</td>
		</tr>
		<tr>
			<td>Biomek FXP Laboratory Automation Workstation Customer Startup Guide</td>
			<td>Beckman Coulter</td>
			<td>PN B32335AB</td>
			<td>Used to aid in setting up the Biomek FX</td>
		</tr>
		<tr>
			<td>Biomek Software User&#39;s Manual</td>
			<td>Beckman Coulter</td>
			<td>PN 987835</td>
			<td>Used to set up and understand the Software</td>
		</tr>
	</tbody>
</table>

optimizing the use of a liquid handling robot to conduct a high throughput forward chemical genetics screen of arabidopsis thaliana

Kimyasal genetik giderek gen artıklık veya ölümcül nedeniyle geleneksel genetik inatçı olabilir bitkiler özellikleri kodunu çözmek için istihdam olmak. Ancak, sentetik bağlanabilecek küçük bir moleküldür biyoaktif olma olasılığı düşüktür; Bu nedenle, moleküller binlerce ilgi bulmak için test gerekir. Robot sistemleri çok sayıda örnekleri, hangi ile kimyasal bir kütüphane en aza/hata standartlaştırılması ek olarak taranması hızı artan işlemek için tasarlanmıştır işleme sıvı. Bir tezgah üstü çok kanallı sıvı kullanarak kitaplığa Arabidopsis thaliana (Arabidopsis), üzerinde 50.000 küçük moleküllerin yüksek üretilen iş ileri kimyasal genetik ekranı ulaşmak için protokolleri robot işleme geliştirilmiş olup, tüm en az gerektiren teknisyen katılımı. Bu protokoller ile 3,271 küçük moleküller görünür fenotipik değişiklikler neden olduğu keşfedildi. 1,563 bileşikleri kısa kök, 1,148 bileşikler değişmiş renklendirme, 383 bileşikler neden kök saç ve diğer, Sigara kategorize, değişiklikler ve 177 bileşikler inhibe çimlenme indüklenen.

Doğru ve verimli bir şekilde fenotipleri konsantrasyonları diseksiyon mikroskop altında eleme, küçük moleküller ilavesi dayalı karakterize bu yöntem Arabidopsis. üzerinde ileri kimyasal genetiğin sonu hedefi yeteneğidir Ne zaman tüm 50.000 bileşikler ekranlı gözlenen fenotipleri farklı ve çeşitli farklı sınıflar (Şekil 2) kırık. Şekil 3A -F diseksiyon mikroskop altında düşü...

Watch this Scientific Journal Video about Optimizing the Use of a Liquid Handling Robot to Conduct a High Throughput Forward Chemical Genetics Screen of Arabidopsis thaliana at JoVE.com

Optimizing the Use of a Liquid Handling Robot to Conduct a High Throughput Forward Chemical Genetics Screen of Arabidopsis thaliana

Sıvı işleme bir yüksek üretilen iş ileri kimyasal genetik ekran Arabidopsis thaliana yapmak için Robot kullanımı en iyi duruma getirme

Sentetik küçük moleküllerin yüksek üretilen iş ekran modeli bitki türleri, Arabidopsis thalianayapılmıştır. Sıvı işleme robot için geliştirilen bu iletişim kuralı, ileri kimyasal genetik ekranlar, Bitki fizyolojisi etkileyen roman küçük moleküller keşfi hızlanan hızını artırır.

Chemical genetics is increasingly being employed to decode traits in plants that may be recalcitrant to traditional genetics due to gene redundancy or ...

optimizing-use-liquid-handling-robot-to-conduct-high-throughput

Universidad de Cartagena UDEC

Research

JoVE Journal

Bioengineering

Optimizing the Use of a Liquid Handling Robot to Conduct a High Throughput Forward Chemical Genetics Screen of Arabidopsis thaliana

6.6K Görüntüleme.  University of Kentucky. Sentetik küçük moleküllerin yüksek üretilen iş ekran modeli bitki türleri, Arabidopsis thalianayapılmıştır. Sıvı işleme robot için geliştirilen bu iletişim kuralı, ileri kimyasal genetik ekranlar, Bitki fizyolojisi etkileyen roman küçük moleküller keşfi hızlanan hızını artırır.

