Bu eser NIH (AI119813), Arkansas Üniversitesi start-up ve Arkansas Biosciences Enstitüsü'nden Ödülü tarafından desteklenmiştir.

1. site yönettiği Mutagenesis hedef gen
Not: MDH altında T7 organizatörü pCDF-1 vektör CloDF13 kökeni ve 20-4034kopya sayısı ile ifade edilir.
<ol><li>Amber kodonu gen pozisyonda 140 astar tarafından tanıtmak (ileri astar: GGTGTTTATGACEtiketAACAAACTGTTCGGCG ve ters astar: GGCTTTTTTCAGCACTTCAGCAGCAATTGC), site yönettiği mutagenesis kiti öğretim takip.</li>
	<li>Vahşi-türü malat dehidrataz gen içeren şablonu plazmid yükseltmek ve Polimeraz zincir reaksiyonu (PCR) tepkimesi tarafından stop kodonu mutasyon ekleyin. Reaksiyon karışımı 2 X DNA polimeraz enzimi Mix 12.5 µL, 10 µM ileri astar, 1,25 µL 10 µM ters astar, şablonun DNA 1 µL 1,25 µL içerir (20 ng/µL) (pCDF-1 plazmid vahşi tipi MDH gen içeren) ve 9 nükleaz ücretsiz su µL.<ol>
<li>PCR reaksiyon parametreleri aşağıdaki gibi kullanın: ilk denatürasyon 30 98 ° C'de s; 10 25 döngüleri 98 ° c, 30 s s 55 ° C ve 3 dak 72 ° c; 3 dk 72 ° C'de son uzantısı. PCR sonra güçlendirilmiş malzeme kiti daireselleşmesi ve şablonu kaldırma için oda sıcaklığında 1 h için doğrudan kinaz-ligaz-DpnI enzim karışıma ekleyin. Not: 1 µL PCR ürün, 5 µL 2 reaksiyon arabellek X, 10 X kinaz-ligaz-DpnI enzim karışımı 1 µL ve nükleaz ücretsiz su 3 µL tepki karışımı içerir.</li>
	</ol>
</li>
	<li>25 µL tüp tepki karışımı 5 µL çözdürülen yetkili E. coli eklemek DH5α hücreleri Takımı'ndan. Dikkatle karışımı için tüp fiske ve buz 30 dk. ısı şok 30 s ve ek 5 min için buz üzerinde yer için 42 ° C'de karışımı karışımı yerleştirin.<ol>
<li>Oda sıcaklığında Catabolite baskı (SOC) medya kiti ile süper en iyi suyu 600 µL karışım içine pipet, 250 devir / dakikada sallayarak ile 60 dk 37 ° C'de kuluçkaya, karşılık gelen antibiyotik ile bir lysogeny suyu (LB) agar plaka üzerine 100 µL yaymak ve gecede 37 ° C'de 250 devir / dakikada sallayarak ile kuluçkaya.</li>
	</ol>
</li>
	<li>4-6 tek kolonileri 6 mL taze LB ortamla ilgili antibiyotik içine almak ve 37 ° C'de gecede 250 devir / dakikada sallayarak ile kuluçkaya. Plazmid üreticinin el kitabı, sonra Gönder plazmid DNA sıralama stop kodonu mutasyon doğru pozisyonlarda onaylamak için servis saðlayýcýnýzýn protokolüne göre için takip plazmid arıtma seti tarafından her gece kültüründen ayıklayın.</li>
	<li>Gerilme-80 ° C'de doğru sırada ile 1 mL gecede kültür ve 300 µL % 100 DMSO karıştırarak saklayın.</li>
</ol>2. ifade Acetylated protein
<ol><li>En iyi duruma getirilmiş acetyllysyl-tRNA sentetaz33 genler takın ve tRNA optimizePyl 32 pTech plazmid içine. TRNA sentetaz gen bünye lpp organizatörü altında yerleştirin. TRNA gen bünye proK organizatörü34altında yerleştirin.<ol>
<li>Malat dehidrogenaz, mutasyona uğramış etiketi içeren gen içeren ifade vektör34 co dönüştürmek ve en iyi duruma getirilmiş acetyllysine birleşme sistem yataklık plazmid, 25 µL tarafından çözülmüş yetkili E. coli BL21(DE3) hücreleri 10 s ve ek 5 min için buz üzerinde yer için 42 ° C'de ısı şok.</li>
		<li>Oda sıcaklığında SOC kitle 600 µL karışım içine pipet, 275 rpm'de sallayarak ile 60 dk 37 ° C'de kuluçkaya, 100 µg/mL streptomisin ve 50 µg/mL kloramfenikol ile bir plaka üzerine 100 µL yaymak ve gecede 37 ° C'de 275 rpm'de sallayarak ile kuluçkaya.</li>
	</ol>
</li>
	<li>Bir koloni plaka almak ve 100 µg/mL streptomisin ve 50 µg/mL kloramfenikol 250 rpm hızında sallayarak ile 50 mL tüp gecede 37 ° C'de 15 mL taze LB medya içine aşılamak. 300 mL taze LB medya bir 1 L şişesi antibiyotik ile 15 mL gecede kültür aktarmak ve 37 ° C'de 250 devir / dakikada sallayarak ile kuluçkaya.</li>
	<li>Acetyllysine 4 ° C'de depolamak 100 mM hisse senedi çözüm yapmak için su ile dağıtılması Absorbans 0,5 600 ulaştığında 5 mM acetyllysine ve 20 mM Nikotinamid (uyku inhibitörü) büyüme ortamına Ekle nm.<ol>
<li>250 devir / dakikada sallayarak 37 ° C'de bir ek 1 h için hücrelerin büyümesine sonra 0,5 mM IPTG protein ifade ekleyin ve 25 ° C'de hücreler gecede, 180 rpm'de sallayarak ile büyümek. Not: İfade koşulları farklı proteinler için en iyi duruma getirme gerekebilir.</li>
	</ol>
</li>
