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E. 1. izolasyonu E. coli Eş transformantlar <ol><li> Uygun E. elektro-yetkili hücreleri hazırlayın coli suşu transforme edilecek. LB ortamı, 1 ml (tripton, maya ekstresi, 10 NaCl, 5, ve 10 g / l&#39;lik konsantrasyonlarda kullanılır) tercih edilen soyunun tek bir koloni, ve transfer 180 rpm çalkalama koşulları altında, 37 ° C&#39;de inkübe edin. Ön-kültür 1 seyreltin: 500, taze LB ortamında 25 ml ve 37 ° C&#39;de kültür inkübe edin. </li><li> Log-faz (0.6 OD) içinde 20 dakika için 5000 xg) bir hücre süspansiyonu santrifüj ve orijinal kültür hacminin yarısı% 10, h / h buz soğukluğunda steril gliserol-su içinde pelletini. Her seferinde tabanda hacmi yarıya, bu adımı 4 kez tekrarlayın. Son olarak, gliserol / su içinde tekrar süspansiyon pelet ve 50 ul alikolar içinde hücre süspansiyonu bölün. 6 ay -80 ° C de alikotları saklayın. </li><li> 0.5 mM EDTA ile takviye edilmiş, steril su içinde arzu edilen plazmid çözülür. Ic üzerinde çözülmeE elektro-kompetan hücrelerin bir tümböleni ve vektör uygun bir miktar (2.5-5 ng) ile karıştırılır. Elektroporasyon için uygun olan bir 0.1 cm küvet içine karışımı koyun ve 1.8 kV uygulanır. </li><li> Hemen SOC ortamında 1 ml &#39;ye elektroporatlanmıştır hücreleri transferi (LB ortamı% 0.2 / h glukoz, 10 mM MgCl2, 2.5 mM KCI ile takviye edilmiş), çalkalama altında 1 saat boyunca inkübe edilir ve son olarak da içeren Petri kapları 100 ul alikotları aktarmak Uygun antibiyotik ile LB agar. 37 ° C&#39;de O / N inkübe edin. </li><li> Petri kapları tek koloniler çizgiler ile transformantlann arındırın. </li><li> Birincil Transformantların elektro-yetkili hücreleri hazırlayın ve tekrar ileri plazmidlerle dönüştürmek için 1,1-1,5 adımları. </li><li> Ko-transformantların gliserol stokları hazırlayın. Uygun antibiyotikler ile takviye edilmiş LB içinde tek bir koloni transfer, çalkalanarak 37 ° C&#39;de inkübe ve log-faz (0.6 OD) santrifüj 20 dakika boyunca 5000 x g&#39;de hücre süspansiyonu. ROrta% 15 h / h gliserol ihtiva eden LB-antibiyotikler pelet esuspend. Alikotları koyun ve en saklayın - 80 ° C. </li></ol> E. α ve ε Altbirimlerin 2. birlikte ifadesi, E. coli DNA polimeraz III <ol><li> Steril bir çerçevenin bulunduğu uygun soy gliserol stokundan bir miktar transfer LB ortamı ve antibiyotikler içeren bir Petri kutusu içine (örneğin, TOP10 / pGOOD-ε243 ve TOP10 / pGOOD-ε243-θ / pBADα1160, Şekil 1 &#39;e bakınız). Hücreler damlacık hücreler süspansiyon Petri kabı üzerine kuru ve çizgi edelim. 37 ° CO / N inkübe edin. </li><li> Steril kürdan LB-antibiyotikler ortamı, 1 ml için tek bir koloni ile Aktarım. 37 ° C&#39;de 8 saat inkübe edin. Taze ortam içinde 500 ve 15 saat boyunca 30 ° C&#39;de inkübe: kültür öncesi 1 seyreltin. </li><li> IPTG, arabinoz veya arabinoz ve IPTG, 1 mM her ekleyin. 2.5 saat boyunca 30 ° C&#39;de inkübe edilir. Hücreler toplanır ve -20 ° C &#39;de pelet saklayın. </li><li> Liziz tamponu (50 mM Tris-HCI, 50 mM NaCI, 1 mM EDTA, 2.5 mM ditiyotreitol, 1 mM fenilmetilsülfonil florür (PMSF), pH 8) &#39;de buz ve tekrar süspansiyon ile çözülme peletler. Yavaşça soğuk cam çömlekçi olan hücreler süspansiyon homojenize. </li><li> 15 sn soğutma aralığı (4 kür) takip 15 sn 15 W, bir sonikasyon. </li><li> Çözünür fraksiyonu geri kazanmak için, 4 ° C&#39;de, 20 dakika boyunca 10,000 x g&#39;de toplam protein ekstreleri santrifüjleyin. </li><li> SDS-PAGE (% 12.5 akrilamid) her çözünür protein ekstresi alikolar ile analiz edin. Bu amaçla, H2O 60 ul ve yükleme tamponu (250 mM Tris-HCI, pH 20 ul ihtiva eden Eppendorf tüplerine her çözünür protein ekstresi 20 ul transfer 6.8, 10 mM β-merkaptoetanol,% 10 (ağ / hac) SDS,% 50 (h / h) gliserol,% 0.25, mavi (ağ / hac) bromofenol) ve 5 dakika kaynatılır. Yük, her numune için 18 ul ila yaklaşık 1.5 saat için 140 V&#39;ta elektroforez gerçekleştirin. </li></ol> Deinococ arasında α alt birimi 3. birlikte ifadesi,cus radiodurans DNA Polimeraz III ε E. Alt Birim E. coli DNA polimeraz