Video: Optimizing the Use of a Liquid Handling Robot to Conduct a High Throughput Forward Chemical Genetics Screen of Arabidopsis thaliana

Adaptation of a Haptic Robot in a 3T fMRI

Functional magnetic resonance imaging (fMRI) provides excellent functional brain imaging via the BOLD signal 1 with advantages including non-ionizing radiation, millimeter spatial accuracy of anatomical and functional data 2, and nearly real-time analyses 3. Haptic robots provide precise measurement and control of position and force of a cursor in a reasonably confined space. Here we combine these two technologies to allow precision experiments involving motor control with haptic/tactile environment interaction such as reaching or grasping. The basic idea is to attach an 8 foot end effecter supported in the center to the robot 4 allowing the subject to use the robot, but shielding it and keeping it out of the most extreme part of the magnetic field from the fMRI machine (Figure 1).
The Phantom Premium 3.0, 6DoF, high-force robot (SensAble Technologies, Inc.) is an excellent choice for providing force-feedback in virtual reality experiments 5, 6, but it is inherently non-MR safe, introduces significant noise to the sensitive fMRI equipment, and its electric motors may be affected by the fMRI's strongly varying magnetic field. We have constructed a table and shielding system that allows the robot to be safely introduced into the fMRI environment and limits both the degradation of the fMRI signal by the electrically noisy motors and the degradation of the electric motor performance by the strongly varying magnetic field of the fMRI. With the shield, the signal to noise ratio (SNR: mean signal/noise standard deviation) of the fMRI goes from a baseline of &tilde;380 to &tilde;330, and &tilde;250 without the shielding. The remaining noise appears to be uncorrelated and does not add artifacts to the fMRI of a test sphere (Figure 2). The long, stiff handle allows placement of the robot out of range of the most strongly varying parts of the magnetic field so there is no significant effect of the fMRI on the robot. The effect of the handle on the robot's kinematics is minimal since it is lightweight (&tilde;2.6 lbs) but extremely stiff 3/4" graphite and well balanced on the 3DoF joint in the middle. The end result is an fMRI compatible, haptic system with about 1 cubic foot of working space, and, when combined with virtual reality, it allows for a new set of experiments to be performed in the fMRI environment including naturalistic reaching, passive displacement of the limb and haptic perception, adaptation learning in varying force fields, or texture identification 5, 6.

The adaptation and use of a haptic robot in a 3T fMRI is described.

3T fMRI Haptik Robot Adaptasyon

Functional magnetic resonance imaging (fMRI) provides excellent functional brain imaging via the BOLD signal 1 with advantages including non-ionizing ...

Biyomühendislik

High-throughput Protein Expression Generator Using a Microfluidic Platform

Rapidly increasing fields, such as systems biology, require the development and implementation of new technologies, enabling high-throughput and high-fidelity measurements of large systems. Microfluidics promises to fulfill many of these requirements, such as performing high-throughput screening experiments on-chip, encompassing biochemical, biophysical, and cell-based assays1. Since the early days of microfluidics devices, this field has drastically evolved, leading to the development of microfluidic large-scale integration2,3. This technology allows for the integration of thousands of micromechanical valves on a single device with a postage-sized footprint (Figure 1). We have developed a high-throughput microfluidic platform for generating in vitro expression of protein arrays (Figure 2) named PING (Protein Interaction Network Generator). These arrays can serve as a template for many experiments such as protein-protein 4, protein-RNA5 or protein-DNA6 interactions.
The device consist of thousands of reaction chambers, which are individually programmed using a microarrayer. Aligning of these printed microarrays to microfluidics devices programs each chamber with a single spot eliminating potential contamination or cross-reactivity Moreover, generating microarrays using standard microarray spotting techniques is also very modular, allowing for the arraying of proteins7, DNA8, small molecules, and even colloidal suspensions. The potential impact of microfluidics on biological sciences is significant. A number of microfluidics based assays have already provided novel insights into the structure and function of biological systems, and the field of microfluidics will continue to impact biology.

We present a microfluidic approach for the expression of protein arrays. The device consists of thousands of reaction chambers controlled by micro-mechanical valves. The microfluidic device is mated to a microarray-printed gene library. These genes are then transcribed and translated on-chip, resulting in a protein array ready for experimental use.