	<li>3000 x g 15dk için 4 ° c de centrifuging tarafından hücreleri toplamak, süpernatant atmak ve hücre topakları fosfat tamponlu tuz (PBS) arabellek (10 mM Na2HPO4, 1.8 mM KH2PO4, 137 mM NaCl, 2.7 mM KCl) ile yıkayın. 10.000 x g 5 min için 4 ° C'de yıkanmış hücreleri toplamak, süpernatant atmak ve hücre granül-80 ° C'de depolayın</li>
</ol>3. Acetylated Protein saflaştırma
<ol><li>Buzda donmuş hücre topakları çözülme ve 15 mL ile lizis arabellek (50 mM tris (hydroxymethyl) aminomethane (Tris) pH 7.8, 300 mM NaCl, 20 mM imidazole ve 20 mM Nikotinamid) β-mercaptoethanol ve 1 µL Benzonase nükleaz (250 adet) 5 µL yeniden askıya alma.</li>
	<li>Hücreleri tarafından 40 kHz sonication 30 aralıkları tarafından takip 10 s kısa atışlar 10 döngüleri ile çıkış % 70 güçte kırmak için formu ham özü soğutma için s. Santrifüj ham ayıklamak 20.000 x g 4 ° C'de 25 min için de Süpernatant 0,45 µm membran filtre ile filtre ve 1 mL 20 mL su ve lizis arabellek 20 mL equilibrated nikel-nitrilotriacetic asit (Ni-NTA) reçine içeren bir sütun içine yükleyin. Not: Sonication kullanılabilir değilse hücreleri da hafif deterjan tarafından kırık.</li>
	<li>Sütun yıkama arabelleği (50 mM Tris pH 7.8, 300 mM NaCl, 50 mM imidazole ve 20 mM Nikotinamid) 20 mL ile yıkama ve 2 mL elüsyon arabellek (50 mM Tris pH 7.8, 300 mM NaCl, 150 mM imidazole ve 20 mM Nikotinamid) ile elute.</li>
	<li>Elüsyon kesir arabellek (25 mM Tris pH 7.8 ve 10 mM NaCl) desalting ile üreticisinin takip PD-10 sütun tarafından desalt. Eluted protein konsantrasyonu yanında tabi belgili tanımlık öğretim Bradford protein tahlil reaktif ölçmek. Desalted protein daha fazla deneyler için hazır hale geliyor. Not: % 50 gliserol stok protein yapmak ve-80 ° C depolama için tutun.</li>
</ol>4. biyokimyasal Acetylated Protein karakterizasyonu
<ol><li>
SDS-sayfa ve kütle spektrometresi analizleri.
	<ol>
<li>Proteinler 5 min için 105 ° C'de 2 mL tüp içinde Sodyum Lauryl Sülfat (SDS) örnek arabellek (5 µL protein örnek 2 µL 4 X SDS örnek arabellek ile) ile tabiatını, 2000 x g 10 karisimin santrifüj kapasitesi s, 4-%20 Sodyum Lauryl Sülfat polyacrylamide jel electroph üzerine yükleme oresis (SDS-sayfa) jel ve koşmak vasıl 200 V 30 dk için.</li>
		<li>Jel distile su ile yıkayın ve yavaşça 5 min için işlemi 3 kez tekrar çalkalanır. Su atın ve jel ile nazik sallayarak ile 1 h için Coomassie mavi leke leke. Distile su ile jel de-leke, 30 dk ve bu de-leke 3 kez tekrar yavaşça salla.</li>
		<li>Coomassie mavi lekeli SDS-sayfa jel üzerinde 33 kDa adlı grup kesmek ve kütle spektrometresi tesis veya şirketler acetyllysine tasarlanmış konumda kurulmuştur onaylamak için gönderin. Not: Önceki deneme34kütle spektrometresi çözümleme protokolü takip ettim.</li>
	</ol>
</li>
	<li>
Batı kurutma
	<ol>
<li>SDS-sayfa jel adım 4.1 aynı iletişim kuralını çalıştırmak. Jel çalıştırmak sonra jel 15dk için Aktarım arabellek (25 mM Tris, 192 mM glisin, pH 8.3 ve % 20 metanol) ile ıslatın.</li>
		<li>0.2 µm, 7 cm x 8,5 cm polivinilidin difluoride (PVDF) membran metanol 1 dakika ile etkinleştirin ve transfer sandviç hazırlamadan önce Aktarım arabellek ile durulayın. Not: Metanol ciltle temas, göz teması, yenmesi veya inhalasyon durumunda zararlıdır. Şiddetli aşırı pozlama ölüme neden olabilir.</li>
		<li>Katot anot (sünger, filtre kağıdı, SDS-sayfa jel, PVDF membran, filtre kağıdı ve sünger) transfer sandviç olun. Sürekli mevcut 350 çalıştırın transfer tankında koydum mA 45 dk için. Not: Transfer süresi optimizasyonu gerekebilir.</li>
		<li>PVDF membran 25 mL Tris arabelleğe alınmış serum fizyolojik, % 0,1 ile yıkayın ara 20 (TBST) (137 mM NaCl, 20 mM Tris, %0.1 ara-20, pH 7,6) arabellek nazik sallayarak ile 5 min için. Membran ile %5 sığır serum albümin (BSA) oda sıcaklığında 1 h için TBST arabellekte engelleyin.</li>
		<li>HRP-Birleşik acetyllysine-antikor 1 µg/ml TBST %5 BSA ile 4 ° C'de gecede nazik sallayarak ile seyreltilmiş son bir konsantrasyon ile ile membran kuluçkaya. Not: Daha hızlı sonuç için bu adım 1 h için oda sıcaklığında gerçekleştirilebilir. Antikor seyreltme optimizasyonu gerekebilir.</li>
		<li>Membran ile 20 mL TBST arabellek ile nazik sallayarak, 5 min için adım 4 kez tekrar yıkayın. Kemiluminesan substrat membran için üreticinin yönergelerini izleyerek uygulanır. Kamera tabanlı bir şarj kuplajlı cihaz (CCD) Imager sinyali yakalamak.</li>
	</ol>
</li>
</ol>