III <ol><li> E. ön kültür hazırlanması E. coli suşu TOP 10 / Dr pBAD-α / LB-ampisilin, tetrasiklin ortamı, 1 ml pGOOD-ε243 ve 37 ° C &#39;de 8 saat daha inkübe edilir. Taze ortam içinde, 1.000 ve 37 ° C &#39;de O / N inkübe: 1 seyreltin. </li><li> 1 seyreltin: taze ortam, 100, 200 içine mi, daha sonra arabinoz ve IPTG, 1 mM her biri 3 saat; özellikle, 3 saat süre ile 37 ° C&#39;de inkübe edin. Hücreler hasat ve -20 ° C &#39;de pelet saklayın. </li><li> 50 mM Tris-HCI, pH 8, 150 mM NaCI, 1 mM EDTA, 1 mM PMSF içinde pelletini. Homojenize ve 2.4 ve 2.5 de tarif edildiği gibi hücreler bozabilir. </li><li> Hemen hücreler 20 dakika boyunca 10,000 x g&#39;de santrifüj lizata. Pelet atın. Kağıt filtre 3 kat ile donatılmış bir Buchner hunisi ile süpernatant filtre. Nazik vakum uygulayın. Aşırı köpüklenme önlemek için, filtrasyon sırasında buz üzerinde vakum Erleni tutun. </li><li> Bradford 14 göre protein konsantrasyonu belirleyin. </li></ol> 4. Jel Filtrasyon Kromatoarafisi <ol><li> 50 mM Tris-HCI, 150 mM NaCI, 1 mM EDTA, pH kolonuna 8. Yük çözünür protein ekstresi ile bir su ceketli 16x70 (200) jel süzme sütunu dengelenmesi. En uygun çözünürlük için, 1 ml örnek döngü kullanmak. / Dakika 0.6 ml kromatografisi yapın. Boyunca 4 ° C&#39;de kolon sıcaklığı muhafaza edin. </li><li> 0.9 ml kesirler toplayın ve SDS-PAGE ile analiz. Aktarım H2O 60 ul ve yükleme tamponu (250 mM Tris-HCI pH 6.8, 10 mM β-merkaptoetanol,% 10 (a / h) SDS, w% 50 20 ul ihtiva eden Eppendorf tüplerine ilgili her fraksiyonun 20 ul ( h / h) gliserol,% 0.25 (mavi ağırlık / hacim) bromofenol) ve 5 dakika kaynatılır. Yük, her numune için 18 ul ila yaklaşık 1.5 saat için 140 V&#39;ta elektroforez gerçekleştirin. </li><li> 3&#39;-5 &#39;ekzonükleaz ve her bir fraksiyonun DNA polimeraz aktivitesi 96 biz de belirlerll mikroplakalar. Alt-tabaka 15 olarak timidin 5&#39;-monofosfat, p-nitrofenil ester (p NP TMP) ile eksonükleaz aktivitesi deneyi. PPX (pirofosfataz, purin nükleosit fosforilaz, ksantin oksidaz) enzim-bağlı deney 16 ve DNA polimeraz aktivitesi tahmin 2- (4-İyodofenil) -3- (4-nitrofenil) -5-fenil-2H-tetrazolyum klorid (INT) Elektron alıcı 16 gibi. </li></ol> Popülasyonlarının E. Yabani tip ε alt biriminin birlikte ifade 5. Mutasyon Analizi E. coli DNA Polimeraz III ve mutajenik εD12A Variant <ol><li> E. tek bir koloni transfer pBAD-ε ve LB-antibiyotikler ortamın 1 ml pGOOD1-εD12A 11 vektörleri içeren E. coli TOP 10. 37 ° C&#39;de O / N inkübe edin. </li><li> (Arabinoz, IPTG veya arabinoz ve IPTG, 1 mM), uygun indükleyicileri eklemek, taze ortam (10 mi) ihtiva eden 250 3 şişeleri ve 37 ° C&#39;de inkübe: Ön-kültür 1 seyreltin8 saat karıştırıldı. Paralel olmayan kaynaklı kültürlerde hazırlayın. </li><li> 1 ml&#39;lik alikolar toplamak, daha sonra 1 sulandırmak taze ortam (10 mi) ihtiva eden yeni şişelere 500 uygun indükleyicileri ile takviye edilmiş ya da. 37 ° C&#39;de O / N inkübe edin. </li><li> Tekrar 1 ml 5.2 ve 5.3 ve toplamak alikotları adımları. </li><li> Nesillerin sayısı her kültürde meydana belirleyin. LB plakaları, inokulum ve kültür uygun seri dilüsyonları 100 ul üzerine aktarın. 37 ° C&#39;de O / N inkübe edin. LB plakaları üzerine kolonileri sayın ve büyüme (C log) sonunda aşı içinde mevcut (I log) ve kültürde hücre sayısının kaydını hesaplar. Nesillerin sayısını belirlemek için, formülü kullanın: (C log - I log) /0.301. </li><li> 20 dakika boyunca 5000 x g&#39;de santrifüj ve 50 mM Tris-HCI, pH 7.6, 50 mM NaCI, 1 ml süspansiyon hücrelerin. Hücreleri geçirgenliği, 20 saniye kloroform ve vorteks 2-3 damla ekleyin. </li><li> Β- belirleyinSubstrat olarak p-nitrofenil-β-D-glukopiranosid (PNPGluc) kullanılarak 96 çukurlu bir mikrolevhadaki her bir kısım bir glukosidaz aktivitesi. Her çukuruna geçirgen hücrelerin 100 ul ve alt-tabaka 100 ul (16 mg / H2 O ml stok çözelti) eklenir. Kuyularda hava kabarcıklarını önlemek için özen gösterin. Bir mikrolevha okuyucu ve uygun bir filtre kullanılarak, 420 nm&#39;de absorbansı okumak. </li></ol>