Bir mikroakışkan Platform kullanarak Yüksek throughput Protein Ekspresyonu Jeneratör

Rapidly increasing fields, such as systems biology, require the development and implementation of new technologies, enabling high-throughput and ...

High-throughput Synthesis of Carbohydrates and Functionalization of Polyanhydride Nanoparticles

Transdisciplinary approaches involving areas such as material design, nanotechnology, chemistry, and immunology have to be utilized to rationally design efficacious vaccines carriers. Nanoparticle-based platforms can prolong the persistence of vaccine antigens, which could improve vaccine immunogenicity1. Several biodegradable polymers have been studied as vaccine delivery vehicles1; in particular, polyanhydride particles have demonstrated the ability to provide sustained release of stable protein antigens and to activate antigen presenting cells and modulate immune responses2-12.
The molecular design of these vaccine carriers needs to integrate the rational selection of polymer properties as well as the incorporation of appropriate targeting agents. High throughput automated fabrication of targeting ligands and functionalized particles is a powerful tool that will enhance the ability to study a wide range of properties and will lead to the design of reproducible vaccine delivery devices.
The addition of targeting ligands capable of being recognized by specific receptors on immune cells has been shown to modulate and tailor immune responses10,11,13 C-type lectin receptors (CLRs) are pattern recognition receptors (PRRs) that recognize carbohydrates present on the surface of pathogens. The stimulation of immune cells via CLRs allows for enhanced internalization of antigen and subsequent presentation for further T cell activation14,15. Therefore, carbohydrate molecules play an important role in the study of immune responses; however, the use of these biomolecules often suffers from the lack of availability of structurally well-defined and pure carbohydrates. An automation platform based on iterative solution-phase reactions can enable rapid and controlled synthesis of these synthetically challenging molecules using significantly lower building block quantities than traditional solid-phase methods16,17.
Herein we report a protocol for the automated solution-phase synthesis of oligosaccharides such as mannose-based targeting ligands with fluorous solid-phase extraction for intermediate purification. After development of automated methods to make the carbohydrate-based targeting agent, we describe methods for their attachment on the surface of polyanhydride nanoparticles employing an automated robotic set up operated by LabVIEW as previously described10. Surface functionalization with carbohydrates has shown efficacy in targeting CLRs10,11 and increasing the throughput of the fabrication method to unearth the complexities associated with a multi-parametric system will be of great value (Figure 1a).

In this article, a high throughput method is presented for the synthesis of oligosaccharides and their attachment to the surface of polyanhydride nanoparticles for further use in targeting specific receptors on antigen presenting cells.

Polianhidrid Nanopartiküller Yüksek throughput Karbonhidrat sentezi ve işlevsel

Transdisciplinary approaches involving areas such as material design, nanotechnology, chemistry, and immunology have to be utilized to rationally design ...

Designing a Bio-responsive Robot from DNA Origami

Nucleic acids are astonishingly versatile. In addition to their natural role as storage medium for biological information1, they can be utilized in parallel computing2,3 , recognize and bind molecular or cellular targets4,5 , catalyze chemical reactions6,7 , and generate calculated responses in a biological system8,9. Importantly, nucleic acids can be programmed to self-assemble into 2D and 3D structures10-12, enabling the integration of all these remarkable features in a single robot linking the sensing of biological cues to a preset response in order to exert a desired effect.
Creating shapes from nucleic acids was first proposed by Seeman13, and several variations on this theme have since been realized using various techniques11,12,14,15 . However, the most significant is perhaps the one proposed by Rothemund, termed scaffolded DNA origami16. In this technique, the folding of a long (&gt;7,000 bases) single-stranded DNA 'scaffold' is directed to a desired shape by hundreds of short complementary strands termed 'staples'. Folding is carried out by temperature annealing ramp. This technique was successfully demonstrated in the creation of a diverse array of 2D shapes with remarkable precision and robustness. DNA origami was later extended to 3D as well17,18 .
 