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M., Soll, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rationally+evolving+tRNAPyl+for+efficient+incorporation+of+noncanonical+amino+acids.">Rationally evolving tRNAPyl for efficient incorporation of noncanonical amino acids.</a> Nucleic Acids Res. 43 (22), e156 (2015).</li><li>Bryson, D., 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=Continuous+directed+evolution+of+aminoacyl-tRNA+synthetases+to+alter+amino+acid+specificity+and+enhance+activity.">Continuous directed evolution of aminoacyl-tRNA synthetases to alter amino acid specificity and enhance activity.</a> Nat Chem Biol. , (2017).</li><li>Venkat, S., Gregory, C., Sturges, J., Gan, Q., Fan, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studying+the+Lysine+Acetylation+of+Malate+Dehydrogenase.">Studying the Lysine Acetylation of Malate Dehydrogenase.</a> J Mol Biol. 429 (9), 1396-1405 (2017).</li><li>Venkat, S., Gregory, C., Gan, Q., Fan, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biochemical+characterization+of+the+lysine+acetylation+of+tyrosyl-tRNA+synthetase+in+Escherichia+coli.">Biochemical characterization of the lysine acetylation of tyrosyl-tRNA synthetase in Escherichia coli.</a> Chembiochem. 18 (19), 1928-1934 (2017).</li><li>Liu, C. C., Schultz, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adding+new+chemistries+to+the+genetic+code.">Adding new chemistries to the genetic code.</a> Annu Rev Biochem. 79, 413-444 (2010).</li><li>Mukai, T., Lajoie, M. J., Englert, M., Soll, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rewriting+the+Genetic+Code.">Rewriting the Genetic Code.</a> Annu Rev Microbiol. , (2017).</li><li>O'Donoghue, P., Ling, J., Wang, Y. S., Soll, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Upgrading+protein+synthesis+for+synthetic+biology.">Upgrading protein synthesis for synthetic biology.</a> Nat Chem Biol. 9 (10), 594-598 (2013).</li><li>Chin, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expanding+and+reprogramming+the+genetic+code+of+cells+and+animals.">Expanding and reprogramming the genetic code of cells and animals.</a> Annu Rev Biochem. 83, 379-408 (2014).</li><li>O'Donoghue, P., 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=Near-cognate+suppression+of+amber,+opal+and+quadruplet+codons+competes+with+aminoacyl-tRNAPyl+for+genetic+code+expansion.">Near-cognate suppression of amber, opal and quadruplet codons competes with aminoacyl-tRNAPyl for genetic code expansion.</a> FEBS Lett. 586 (21), 3931-3937 (2012).</li><li>Aerni, H. R., Shifman, M. A., Rogulina, S., O'Donoghue, P., Rinehart, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Revealing+the+amino+acid+composition+of+proteins+within+an+expanded+genetic+code.">Revealing the amino acid composition of proteins within an expanded genetic code.</a> Nucleic Acids Res. 43 (2), e8 (2015).</li><li>Huang, 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=A+convenient+method+for+genetic+incorporation+of+multiple+noncanonical+amino+acids+into+one+protein+in+Escherichia+coli.">A convenient method for genetic incorporation of multiple noncanonical amino acids into one protein in Escherichia coli.</a> Mol Biosyst. 6 (4), 683-686 (2010).</li><li>Venkat, 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=Genetically+encoding+thioacetyl-lysine+as+a+nondeacetylatable+analog+of+lysine+acetylation+in+Escherichia+coli.">Genetically encoding thioacetyl-lysine as a nondeacetylatable analog of lysine acetylation in Escherichia coli.</a> FEBS Open. , (2017).</li><li>Wan, W., Tharp, J. M., Liu, W. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pyrrolysyl-tRNA+synthetase:+an+ordinary+enzyme+but+an+outstanding+genetic+code+expansion+tool.">Pyrrolysyl-tRNA synthetase: an ordinary enzyme but an outstanding genetic code expansion tool.</a> Biochim Biophys Acta. 1844 (6), 1059-1070 (2014).</li><li>Gregoretti, I. V., Lee, Y. M., Goodson, H. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+evolution+of+the+histone+deacetylase+family:+functional+implications+of+phylogenetic+analysis.">Molecular evolution of the histone deacetylase family: functional implications of phylogenetic analysis.</a> J Mol Biol. 338 (1), 17-31 (2004).</li><li>Weinert, B. 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=Acetyl-phosphate+is+a+critical+determinant+of+lysine+acetylation+in+E.+coli.">Acetyl-phosphate is a critical determinant of lysine acetylation in E. coli.</a> Mol cell. 51 (2), 265-272 (2013).</li></ol>

Yazarlar ifşa gerek yok.

Meşru amino asitler (ncAAs) genetik birleşme ncAA tahsil tRNA tarafından atanan bir kodon, çoğunlukla sarı stop kodonu UAG36,37,38,39, bastırılması dayanmaktadır karşılık gelen antikodon içeren. Bilindiği gibi UAG kodon tarafından yayın faktör-1 (RF1) bakterilerde tanınır ve bu da tarafından soydaş tRNA kurallı amino asitler (cAAs) tarafından tahsil ana bilgisayarlardan ...