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D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+set+of+ligation-independent+expression+vectors+for+co-expression+of+proteins+in+Escherichia+coli.">A set of ligation-independent expression vectors for co-expression of proteins in Escherichia coli.</a> Protein Expr. Purif. 47 (1), 217-224 (2006).</li><li>Sun, J., Hopkins, R. C., Jenney, F. E., McTernan, P. M., Adams, M. W. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterologous+expression+and+maturation+of+an+NADP-dependent+[NiFe]-hydrogenase:+a+key+enzyme+in+biofuel+production.">Heterologous expression and maturation of an NADP-dependent [NiFe]-hydrogenase: a key enzyme in biofuel production.</a> PloS One. 5 (5), (2010).</li></ol>

The authors declare that they have no competing financial interests.

Proteinler olan üçüncül yapı uyumlarının 25 arasında, sınırlı sayıda sınırlı değildir bölgeleri içeren, doğal olarak düzensiz olabilir. Bu düzensiz proteinler toplama 25 genellikle eğilimli, ve onların izolasyon ve karakterizasyonu zor bir görev temsil edebilir. E. ε alt birimi coli DNA polimeraz III iki farklı etki yani 26,27, özellikleri: i) N-ter etki, θ alt birimini bağlama 3&#39;-5 &#39;ekzonükleaz aktivitesini taşıyan ve yetkin; ii)...