The current paper will focus on the caDNAno 2.0 software19 developed by Douglas and colleagues. caDNAno is a robust, user-friendly CAD tool enabling the design of 2D and 3D DNA origami shapes with versatile features. The design process relies on a systematic and accurate abstraction scheme for DNA structures, making it relatively straightforward and efficient.
In this paper we demonstrate the design of a DNA origami nanorobot that has been recently described20. This robot is 'robotic' in the sense that it links sensing to actuation, in order to perform a task. We explain how various sensing schemes can be integrated into the structure, and how this can be relayed to a desired effect. Finally we use Cando21 to simulate the mechanical properties of the designed shape. The concept we discuss can be adapted to multiple tasks and settings.

DNA origami is a powerful method for fabricating precise nanoscale objects by programming the self-assembly of DNA molecules. Here, we describe how DNA origami can be utilized to design a robotic robot capable of sensing biological cues and responding by shape shifting, subsequently relayed to a desired effect.

DNA Origami bir Bio-duyarlı Robot Tasarımı

Nucleic acids are astonishingly versatile. In addition to their natural role as storage medium for biological information1, they can be utilized in ...

High-throughput, Automated Extraction of DNA and RNA from Clinical Samples using TruTip Technology on Common Liquid Handling Robots

TruTip is a simple nucleic acid extraction technology whereby a porous, monolithic binding matrix is inserted into a pipette tip. The geometry of the monolith can be adapted for specific pipette tips ranging in volume from 1.0 to 5.0 ml. The large porosity of the monolith enables viscous or complex samples to readily pass through it with minimal fluidic backpressure. Bi-directional flow maximizes residence time between the monolith and sample, and enables large sample volumes to be processed within a single TruTip. The fundamental steps, irrespective of sample volume or TruTip geometry, include cell lysis, nucleic acid binding to the inner pores of the TruTip monolith, washing away unbound sample components and lysis buffers, and eluting purified and concentrated nucleic acids into an appropriate buffer. The attributes and adaptability of TruTip are demonstrated in three automated clinical sample processing protocols using an Eppendorf epMotion 5070, Hamilton STAR and STARplus liquid handling robots, including RNA isolation from nasopharyngeal aspirate, genomic DNA isolation from whole blood, and fetal DNA extraction and enrichment from large volumes of maternal plasma (respectively).

TruTip is a simple nucleic acid extraction technology whereby a porous, monolithic binding matrix is inserted into a pipette tip. Consequently, the sample preparation format is compatible with most liquid handling instruments, and can be used for many medium to high-throughput clinical applications and sample types.

Ortak Liquid Handling Robotlar TruTip Teknoloji kullanarak Klinik Örneklerden DNA ve RNA yüksek verimlilik, Otomatik Ekstraksiyon

TruTip is a simple nucleic acid extraction  technology whereby a porous, monolithic binding matrix is inserted into a  pipette tip. The geometry of the ...

Optimizing Attachment of Human Mesenchymal Stem Cells on Poly(&#949;-caprolactone) Electrospun Yarns

Research into biomaterials and tissue engineering often includes cell-based in vitro investigations, which require initial knowledge of the starting cell number. While researchers commonly reference their seeding density this does not necessarily indicate the actual number of cells that have adhered to the material in question. This is particularly the case for materials, or scaffolds, that do not cover the base of standard cell culture well plates. This study investigates the initial attachment of human mesenchymal stem cells seeded at a known number onto electrospun poly(&#949;-caprolactone) yarn after 4 hr in culture. Electrospun yarns were held within several different set-ups, including bioreactor vessels rotating at 9 rpm, cell culture inserts positioned in low binding well plates and polytetrafluoroethylene (PTFE) troughs placed within petri dishes. The latter two were subjected to either static conditions or positioned on a shaker plate (30 rpm). After 4 hr incubation at 37&#160;oC, 5% CO2, the location of seeded cells was determined by cell DNA assay. Scaffolds were removed from their containers and placed in lysis buffer. The media fraction was similarly removed and centrifuged &#8211; the supernatant discarded and pellet broken up with lysis buffer. Lysis buffer was added to each receptacle, or well, and scraped to free any cells that may be present. The cell DNA assay determined the percentage of cells present within the scaffold, media and well fractions. Cell attachment was low for all experimental set-ups, with greatest attachment (30%) for yarns held within cell culture inserts and subjected to shaking motion. This study raises awareness to the actual number of cells attaching to scaffolds irrespective of the stated cell seeding density.