Proteinlerin translasyonel modifikasyonlar (PTMs) çeviri işleminden sonra ortaya çıkan ve Fonksiyonel grupların kovalent ilavesi amino asit kalıntıları, gene de dahil olmak üzere hemen hemen tüm biyolojik süreçler içinde önemli roller oynayan ortaya çıkan Transkripsiyon, stres tepkisi, hücresel başkalaşım ve metabolizma1,2,3. Bugüne kadar yaklaşık 400 farklı PTMs saptanan4olmuştur. Onlar protein etkinlik ve yerelleştirme düzenleyen ve proteinler, nükleik asitler, lipidler ve Kofaktörler5gibi diğer molekülleri ile etkileşimi etkileyen genom ve Proteom karışıklık protein PTMs tarafından büyük ölçüde güçlendirilmiş.
Protein asetilasyon son yirmi yılda6,7,8,9,10,11,12PTMs çalışmalarda ön planda olmuştur. Lizin asetilasyon Histon içinde daha 50 yıl önce13,14, iyi incelemiş ve 80'den fazla transkripsiyon faktörleri, düzenleyiciler ve çeşitli proteinler15bulunduğunun bilinmesi was ilk kâşif, 16,17. Protein asetilasyon üzerinde çalışmalar sadece bize onun düzenleyici mekanizmaları daha derin bir anlayış ile sağlanan, ancak da işlevsiz asetilasyon18,19tarafından, neden olduğu hastalıkların bir dizi için Tedaviler güdümlü 20 , 21 , 22 , 23. bu lizin asetilasyon sadece ökaryotlarda olur, ancak son yıllarda yapılan çalışmalarda bu protein asetilasyon de Kemotaksis, asit direnci, harekete geçirmek ve istikrar gibi bakteri fizyolojisi, anahtar rol oynar göstermiştir inanılıyordu patojen Adaları ve diğer virülans proteinler24,25,26,27,28,29ile ilgili.
Biyokimyasal olarak lizin asetilasyon karakterize etmek için yaygın olarak kullanılan bir yöntem sitesi yönetmen mutagenesis kullanıyor. Glutamin benzer büyüklüğü ve polarite nedeniyle acetyllysine bir taklit kullanılır. Pozitif býrakýldýðýnda fizyolojik koşullar altında korur ancak acetylated olamaz beri arginin acetylated lizin taklit kullanılmaktadır. Ancak, her iki taklit eder gerçek isosteres değildir ve her zaman beklenen sonuçları30verir. En titiz yaklaşım olduğu zor veya imkansız en klasik yöntemlerle lizin asetilasyon doğa7,11düşük stoichiometry nedeniyle, belirli lizin artıkları, homojen acetylated proteinler oluşturmaktır. Bu meydan okuma bir mühendislik pyrrolysyl-tRNA sentetaz istihdam genetik kod genişleme stratejisi tarafından sökülmüş tRNA şarj etmek için Methanosarcinaceae türlerden varyantPyl ile acetyllysine, kullanır ev sahibi UAG bastırmak için translasyonel makine mRNA kodon durdurmak ve acetyllysine hedef protein31tasarlanmış pozisyonda birleşme yönlendirir. Son zamanlarda, biz bu sistemi geliştirilmiş bir EF-Tu-bağlama tRNA32 ve yükseltilmiş acetyllysyl-tRNA sentetaz33ile optimize. Ayrıca, Gelişmiş birleşme programında küçük bir sistem asetilasyon çalışmalar malat dehidrogenaz34 ve tyrosyl-tRNA sentetaz35başvurdum. Burada, biz tamamen acetylated Proteinlerin moleküler biyokimyasal kullanıcı kimliğini bir demonstrasyon örnek olarak kapsamlı bir şekilde inceledik malat dehidrogenaz (MDH), kullanarak klonlama üzerinden oluşturma protokolü göstermek.

<table border="0" cellpadding="0" cellspacing="0" width="615">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Bradford protein assay</td>
			<td style="width:113px;">Bio-Rad</td>
			<td style="width:119px;">5000006</td>
			<td style="width:167px;">Protein concentration</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">4x Laemmli Sample Buffer</td>
			<td style="width:113px;">Bio-Rad</td>
			<td style="width:119px;">1610747</td>
			<td style="width:167px;">SDS sample buffer</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Coomassie G-250 Stain</td>
			<td style="width:113px;">Bio-Rad</td>
			<td style="width:119px;">1610786</td>
			<td style="width:167px;">SDS-PAGE gel staining</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">4-20% SDS-PAGE ready gel</td>
			<td style="width:113px;">Bio-Rad</td>
			<td style="width:119px;">4561093</td>
			<td style="width:167px;">Protein determination</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Ac-K-100 (HRP Conjugate)</td>
			<td style="width:113px;">Cell Signaling</td>
			<td style="width:119px;">6952</td>
			<td style="width:167px;">Antibody</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">IPTG</td>
			<td style="width:113px;">CHEM-IMPEX</td>
			<td style="width:119px;">194</td>
			<td style="width:167px;">Expression inducer</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:216px;">N&#949;-Acetyl-L-lysine</td>
			<td style="width:113px;">CHEM-IMPEX</td>
			<td style="width:119px;">5364</td>
			<td style="width:167px;">Noncanonical amino acid</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">PD-10 desalting column</td>
			<td style="width:113px;">GE Healthcare</td>
			<td style="width:119px;">17085101</td>
			<td style="width:167px;">Desalting</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Q5 Site-Directed Mutagenesis Kit</td>
			<td style="width:113px;">NEB</td>
			<td style="width:119px;">E0554</td>
			<td style="width:167px;">Introducing the stop codon</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">BL21 (DE3) cells</td>
			<td style="width:113px;">NEB</td>
			<td style="width:119px;">C2527</td>
			<td style="width:167px;">Expressing strain</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">QIAprep Spin Miniprep Kit</td>
			<td style="width:113px;">QIAGEN</td>
			<td style="width:119px;">27106</td>
			<td style="width:167px;">Extracting plasmids</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Ni-NTA resin</td>
			<td style="width:113px;">QIAGEN</td>
			<td style="width:119px;">30210</td>
			<td style="width:167px;">Affinity purification resin</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">nicotinamide</td>
			<td style="width:113px;">Sigma-Aldrich</td>
			<td style="width:119px;">N3376</td>
			<td style="width:167px;">Deacetylase inhibitor</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">&#946;-Mercaptoethanol</td>
			<td style="width:113px;">Sigma-Aldrich</td>
			<td style="width:119px;">M6250</td>
			<td style="width:167px;">Reducing agent</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">BugBuster Protein Extraction Reagent</td>
			<td style="width:113px;">Sigma-Aldrich</td>
			<td style="width:119px;">70584</td>
			<td style="width:167px;">Breaking cells</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Benzonase nuclease</td>
			<td style="width:113px;">Sigma-Aldrich</td>
			<td style="width:119px;">E1014</td>
			<td style="width:167px;">DNase</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">ECL Western Blotting Substrate</td>
			<td style="width:113px;">ThermoFisher</td>
			<td style="width:119px;">32106</td>
			<td style="width:167px;">Chemiluminescence</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Premixed LB Broth</td>
			<td style="width:113px;">VWR</td>
			<td style="width:119px;">97064</td>
			<td style="width:167px;">Cell growth medium</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Bovine serum albumin</td>
			<td style="width:113px;">VWR</td>
			<td style="width:119px;">97061-416</td>
			<td style="width:167px;">western blots blocking</td>
		</tr>
	</tbody>
</table>