overekspresyonu teknolojilerin tanıtılması düşük kopya sayısı enzimlerin biyokimyasal çalışmalar ve farmakolojik olarak aktif proteinlerin (örneğin, insülin) sanayi üretimi ya artırdı. Bu teknolojilerin gelişiyle bu yana, önemli gelişmeler rekombinant proteinlerin verim ve kaliteyi artırmak için elde edilmiştir. Buna ek olarak, prokaryotik ve ökaryotik 1,2 3 ekspresyonu sistemleri, protein biyoteknoloji, yani, Escherichia coli &quot;iş atı&quot; yararlı alternatifler sunan, yıllar içinde geliştirilmiştir. E. alternatif platformların Özellikle, kullanılabilirlik E. coli rekombinant peptitler veya post-translasyonel modifikasyonlar taşıyan proteinlerin üretimine yol açtı. Bununla birlikte, söz edilmelidir E. E. coli halen yeniden birleştirici protein üretimi için tercih edilen bir organizmayı ifade eder. Bu en alakalı olabilir aralarında çeşitli faktörler, nedeniylekabul: i) ekspresyonu sistemleri (ifade vektörleri ve suşları oldukça bir dizi kullanılabilirlik) E. için E. coli 1,2; E. ii) Kısa nesil süreleri ve yüksek biyokütle verimi, Zengin ve sentetik medya çeşitli coli; iii) biyokimyasal olarak ve bu mikroorganizmanın genetik düzeyde ya uyduruk manipülasyon; iv), toksik proteinlerin 4 üretebilen suşlarının izole edilmesi; v) nüfus düzeyinde 5,6 homojen indüksiyon sahip suşlar yapımı. Buna ek olarak, son zamanlarda, E. üretimi için uygun olan bu ifade sistemleri gördü translasyon sonrası modifiye edilmiş proteinlerin E. coli tasarlanmış ve 2 yapılabilir. Şu anda, proteini aşırı ekspresyonu esas olan hypersynthesis uygun bir plasmid içine klonlanmış tek bir gen ile yapılabilir monomerik ya da homo-oligomerik proteinler elde etmek için kullanılır. Bununla birlikte, dikkat son zamanlardaE. inşaat ödenen coli proteini birlikte ifade sistemleri, bir hetero-oligomerik kompleksleri 2, in vivo olarak, üretim zorlu. İlginç bir şekilde, protein, birlikte-ifade edilme daha önceki deneylerde, siyanobakteri ribuloz-1,5-bisfosfat karboksilazın / oksijenaz 7,8 arasında büyük ve küçük alt birimleri ve türler arası düzeneği, ve HIV-1 kesik ve tam uzunluktaki formları arasındaki ilişkiyi ele transkriptaz 9 ters. Bu öncü çalışmalar, protein ko-ifadesi in vitro yeniden geleneksel güçlü bir alternatif temsil ettiğini göstermiştir. E. koli içinde ilave olarak, protein, ko-ekspresyonu E. coli doğal olmayan amino asitler 2 ihtiva eden proteinler elde etmek için, post-translasyonel modifikasyonlar 2 taşıyan çeşitli proteinleri üretmek için ve Süreksprese membran proteinlerinin 2 verimini artırmak için kullanılmıştır. Ayrıca, bir araç olarak bir protein birlikte-ifade edilme potansiyel E. kazandırma coli rekabetProtein salgılanması nce aktif soruşturma 2 altında. E. Protein birlikte-ifade edilme iki temel strateji coli takip edilebilir: Farklı genleri barındırmak için tek bir plazmid kullanımı aşın i); ii) tek bir hücre içinde birden fazla plasmidlerin kullanımı hedef proteinlerin yardımcı şekilde ifade etmek. Tandem promotör / operatör elemanları ihtiva eden, özellikle plazmidler ko-ekspresyonu 10 oluşturulmuştur, ancak ilk durumda, plazmid seçimi için kriterler, geleneksel tek bir protein aşın deney olanlardan farklı değildir. Bu ilk yaklaşım bu nedenle oldukça basittir. Bununla birlikte, farklı proteinler yardımcı şekilde ifade etmek tek plazmidin kullanılması, sağlamanın iki büyük bir zorlukla karşı karşıya olduğu belirtilmelidir: i) eş ifade edilen proteinlerin sayısının sınırlandırılması barındırılan genlerin sayısı ile vektör artar, moleküler kütlesi; ii) birden fazla gen, tek bir promotörün kontrolü altında klonlandı zaman, polarite gerilemenin olabilirpromoterinden distal genlerin ifadesi e. Tek E., ikili veya çoklu plasmidlerin kullanımı coli hücreleri, bu nedenle plazmid uygun kombinasyonlarla kısıtlamalar getiren, seçilen vektörler uyumluluğunu gerçekleştirmek zorundadır. Bununla birlikte, bu ikinci eş ifade stratejisinin vektörlerinin molekül kütlesi ihtiva eden bir avantaj sahiptir, ve polarite sınırlar. Son zamanlarda birlikte ekspresyon plazmidleri 11 arasında gen mekik kolaylaştırmak için tasarlanmış bir protein ko-ifade sistemi inşa edilmiştir. Özellikle, pGOOD vektörleri serisi inşa ilgili özellikler elde edilmiştir: i) bir replikasyon orijini p15A, ticari vektörler (ör pBAD seri 12) ColE1 orijini içeren pGOOD plazmid uyumluluğunu sağlamak; ii) bir tetrasiklin direnç kasedi; iii) lac varlığı düzenleyici unsurları, yani Yarışmayı-Operatörü (O 1) çift ve IacI -türevli </em&gt; q geni. Uygun bir pBAD-pGOOD çift kullanarak, E. katalitik çekirdeğini aşırı ifade başardık üç farklı alt üniteden oluşan coli DNA polimeraz III, yani α (5&#39;-3 &#39;polimeraz), ε (3&#39;-5&#39; eksonükleaz) ve (stabilize edici ε) 13 θ. Özellikle, αεθ kompleksinin birlikte ifadesi, E. ek sıkı sıkıya bağlı olduğunu göstermiştir IPTG ve arabinoz hem de E. coli kültür ortamı, pGOOD ve pBAD sırasıyla (Şekil 1A), tetikleyici aşırı ekspresyonu. Mevcut yazıda, proteinin birlikte ifadesi, etkin bir şekilde bir protein kompleksi alt biriminin çözünürlüğünün kötü bağlantılı zorlukların üstesinden nasıl göstermektedir. Buna ek olarak, ne kadar in vivo proteinin tamamlama testlerinde gerçekleştirilebilir göstermektedir, ve nihayet E. ayar mutasyon sıklığı protein ko-ifade edilmesi için rapor dağ geçidii. Bu amaçla, her vaka çalışması ilgili örnekler göstermek için uygun pGOOD-pBAD çiftler kullanılır.

<table border="0" cellpadding="0" cellspacing="0" width="627">
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the multifaceted benefits of protein co-expression in escherichia coli

We report here that the expression of protein complexes in vivo in Escherichia coli can be more convenient than traditional reconstitution experiments in vitro. In particular, we show that the poor solubility of Escherichia coli DNA polymerase III &#949; subunit (featuring 3&#8217;-5&#8217; exonuclease activity) is highly improved when the same protein is co-expressed with the &#945; and &#952; subunits (featuring DNA polymerase activity and stabilizing &#949;, respectively). We also show that protein co-expression in E. coli can be used to efficiently test the competence of subunits from different bacterial species to associate in a functional protein complex. We indeed show that the &#945; subunit of Deinococcus radiodurans DNA polymerase III can be co-expressed in vivo with the &#949; subunit of E. coli. In addition, we report on the use of protein co-expression to modulate mutation frequency in E. coli. By expressing the wild-type &#949; subunit under the control of the araBAD promoter (arabinose-inducible), and co-expressing the mutagenic D12A variant of the same protein, under the control of the lac promoter (inducible by isopropyl-thio-&#946;-D-galactopyranoside, IPTG), we were able to alter the E. coli mutation frequency using appropriate concentrations of the inducers arabinose and IPTG. Finally, we discuss recent advances and future challenges of protein co-expression in E. coli.