This article describes a range of set-ups for seeding human mesenchymal stem cells onto materials, in this case electrospun yarns, that do not cover the base of standard culture well plates in order to maximize and quantify the number of cells that initially attach compared to the known seeding density.

Poli (ε-kaprolakton) electrospun İplik İnsan Mezenkimal Kök Hücre Ek Optimize

Research into biomaterials and tissue engineering often includes cell-based in vitro investigations, which require initial knowledge of the starting cell ...

Use of a High-throughput In Vitro Microfluidic System to Develop Oral Multi-species Biofilms

There are few high-throughput in vitro systems which facilitate the development of multi-species biofilms that contain numerous species commonly detected within in vivo oral biofilms. Furthermore, a system that uses natural human saliva as the nutrient source, instead of artificial media, is particularly desirable in order to support the expression of cellular and biofilm-specific properties that mimic the in vivo communities. We describe a method for the development of multi-species oral biofilms that are comparable, with respect to species composition, to supragingival dental plaque, under conditions similar to the human oral cavity. Specifically, this methods article will describe how a commercially available microfluidic system can be adapted to facilitate the development of multi-species oral biofilms derived from and grown within pooled saliva. Furthermore, a description of how the system can be used in conjunction with a confocal laser scanning microscope to generate 3-D biofilm reconstructions for architectural and viability analyses will be presented. Given the broad diversity of microorganisms that grow within biofilms in the microfluidic system (including Streptococcus, Neisseria, Veillonella, Gemella, and Porphyromonas), a protocol will also be presented describing how to harvest the biofilm cells for further subculture&#160;or DNA extraction and analysis. The limits of both the microfluidic biofilm system and the current state-of-the-art data analyses will be addressed. Ultimately, it is envisioned that this article will provide a baseline technique that will improve the study of oral biofilms and aid in the development of additional technologies that can be integrated with the microfluidic platform.

Use of a High-throughput In Vitro Microfluidic System to Develop Oral Multi-species Biofilms

The goal of this methods paper is to describe the use of a microfluidic system for the development of multi-species biofilms that contain species typically identified in human supragingival dental plaque.&#160;Methods to describe biofilm architecture, biofilm viability, and an approach to harvest biofilm for culture-dependent or culture-independent analyses are highlighted.

Yüksek verimli bir kullanımı<em&gt; İn Vitro</em&gt; Mikroakışkan Sistemi Ağız Çok türler biyofilm Geliştirmek İçin

There are few high-throughput in vitro systems which facilitate the development of multi-species biofilms that contain numerous species commonly detected ...

Generation of Scalable, Metallic High-Aspect Ratio Nanocomposites in a Biological Liquid Medium

The goal of this protocol is to describe the synthesis of two novel biocomposites with high-aspect ratio structures. The biocomposites consist of copper and cystine, with either copper nanoparticles (CNPs) or copper sulfate contributing the metallic component. Synthesis is carried out in liquid under biological conditions (37 &#176;C) and the self-assembled composites form after 24 hr. Once formed, these composites are highly stable in both liquid media and in a dried form. The composites scale from the nano- to micro- range in length, and from a few microns to 25 nm in diameter. Field emission scanning electron microscopy with energy dispersive X-ray spectroscopy (EDX) demonstrated that sulfur was present in the NP-derived linear structures, while it was absent from the starting CNP material, thus confirming cystine as the source of sulfur in the final nanocomposites. During synthesis of these linear nano- and micro-composites, a diverse range of lengths of structures is formed in the synthesis vessel. Sonication of the liquid mixture after synthesis was demonstrated to assist in controlling average size of the structures by diminishing the average length with increased time of sonication. Since the formed structures are highly stable, do not agglomerate, and are formed in liquid phase, centrifugation may also be used to assist in concentrating and segregating formed composites.

Here we present a protocol to synthesize novel, high-aspect ratio biocomposites under biological conditions and in liquid media. The biocomposites scale from nanometers to micrometers in diameter and length, respectively. Copper nanoparticles (CNPs) and copper sulfate combined with cystine are the key components for the synthesis.