a facile protocol to generate site-specifically acetylated proteins in escherichia coli

Proteinlerin belirli konumlarda meydana translasyonel modifikasyonlar hücresel çeşitli önemli rol oynarlar gösterilmiştir. Bunlar arasında tersinir lizin asetilasyon en çok dağıtılmış hayat tüm etki alanlarında biridir. Daha da çok sayıda acetylome kütle spektrometresi tabanlı çalışma gerçekleştirilmiş olsa da, bu sözde asetilasyon hedefler karakterizasyonu sınırlı olmuştur. Olası bir nedeni çoğu klasik biyokimyasal yaklaşım tarafından istenen pozisyonlarda tamamen acetylated proteinleri üretmek zor olmasıdır. Bu zorluğu aşmak için genetik kod genişletme tekniği cotranslational birleşme yönlendirmek için bir mühendislik pyrrolysyl-tRNA sentetaz varyant ve onun soydaş tRNA Methanosarcinaceae türlerden çifti kullanın uygulandı protein ilgi belirli sitesinde acetyllysine. Histon asetilasyon çalışmada ilk uygulama sonra bu yaklaşım asetilasyon çalışmalar proteinler çeşitli kolaylaştırdı. Bu çalışmada, ev sahibi olarak modeli bakteri Escherichia coli kullanarak site-specifically acetylated proteinleri üretmek için facile bir protokol gösterdi. Malat dehidrogenaz gösteri örnek bu eser olarak kullanıldı.

Bu vahşi tipi MDH 31 mg-1 L kültür iken acetylated MDH protein verimini 15 mg-1 L kültür, oldu. Şekil 1' de gösterildiği gibi saf protein SDS-PAGE tarafından analiz edildi. Vahşi-türü MDH olumlu denetim34kullanıldı. Acetyllysine (AcK) birleşme sistem ve mutant mdh gen yataklık hücrelerden ama AcK olmadan büyüme medya, saf protein bir negatif kontrol kullanıldı. Lizin asetilasyon saf protein western Blot ac...

Watch this Scientific Journal Video about A Facile Protocol to Generate Site-Specifically Acetylated Proteins in Escherichia Coli at JoVE.com

A Facile Protocol to Generate Site-Specifically Acetylated Proteins in Escherichia Coli

Genetik kod genişleme biyolojik süreçler, protein asetilasyon dahil olmak üzere geniş bir çalışma için güçlü bir araç olarak hizmet vermektedir. Burada biz homojen oluşturmak için bu teknik yararlanma facile bir protokol proteinler, Escherichia coli hücreleri belirli sitelerdeki acetylated göstermek.

Escherichia Coli proteinler Site-Specifically oluşturmak için Facile iletişim kuralı Acetylated

Post-translational modifications that occur at specific positions of proteins have been shown to play important roles in a variety of cellular processes. ...

a-facile-protocol-to-generate-site-specifically-acetylated-proteins

Research

JoVE Journal

Immunology and Infection

A Facile Protocol to Generate Site-Specifically Acetylated Proteins in Escherichia Coli

6.8K Görüntüleme.  University of Arkansas. Genetik kod genişleme biyolojik süreçler, protein asetilasyon dahil olmak üzere geniş bir çalışma için güçlü bir araç olarak hizmet vermektedir. Burada biz homojen oluşturmak için bu teknik yararlanma facile bir protokol proteinler, Escherichia coli hücreleri belirli sitelerdeki acetylated göstermek.

Video: A Facile Protocol to Generate Site-Specifically Acetylated Proteins in Escherichia Coli

Using Fluorescent Proteins to Visualize and Quantitate Chlamydia Vacuole Growth Dynamics in Living Cells

The obligate intracellular bacterium Chlamydia elicits a great burden on global public health. C.&#160;trachomatis is the leading bacterial cause of sexually transmitted infection and also the primary cause of preventable blindness in the world. An essential determinant for successful infection of host cells by Chlamydia is the bacterium's ability to manipulate host cell signaling from within a novel, vacuolar compartment called the inclusion. From within the inclusion, Chlamydia acquire nutrients required for their 2-3 day developmental growth, and they additionally secrete a panel of effector proteins onto the cytosolic face of the vacuole membrane and into the host cytosol. Gaps in our understanding of Chlamydia biology, however, present significant challenges for visualizing and analyzing this intracellular compartment. Recently, a reverse-imaging strategy for visualizing the inclusion using GFP expressing host cells was described. This approach rationally exploits the intrinsic impermeability of the inclusion membrane to large molecules such as GFP. In this work, we describe how GFP- or mCherry-expressing host cells are generated for subsequent visualization of chlamydial inclusions. Furthermore, this method is shown to effectively substitute for costly antibody-based enumeration methods, can be used in tandem with other fluorescent labels, such as GFP-expressing Chlamydia, and can be exploited to derive key quantitative data about inclusion membrane growth from a range of Chlamydia species and strains.