E. ε alt birimi E. coli DNA polimeraz III 243 amino asitten oluşur ve kalıntılar 187-243 17 silinir sürece, zayıf çözünürlüğe 17,18 bulunmaktadır. Bununla birlikte, daha önce tam uzunlukta α, ε ve θ alt birimlerinin birlikte ifadesi, çözünür bir DNA polimeraz III katalitik çekirdek (Şekil 1) elde edilir ki 11 göstermiştir. I) α ve ε alt birimden fazla sentezlenmesi, bağımsız olarak arabinoz ve IPTG sırasıyla (Şek...

Watch this Scientific Journal Video about The Multifaceted Benefits of Protein Co-expression in Escherichia coli at JoVE.com

The Multifaceted Benefits of Protein Co-expression in Escherichia coli

Protein co-expression is a powerful alternative to the reconstitution in vitro of protein complexes, and is of help in performing biochemical and genetic tests in vivo. Here we report on the use of protein co-expression in Escherichia coli to obtain protein complexes, and to tune the mutation frequency of cells.

Protein birlikte-ifade edilme yönlü Faydalar<em&gt; Escherichia coli</em

We report here that the expression of protein complexes in vivo in Escherichia coli can be more convenient than traditional reconstitution experiments in ...

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Biology

The Multifaceted Benefits of Protein Co-expression in Escherichia coli

11.8K Görüntüleme.  University of Bologna. Protein co-expression is a powerful alternative to the reconstitution in vitro of protein complexes, and is of help in performing biochemical and genetic tests in vivo. Here we report on the use of protein co-expression in Escherichia coli to obtain protein complexes, and to tune the mutation frequency of cells. 

Video: The Multifaceted Benefits of Protein Co-expression in Escherichia coli

Purification of the M. magneticum Strain AMB-1 Magnetosome Associated Protein MamA&Delta;41

Magnetotactic bacteria comprise a diverse group of aquatic microorganisms that are able to orientate themselves along geomagnetic fields. This behavior is believed to aid their search for suitable environments (1). This capability is conferred by the magnetosome, a subcellular organelle that consists of a linear-chain assembly of lipid vesicles each able to biomineralize and enclose a ~50-nm crystal of magnetite or greigite. A principle component of the magnetosome that was shown to be required for the formation of functional vesicles is MamA. MamA is a highly abundant magnetosome-associated protein which is one of the most characterized magnetosome-associated proteins in vivo (2-6). This article focuses on the purification of MamA, which despite being studied in vivo, no clear functional or structural details have been identified for it. Bioinformatics analysis suggested that MamA is a tetra-tricopeptide repeat (TPR) containing protein. TPR is a structural motif found as such or forming part of a bigger fold in a wide range of proteins, it serves as a template for protein-protein interactions and mediates multi-protein complexes (7). TPRs are involved in many crucial tasks in eukaryotic cell organelle processes and many bacterial pathways (8-14). In order to understand MamA, a unique TPR containing protein, highly purified protein is required as a first step. In this article, we present the purification protocol for a stable MamA deletion mutant (MamA&Delta;41) from M. magneticum AMB-1.

MamA is a unique Magnetosome associated protein which was shown to be involved in magnetosome activation. Here we present the purification protocol of MamA deletion mutant (MamA&Delta;41) from M. magneticum AMB-1.

Saflaştırılması M. magneticum Deformasyon AMB-1 Magnetosome İlişkili Protein MamAΔ41

Magnetotactic bacteria comprise a diverse group of aquatic microorganisms that are able to orientate themselves along geomagnetic fields. This behavior is ...

Biyoloji

An Assay for Measuring the Activity of Escherichia coli Inducible Lysine Decarboxyase

Escherichia coli is an enteric bacterium that is capable of growing over a wide range of pH values (pH 5 - 9)1 and, incredibly, is able to survive extreme acid stresses including passage through the mammalian stomach where the pH can fall to as low as pH 1 - 22. To enable such a broad range of acidic pH survival, E. coli possesses four different inducible amino acid decarboxylases that decarboxylate their substrate amino acids in a proton-dependent manner thus raising the internal pH. The decarboxylases include the glutamic acid decarboxylases GadA and GadB3, the arginine decarboxylase AdiA4, the lysine decarboxylase LdcI5, 6 and the ornithine decarboxylase SpeF7. All of these enzymes utilize pyridoxal-5'-phospate as a co-factor8 and function together with inner-membrane substrate-product antiporters that remove decarboxylation products to the external medium in exchange for fresh substrate2. In the case of LdcI, the lysine-cadaverine antiporter is called CadB. Recently, we determined the X-ray crystal structure of LdcI to 2.0 &#197;, and we discovered a novel small-molecule bound to LdcI the stringent response regulator guanosine 5'-diphosphate,3'-diphosphate (ppGpp) 14. The stringent response occurs when exponentially growing cells experience nutrient deprivation or one of a number of other stresses9. As a result, cells produce ppGpp which leads to a signaling cascade culminating in the shift from exponential growth to stationary phase growth10. We have demonstrated that ppGpp is a specific inhibitor of LdcI 14. Here we describe the lysine decarboxylase assay, modified from the assay developed by Phan et al.11, that we have used to determine the activity of LdcI and the effect of pppGpp/ppGpp on that activity. The LdcI decarboxylation reaction removes the &alpha;-carboxy group of L-lysine and produces carbon dioxide and the polyamine cadaverine (1,5-diaminopentane)5. L-lysine and cadaverine can be reacted with 2,4,6-trinitrobenzensulfonic acid (TNBS) at high pH to generate N,N'-bistrinitrophenylcadaverine (TNP-cadaverine) and N,N&#8242;-bistrinitrophenyllysine (TNP-lysine), respectively11. The TNP-cadaverine can be separated from the TNP-lysine as the former is soluble in organic solvents such as toluene while the latter is not (See Figure 1). The linear range of the assay was determined empirically using purified cadaverine.