Biyolojik Sıvı Ortamda Ölçeklenebilir, Metalik Yüksek Aspect Ratio Nanokompositlerin Üretimi

The goal of this protocol is to describe the synthesis of two novel biocomposites with high-aspect ratio structures. The biocomposites consist of copper ...

Automated Robotic Liquid Handling Assembly of Modular DNA Devices

Recent advances in modular DNA assembly techniques have enabled synthetic biologists to test significantly more of the available &#34;design space&#34; represented by &#34;devices&#34; created as combinations of individual genetic components. However, manual assembly of such large numbers of devices is time-intensive, error-prone, and costly. The increasing sophistication and scale of synthetic biology research necessitates an efficient, reproducible way to accommodate large-scale, complex, and high throughput device construction.
Here, a DNA assembly protocol using the Type-IIS restriction endonuclease based Modular Cloning (MoClo) technique is automated on two liquid-handling robotic platforms. Automated liquid-handling robots require careful, often times tedious optimization of pipetting parameters for liquids of different viscosities (e.g. enzymes, DNA, water, buffers), as well as explicit programming to ensure correct aspiration and dispensing of DNA parts and reagents. This makes manual script writing for complex assemblies just as problematic as manual DNA assembly, and necessitates a software tool that can automate script generation. To this end, we have developed a web-based software tool, http://mocloassembly.com, for generating combinatorial DNA device libraries from basic DNA parts uploaded as Genbank files. We provide access to the tool, and an export file from our liquid handler software which includes optimized liquid classes, labware parameters, and deck layout. All DNA parts used are available through Addgene, and their digital maps can be accessed via the Boston University BDC ICE Registry. Together, these elements provide a foundation for other organizations to automate modular cloning experiments and similar protocols.
The automated DNA assembly workflow presented here enables the repeatable, automated, high-throughput production of DNA devices, and reduces the risk of human error arising from repetitive manual pipetting. Sequencing data show the automated DNA assembly reactions generated from this workflow are ~95% correct and require as little as 4% as much hands-on time, compared to manual reaction preparation.

Here, an automated workflow to perform modular DNA &#34;device&#34; assembly using a modular cloning DNA assembly method on liquid-handling robots is presented. The protocol uses an intuitive software tool for generating liquid handler picklists for combinatorial DNA device library generation, which we demonstrate using two liquid handling platforms.

Robot sıvı işleme derleme modüler DNA cihazların otomatik

Recent advances in modular DNA assembly techniques have enabled synthetic biologists to test significantly more of the available &#34;design space&#34; ...

Synthesis of Biocompatible Liquid Crystal Elastomer Foams as Cell Scaffolds for 3D Spatial Cell Cultures

Here, we present a step-by-step preparation of a 3D, biodegradable, foam-like cell scaffold. These scaffolds were prepared by cross-linking star block co-polymers featuring cholesterol units as side-chain pendant groups, resulting in smectic-A (SmA) liquid crystal elastomers (LCEs). Foam-like scaffolds, prepared using metal templates, feature interconnected microchannels, making them suitable as 3D cell culture scaffolds. The combined properties of the regular structure of the metal foam and of the elastomer result in a 3D cell scaffold that promotes not only higher cell proliferation compared to conventional porous templated films, but also better management of mass transport (i.e., nutrients, gases, waste, etc.). The nature of the metal template allows for the easy manipulation of foam shapes (i.e., rolls or films) and for the preparation of scaffolds of different pore sizes for different cell studies while preserving the interconnected porous nature of the template. The etching process does not affect the chemistry of the elastomers, preserving their biocompatible and biodegradable nature. We show that these smectic LCEs, when grown for extensive time periods, enable the study of clinically relevant and complex tissue constructs while promoting the growth and proliferation of cells.

This study presents a methodology to prepare 3D, biodegradable, foam-like cell scaffolds based on biocompatible side-chain liquid crystal elastomers (LCEs). Confocal microscopy experiments show that foam-like LCEs allow for cell attachment, proliferation, and the spontaneous alignment of C2C12s myoblasts.

3D Mekansal Hücre Kültürleri için hücre yapı iskeleleri olarak biyo-uyumlu sıvı kristal Elastomer Köpüklerin sentezi

Here, we present a step-by-step preparation of a 3D, biodegradable, foam-like cell scaffold. These scaffolds were prepared by cross-linking star block ...