Using Fluorescent Proteins to Visualize and Quantitate Chlamydia Vacuole Growth Dynamics in Living Cells

A live cell fluorescent protein based method for illuminating cellular vacuoles (inclusions) containing Chlamydia is described. This strategy enables rapid, automated determination of Chlamydia infectivity in samples and can be used to quantitatively investigate inclusion growth dynamics.

Görselleştirme ve Miktarının Belirlenmesi için floresan proteinleri kullanarak<em&gt; Chlamydia</em&gt; Vakuol Büyüme Dinamikleri Yaşayan Hücrelerde

The obligate intracellular bacterium Chlamydia elicits a great burden on global public health. C.&#160;trachomatis is the leading bacterial cause of ...

İmmünoloji ve Enfeksiyon

A Protocol to Infect Caenorhabditis elegans with Salmonella typhimurium

In the last decade, C. elegans has emerged as an invertebrate organism to study interactions between hosts and pathogens, including the host defense against gram-negative bacterium Salmonella typhimurium. Salmonella establishes persistent infection in the intestine of C. elegans and results in early death of infected animals. A number of immunity mechanisms have been identified in C. elegans to defend against Salmonella infections. Autophagy, an evolutionarily conserved lysosomal degradation pathway, has been shown to limit the Salmonella replication in C. elegans and in mammals. Here, a protocol is described to infect C. elegans with Salmonella typhimurium, in which the worms are exposed to Salmonella for a limited time, similar to Salmonella infection in humans. Salmonella infection significantly shortens the lifespan of C. elegans. Using the essential autophagy gene bec-1 as an example, we combined this infection method with C. elegans RNAi feeding approach and showed this protocol can be used to examine the function of C. elegans host genes in defense against Salmonella infection. Since C. elegans whole genome RNAi libraries are available, this protocol makes it possible to comprehensively screen for C. elegans genes that protect against Salmonella and other intestinal pathogens using genome-wide RNAi libraries.

A Protocol to Infect Caenorhabditis elegans with Salmonella typhimurium

C. elegans has emerged as a new genetic model to study host-pathogen interactions. Here we describe a protocol to infect C. elegans with Salmonella typhimurium coupled with the double-strand RNAi interference technique to examine the role of host genes in defense against Salmonella infection.

Bulaştırmak için bir protokol<em&gt; Caenorhabditis elegans</em&gt; Ile<em&gt; Salmonella typhimurium</em

In the last decade, C. elegans has emerged as an invertebrate organism to study interactions between hosts and pathogens, including the host defense ...

Non-Invasive Model of Neuropathogenic Escherichia coli Infection in the Neonatal Rat

Investigation of the interactions between animal host and bacterial pathogen is only meaningful if the infection model employed replicates the principal features of the natural infection. This protocol describes procedures for the establishment and evaluation of systemic infection due to neuropathogenic Escherichia coli K1 in the neonatal rat. Colonization of the gastrointestinal tract leads to dissemination of the pathogen along the gut-lymph-blood-brain course of infection and the model displays strong age dependency. A strain of E. coli O18:K1 with enhanced virulence for the neonatal rat produces exceptionally high rates of colonization, translocation to the blood compartment and invasion of the meninges following transit through the choroid plexus. As in the human host, penetration of the central nervous system is accompanied by local inflammation and an invariably lethal outcome. The model is of proven utility for studies of the mechanism of pathogenesis, for evaluation of therapeutic interventions and for assessment of bacterial virulence.

Non-Invasive Model of Neuropathogenic Escherichia coli Infection in the Neonatal Rat

Here, a procedure is described for the establishment of systemic infection in the neonatal rat with cultures of Escherichia coli K1. This non-invasive procedure permits colonization of the gastrointestinal tract, translocation of the pathogen to the systemic circulation, and invasion of the central nervous system at the choroid plexus.

Neuropathogenic olmayan İnvaziv Modeli<em&gt; Escherichia coli</em&gt; Yenidoğan Sıçan Enfeksiyon

Investigation of the interactions between animal host and bacterial pathogen is only meaningful if the infection model employed replicates the principal ...

Phage Phenomics: Physiological Approaches to Characterize Novel Viral Proteins

Current investigations into phage-host interactions are dependent on extrapolating knowledge from (meta)genomes. Interestingly, 60 - 95% of all phage sequences share no homology to current annotated proteins. As a result, a large proportion of phage genes are annotated as hypothetical. This reality heavily affects the annotation of both structural and auxiliary metabolic genes. Here we present phenomic methods designed to capture the physiological response(s) of a selected host during expression of one of these unknown phage genes. Multi-phenotype Assay Plates (MAPs) are used to monitor the diversity of host substrate utilization and subsequent biomass formation, while metabolomics provides bi-product analysis by monitoring metabolite abundance and diversity. Both tools are used simultaneously to provide a phenotypic profile associated with expression of a single putative phage open reading frame (ORF). Representative results for both methods are compared, highlighting the phenotypic profile differences of a host carrying either putative structural or metabolic phage genes. In addition, the visualization techniques and high throughput computational pipelines that facilitated experimental analysis are presented.

Here, we present phenomic approaches for the functional characterization of putative phage genes. Techniques include a developed assay capable of monitoring host anabolic metabolism, the Multi-phenotype Assay Plates (MAPs), in addition to the established method of metabolomics, capable of measuring effects to catabolic metabolism.

Faj Phenomics: Fizyolojik Yaklaşımlar Roman Viral Proteinler karakterize etmek

Current investigations into phage-host interactions are dependent on extrapolating knowledge from (meta)genomes. Interestingly, 60 - 95% of all phage ...