An Assay for Measuring the Activity of Escherichia coli Inducible Lysine Decarboxyase

The activity of the inducible lysine decarboxylase is monitored by reacting the substrate L-lysine and the product cadaverine with 2,4,6-trinitrobenzensulfonic acid to form adducts that have differential solubility in toluene.

Etkinlik Ölçüm Bir Testi Escherichia coli Lizin Decarboxyase Đndüklenebilir

Escherichia coli is an enteric bacterium that is capable of growing over a wide range of pH values (pH 5 - 9)1 and, incredibly, is able to survive extreme ...

High Throughput Screening of Fungal Endoglucanase Activity in Escherichia coli

Cellulase enzymes (endoglucanases, cellobiohydrolases, and &beta;-glucosidases) hydrolyze cellulose into component sugars, which in turn can be converted into fuel alcohols1. The potential for enzymatic hydrolysis of cellulosic biomass to provide renewable energy has intensified efforts to engineer cellulases for economical fuel production2. Of particular interest are fungal cellulases3-8, which are already being used industrially for foods and textiles processing.
Identifying active variants among a library of mutant cellulases is critical to the engineering process; active mutants can be further tested for improved properties and/or subjected to additional mutagenesis. Efficient engineering of fungal cellulases has been hampered by a lack of genetic tools for native organisms and by difficulties in expressing the enzymes in heterologous hosts. Recently, Morikawa and coworkers developed a method for expressing in E. coli the catalytic domains of endoglucanases from H. jecorina3,9, an important industrial fungus with the capacity to secrete cellulases in large quantities. Functional E. coli expression has also been reported for cellulases from other fungi, including Macrophomina phaseolina10 and Phanerochaete chrysosporium11-12. 
We present a method for high throughput screening of fungal endoglucanase activity in E. coli. (Fig 1) This method uses the common microbial dye Congo Red (CR) to visualize enzymatic degradation of carboxymethyl cellulose (CMC) by cells growing on solid medium. The activity assay requires inexpensive reagents, minimal manipulation, and gives unambiguous results as zones of degradation (&#x201C;halos&#x201D;) at the colony site. Although a quantitative measure of enzymatic activity cannot be determined by this method, we have found that halo size correlates with total enzymatic activity in the cell. Further characterization of individual positive clones will determine , relative protein fitness. 
Traditional bacterial whole cell CMC/CR activity assays13 involve pouring agar containing CMC onto colonies, which is subject to cross-contamination, or incubating cultures in CMC agar wells, which is less amenable to large-scale experimentation. Here we report an improved protocol that modifies existing wash methods14 for cellulase activity: cells grown on CMC agar plates are removed prior to CR staining. Our protocol significantly reduces cross-contamination and is highly scalable, allowing the rapid screening of thousands of clones. In addition to H. jecorina enzymes, we have expressed and screened endoglucanase variants from the Thermoascus aurantiacus and Penicillium decumbens (shown in Figure 2), suggesting that this protocol is applicable to enzymes from a range of organisms.

High Throughput Screening of Fungal Endoglucanase Activity in Escherichia coli

We describe a low cost, high throughput method to screen for fungal endoglucanase activity in E. coli. The method relies on a simple visual readout of substrate degradation, does not require enzyme purification, and is highly scalable. This allows for the rapid screening of large libraries of enzyme variants.

Mantar Endoglucanase Faaliyet Yüksek Verimli Tarama Escherichia coli</em

Cellulase enzymes (endoglucanases, cellobiohydrolases, and &beta;-glucosidases) hydrolyze cellulose into component sugars, which in turn can be converted ...