One-step Negative Chromatographic Purification of Helicobacter pylori Neutrophil-activating Protein Overexpressed in Escherichia coli in Batch Mode

Helicobacter pylori neutrophil-activating protein (HP-NAP) is a major virulence factor of Helicobacter pylori (H. pylori). It plays a critical role in H. pylori-induced gastric inflammation by activating several innate leukocytes including neutrophils, monocytes, and mast cells. The immunogenic and immunomodulatory properties of HP-NAP make it a potential diagnostic and vaccine candidate for H. pylori and a new drug candidate for cancer therapy. In order to obtain substantial quantities of purified HP-NAP used for its clinical applications, an efficient method to purify this protein with high yield and purity needs to be established.
In this protocol, we have described a method for one-step negative chromatographic purification of recombinant HP-NAP overexpressed in Escherichia coli (E. coli) by using diethylaminoethyl (DEAE) ion-exchange resins (e.g., Sephadex) in batch mode. Recombinant HP-NAP constitutes nearly 70% of the total protein in E. coli and is almost fully recovered in the soluble fraction upon cell lysis at pH 9.0. Under the optimal condition at pH 8.0, the majority of HP-NAP is recovered in the unbound fraction while the endogenous proteins from E. coli are efficiently removed by the resin.
This purification method using negative mode batch chromatography with DEAE ion-exchange resins yields functional HP-NAP from E. coli in its native form with high yield and purity. The purified HP-NAP could be further utilized for the prevention, treatment, and prognosis of H. pylori-associated diseases as well as cancer therapy.

One-step Negative Chromatographic Purification of Helicobacter pylori Neutrophil-activating Protein Overexpressed in Escherichia coli in Batch Mode

A high yield method for one-step negative purification of recombinant Helicobacter pylori neutrophil-activating protein (HP-NAP) overexpressed in Escherichia coli by using diethylaminoethyl resins in batch mode is described. HP-NAP purified by this method is beneficial for the development of vaccines, drugs, or diagnostics for H. pylori-associated diseases.

Tek adımlı olumsuz Kromatografik arıtma<em&gt; Helicobacter pylori</em&gt; Nötrofil-aktive protein aşın<em&gt; Escherichia coli</em&gt; Toplu Modunda

Helicobacter pylori neutrophil-activating protein (HP-NAP) is a major virulence factor of Helicobacter pylori (H. pylori). It plays a critical role in H. ...

A Protocol to Characterize the Morphological Changes of Clostridium difficile in Response to Antibiotic Treatment

Assessment of antibiotic action with new drug development directed towards anaerobic bacteria is difficult and technically demanding. To gain insight into possible MOA, morphologic changes associated with antibiotic exposure can be visualized using scanning electron microscopy (SEM). Integrating SEM imaging with traditional kill curves may improve our insight into drug action and advance the drug development process. To test this premise, kill curves and SEM studies were conducted using drugs with known but different MOA (vancomycin and metronidazole). C. difficile cells (R20291) were grown with or without the presence of antibiotic for up to 48 h. Throughout the 48 h interval, cells were collected at multiple time points to determine antibiotic efficacy and for imaging on the SEM. Consistent with previous reports, vancomycin and metronidazole had significant bactericidal activity following 24 h of treatment as measured by colony-forming unit (CFU) counting. Using SEM imaging we determined that metronidazole had significant effects on cell length (&#62; 50% reduction in cell length for each antibiotic; P&#60; 0.05) compared to controls and vancomycin. While the phenotypic response to drug treatment has not been documented previously in this manner, they are consistent with the drug&#39;s MOA demonstrating the versatility and reliability of the imaging and measurements and the application of this technique for other experimental compounds.

A Protocol to Characterize the Morphological Changes of Clostridium difficile in Response to Antibiotic Treatment

Antibiotic efficacy is most commonly determined by conducting killing kinetic studies and measuring colony forming units (CFUs). By integrating scanning electron microscopy (SEM) with these standard methods, we can distinguish the pharmacological effects of treatment between different antibiotics.

Morfolojik Değişiklikleri Karakterize Eden Bir Protokol<em&gt; Clostridium difficile</em&gt; Antibiyotik Tedavisine Yanıt Vermek

Assessment of antibiotic action with new drug development directed towards anaerobic bacteria is difficult and technically demanding. To gain insight into ...

Detection of Enterohemorrhagic Escherichia Coli Colonization in Murine Host by Non-invasive In Vivo Bioluminescence System

Enterohemorrhagic E. coli (EHEC) O157:H7, which is a foodborne pathogen that causesdiarrhea, hemorrhagic colitis (HS), and hemolytic uremic syndrome (HUS), colonize to the intestinal tract of humans. To study the detailed mechanism of EHEC colonization in vivo, it is essential to have animal models to monitor and quantify EHEC colonization. We demonstrate here a mouse-EHEC colonization model by transforming the bioluminescent expressing plasmid to EHEC to monitor and quantify EHEC colonization in living hosts. Animals inoculated with bioluminescence-labeled EHEC show intense bioluminescent signals in mice by detection with a non-invasive in vivo imaging system. After 1 and 2 days post infection, bioluminescent signals could still be detected in infected animals, which suggests that EHEC colonize in hosts for at least 2 days. We also demonstrate that these bioluminescent EHEC locate to mouse intestine, specifically in the cecum and colon, from ex vivo images. This mouse-EHEC colonization model may serve as a tool to advance the current knowledge of the EHEC colonization mechanism.

Detection of Enterohemorrhagic Escherichia Coli Colonization in Murine Host by Non-invasive In Vivo Bioluminescence System

A detailed protocol of a mouse model for enterohemorrhagic E. coli (EHEC) colonization by using bioluminescence-labeled bacteria is presented. The detection of these bioluminescent bacteria by a non-invasive in vivo imaging system in live animals can advance our current understanding of EHEC colonization.

Non-invaziv Vivo içinde Bioluminescence sistemi tarafından fare konak Enterohemorrhagic Escherichia Coli kolonizasyon algılama

Enterohemorrhagic E. coli (EHEC) O157:H7, which is a foodborne pathogen that causesdiarrhea, hemorrhagic colitis (HS), and hemolytic uremic syndrome ...