Mapping Bacterial Functional Networks and Pathways in Escherichia Coli using Synthetic Genetic Arrays

Phenotypes are determined by a complex series of physical (e.g. protein-protein) and functional (e.g. gene-gene or genetic) interactions (GI)1. While physical interactions can indicate which bacterial proteins are associated as complexes, they do not necessarily reveal pathway-level functional relationships1. GI screens, in which the growth of double mutants bearing two deleted or inactivated genes is measured and compared to the corresponding single mutants, can illuminate epistatic dependencies between loci and hence provide a means to query and discover novel functional relationships2. Large-scale GI maps have been reported for eukaryotic organisms like yeast3-7, but GI information remains sparse for prokaryotes8, which hinders the functional annotation of bacterial genomes. To this end, we and others have developed high-throughput quantitative bacterial GI screening methods9, 10.
Here, we present the key steps required to perform quantitative E. coli Synthetic Genetic Array (eSGA) screening procedure on a genome-scale9, using natural bacterial conjugation and homologous recombination to systemically generate and measure the fitness of large numbers of double mutants in a colony array format. Briefly, a robot is used to transfer, through conjugation, chloramphenicol (Cm) - marked mutant alleles from engineered Hfr (High frequency of recombination) 'donor strains' into an ordered array of kanamycin (Kan) - marked F- recipient strains. Typically, we use loss-of-function single mutants bearing non-essential gene deletions (e.g. the 'Keio' collection11) and essential gene hypomorphic mutations (i.e. alleles conferring reduced protein expression, stability, or activity9, 12, 13) to query the functional associations of non-essential and essential genes, respectively. After conjugation and ensuing genetic exchange mediated by homologous recombination, the resulting double mutants are selected on solid medium containing both antibiotics. After outgrowth, the plates are digitally imaged and colony sizes are quantitatively scored using an in-house automated image processing system14. GIs are revealed when the growth rate of a double mutant is either significantly better or worse than expected9. Aggravating (or negative) GIs often result between loss-of-function mutations in pairs of genes from compensatory pathways that impinge on the same essential process2. Here, the loss of a single gene is buffered, such that either single mutant is viable. However, the loss of both pathways is deleterious and results in synthetic lethality or sickness (i.e. slow growth). Conversely, alleviating (or positive) interactions can occur between genes in the same pathway or protein complex2 as the deletion of either gene alone is often sufficient to perturb the normal function of the pathway or complex such that additional perturbations do not reduce activity, and hence growth, further. Overall, systematically identifying and analyzing GI networks can provide unbiased, global maps of the functional relationships between large numbers of genes, from which pathway-level information missed by other approaches can be inferred9.

Mapping Bacterial Functional Networks and Pathways in Escherichia Coli using Synthetic Genetic Arrays

Systematic, large-scale synthetic genetic (gene-gene or epistasis) interaction screens can be used to explore genetic redundancy and pathway cross-talk. Here, we describe a high-throughput quantitative synthetic genetic array screening technology, termed eSGA that we developed for elucidating epistatic relationships and exploring genetic interaction networks in Escherichia coli.

Bakteriyel Fonksiyonel Ağlar ve tanımında Haritalama<em&gt; Escherichia Coli</em&gt; Sentetik Genetik Diziler kullanarak

Phenotypes  are determined by a complex series of physical (e.g. protein-protein) and  functional (e.g. gene-gene or genetic) interactions (GI)1. While ...

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the identification of the subunit composition of multi-protein complexes can provide insight into gene function and enhance understanding of biological systems1, 2. Physical interactions can be mapped with high confidence vialarge-scale isolation and characterization of endogenous protein complexes under near-physiological conditions based on affinity purification of chromosomally-tagged proteins in combination with mass spectrometry (APMS). This approach has been successfully applied in evolutionarily diverse organisms, including yeast, flies, worms, mammalian cells, and bacteria1-6. In particular, we have generated a carboxy-terminal Sequential Peptide Affinity (SPA) dual tagging system for affinity-purifying native protein complexes from cultured gram-negative Escherichia coli, using genetically-tractable host laboratory strains that are well-suited for genome-wide investigations of the fundamental biology and conserved processes of prokaryotes1, 2, 7. Our SPA-tagging system is analogous to the tandem affinity purification method developed originally for yeast8, 9, and consists of a calmodulin binding peptide (CBP) followed by the cleavage site for the highly specific tobacco etch virus (TEV) protease and three copies of the FLAG epitope (3X FLAG), allowing for two consecutive rounds of affinity enrichment. After cassette amplification, sequence-specific linear PCR products encoding the SPA-tag and a selectable marker are integrated and expressed in frame as carboxy-terminal fusions in a DY330 background that is induced to transiently express a highly efficient heterologous bacteriophage lambda recombination system10. Subsequent dual-step purification using calmodulin and anti-FLAG affinity beads enables the highly selective and efficient recovery of even low abundance protein complexes from large-scale cultures. Tandem mass spectrometry is then used to identify the stably co-purifying proteins with high sensitivity (low nanogram detection limits).
Here, we describe detailed step-by-step procedures we commonly use for systematic protein tagging, purification and mass spectrometry-based analysis of soluble protein complexes from E. coli, which can be scaled up and potentially tailored to other bacterial species, including certain opportunistic pathogens that are amenable to recombineering. The resulting physical interactions can often reveal interesting unexpected components and connections suggesting novel mechanistic links. Integration of the PPI data with alternate molecular association data such as genetic (gene-gene) interactions and genomic-context (GC) predictions can facilitate elucidation of the global molecular organization of multi-protein complexes within biological pathways. The networks generated for E. coli can be used to gain insight into the functional architecture of orthologous gene products in other microbes for which functional annotations are currently lacking.