Creation of a Dense Transposon Insertion Library Using Bacterial Conjugation in Enterobacterial Strains Such As Escherichia Coli or Shigella flexneri

Transposon mutagenesis is a method that allows gene disruption via the random genomic insertion of a piece of DNA called a transposon. The protocol below outlines a method for high efficiency transfer between bacterial strains of a plasmid harboring a transposon containing a kanamycin resistance marker. The plasmid-borne transposase is encoded by a variant tnp gene that inserts the transposon into the genome of the recipient strain with very low insertional bias. This method thus allows the creation of large mutant libraries in which transposons have been inserted into unique genomic positions in a recipient strain of either Escherichia coli or Shigella flexneri bacteria. By using bacterial conjugation, as opposed to other methods such as electroporation or chemical transformation, large libraries with hundreds of thousands of unique clones can be created. This yields high-density insertion libraries, with insertions occurring as frequently as every 4-6 base pairs in non-essential genes. This method is superior to other methods as it allows for an inexpensive, easy to use, and high efficiency method for the creation of a dense transposon insertion library. The transposon library can be used in downstream applications such as transposon sequencing (Tn-Seq), to infer genetic interaction networks, or more simply, in mutational (forward genetic) screens.

Creation of a Dense Transposon Insertion Library Using Bacterial Conjugation in Enterobacterial Strains Such As Escherichia Coli or Shigella flexneri

Presented here is a simple method for creating a high-density transposon insertion library in Escherichia coli or Shigella flexneri using bacterial conjugation. This protocol allows the creation of a collection of hundreds of thousands of unique mutants in bacteria by the random genomic insertion of a transposon.

Bir yoğun Transposon ekleme kütüphane kullanarak bakteriyel konjugasyon Escherichia Coli Enterobacterial suşları gibi veya Shigella flexneri oluşturulması

Transposon mutagenesis is a method that allows gene disruption via the random genomic insertion of a piece of DNA called a transposon. The protocol below ...

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple condensation reactions. DHDDS (dehydrodolichyl diphosphate synthase) is a eukaryotic long-chain cis-PT (forming cis double bonds from the condensation reaction) that catalyzes chain elongation of farnesyl diphosphate (FPP, an allylic diphosphate) via multiple condensations with isopentenyl diphosphate (IPP). DHDDS is of biomedical importance, as a non-conservative mutation (K42E) in the enzyme results in retinitis pigmentosa, ultimately leading to blindness. Therefore, the present protocol was developed in order to acquire large quantities of purified DHDDS, suitable for mechanistic studies. Here, the usage of protein fusion, optimized culture conditions and codon-optimization were used to allow the overexpression and purification of functionally active human DHDDS in E. coli. The described protocol is simple, cost-effective and time sparing. The homology of cis-PT among different species suggests that this protocol may be applied for other eukaryotic cis-PT as well, such as those involved in natural rubber synthesis.

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

A simple protocol for overexpression and purification of codon-optimized, human cis-prenyltransferase, under non-denaturing conditions, from Escherichia coli, is described, along with an enzymatic activity assay. This protocol can be generalized for production of other cis- prenyltransferase proteins in quantity and quality suitable for mechanistic studies.

Aşırı Ezme ve İnsanın Saflaştırılması<em&gt; Cis</em&gt; -prenıltransferaz in<em&gt; Escherichia coli</em

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple ...

Using Enhanced Green Fluorescence Protein-expressing Escherichia Coli to Assess Mouse Peritoneal Macrophage Phagocytosis

This manuscript describes a simple and reproducible method to perform a phagocytosis assay. The first part of this method involves building a pET-SUMO-EGFP vector (SUMO = small ubiquitin-like modifier) and expressing enhanced green fluorescence protein (EGFP) in Escherichia coli (BL21DE). EGFP-expressing E. coli is coincubated with macrophages for 1 h at 37 &#176;C; the negative control group is incubated on ice for the same amount of time. Then, the macrophages are ready for assessment. The advantages of this technique include its simple and straightforward steps, and phagocytosis can be measured by both flow cytometer and fluorescence microscope. The EGFP-expressing E. coli are stable and display a strong fluorescence signal even after the macrophages are fixed with paraformaldehyde. This method is not only suitable for the assessment of macrophage cell lines or primary macrophages in vitro but also suitable for the evaluation of granulocyte and monocyte phagocytosis in peripheral blood mononuclear cells. The results show that the phagocytic capability of peritoneal macrophages from young (eight-week-old) mice is higher than that of macrophages from aged (16-month-old) mice. In summary, this method measures macrophage phagocytosis and is suitable for studying the innate immune system function.

Using Enhanced Green Fluorescence Protein-expressing Escherichia Coli to Assess Mouse Peritoneal Macrophage Phagocytosis

Here, we present a protocol to assess mouse peritoneal macrophage phagocytosis using enhanced green fluorescence protein-expressing Escherichia coli.

Escherichia Coli proteinler Site-Specifically oluşturmak için Facile iletişim kuralı Acetylated

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Görselleştirme ve Miktarının Belirlenmesi için floresan proteinleri kullanarak

Bulaştırmak için bir protokol

Neuropathogenic olmayan İnvaziv Modeli

Faj Phenomics: Fizyolojik Yaklaşımlar Roman Viral Proteinler karakterize etmek

Tek adımlı olumsuz Kromatografik arıtma

Morfolojik Değişiklikleri Karakterize Eden Bir Protokol

Non-invaziv Vivo içinde Bioluminescence sistemi tarafından fare konak Enterohemorrhagic Escherichia Coli kolonizasyon algılama

Bir yoğun Transposon ekleme kütüphane kullanarak bakteriyel konjugasyon Escherichia Coli Enterobacterial suşları gibi veya Shigella flexneri oluşturulması

Aşırı Ezme ve İnsanın Saflaştırılması

Yeşil floresans Protein-ifade gelişmiş kullanma fare Peritoneal makrofaj fagositoz değerlendirmek için Escherichia Coli