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Affinity purification of tagged proteins in combination with mass spectrometry (APMS) is a powerful method for the systematic mapping of protein interaction networks and for investigating the mechanistic basis of biological processes. Here, we describe an optimized sequential peptide affinity (SPA) APMS procedure developed for the bacterium Escherichia coli that can be used to isolate and characterize stable multi-protein complexes to near homogeneity even starting from low copy numbers per cell.

Protein Kompleksi belirlenmesi<em&gt; Escherichia coli</em&gt; Tandem Kütle Spektrometresi ile Kombine Sıralı Peptid Affinity Arıtma kullanarak

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the ...

Rapid Protocol for Preparation of Electrocompetent Escherichia coli and Vibrio cholerae

Electroporation has become a widely used method for rapidly and efficiently introducing foreign DNA into a wide range of cells. Electrotransformation has become the method of choice for introducing DNA into prokaryotes that are not naturally competent. Electroporation is a rapid, efficient, and streamlined transformation method that, in addition to purified DNA and competent bacteria, requires commercially available gene pulse controller and cuvettes. In contrast to the pulsing step, preparation of electrocompetent cells is time consuming and labor intensive involving repeated rounds of centrifugation and washes in decreasing volumes of sterile, cold water, or non-ionic buffers of large volumes of cultures grown to mid-logarithmic phase of growth. Time and effort can be saved by purchasing electrocompetent cells from commercial sources, but the selection is limited to commonly employed E. coli laboratory strains. We are hereby disseminating a rapid and efficient method for preparing electrocompetent E. coli, which has been in use by bacteriology laboratories for some time, can be adapted to V. cholerae and other prokaryotes. While we cannot ascertain whom to credit for developing the original technique, we are hereby making it available to the scientific community.

Rapid Protocol for Preparation of Electrocompetent Escherichia coli and Vibrio cholerae

Electroporation is a commonly employed method for introducing DNA into bacteria in a process known as transformation. Traditional protocols for the preparation of electrocompetent cells are time consuming and labor intensive. This article describes an alternate, rapid, and efficient method for the preparation of electrocompetent cells presently employed by some laboratories.

Elektrokompetan Hazırlanması için hızlı Protokolü<em&gt; Escherichia coli</em&gt; Ve<em&gt; Vibrio cholerae</em

Electroporation has become a widely used method for rapidly and efficiently introducing foreign DNA into a wide range of cells. Electrotransformation has ...

Detection of Live Escherichia coli O157:H7 Cells by PMA-qPCR

A unique open reading frame (ORF) Z3276 was identified as a specific genetic marker for E. coli O157:H7. A qPCR assay was developed for detection of E. coli O157:H7 by targeting ORF Z3276. With this assay, we can detect as low as a few copies of the genome of DNA of E. coli O157:H7. The sensitivity and specificity of the assay were confirmed by intensive validation tests with a large number of E. coli O157:H7 strains (n = 369) and non-O157 strains (n = 112). Furthermore, we have combined propidium monoazide (PMA) procedure with the newly developed qPCR protocol for selective detection of live cells from dead cells. Amplification of DNA from PMA-treated dead cells was almost completely inhibited in contrast to virtually unaffected amplification of DNA from PMA-treated live cells. Additionally, the protocol has been modified and adapted to a 96-well plate format for an easy and consistent handling of a large number of samples. This method is expected to have an impact on accurate microbiological and epidemiological monitoring of food safety and environmental source.

Detection of Live Escherichia coli O157:H7 Cells by PMA-qPCR

A qPCR assay was developed for detection of Escherichia coli O157:H7 targeting a unique genetic marker, Z3276. The qPCR was combined with propidium monoazide (PMA) treatment for live cell detection. This protocol has been modified and adapted to a 96-well plate format for easy and consistent handling of numerous samples

Live Algılama<em&gt; Escherichia coli</em&gt; O157: PMA-qPCR H7 Hücreler

A unique open reading frame (ORF) Z3276 was identified as a specific genetic marker for E. coli O157:H7. A qPCR assay was developed for detection of E. ...

Non-chromatographic Purification of Recombinant Elastin-like Polypeptides and their Fusions with Peptides and Proteins from Escherichia coli

Elastin-like polypeptides are repetitive biopolymers that exhibit a lower critical solution temperature phase transition behavior, existing as soluble unimers below a characteristic transition temperature and aggregating into micron-scale coacervates above their transition temperature. The design of elastin-like polypeptides at the genetic level permits precise control of their sequence and length, which dictates their thermal properties. Elastin-like polypeptides are used in a variety of applications including biosensing, tissue engineering, and drug delivery, where the transition temperature and biopolymer architecture of the ELP can be tuned for the specific application of interest. Furthermore, the lower critical solution temperature phase transition behavior of elastin-like polypeptides allows their purification by their thermal response, such that their selective coacervation and resolubilization allows the removal of both soluble and insoluble contaminants following expression in Escherichia coli. This approach can be used for the purification of elastin-like polypeptides alone or as a purification tool for peptide or protein fusions where recombinant peptides or proteins genetically appended to elastin-like polypeptide tags can be purified without chromatography. This protocol describes the purification of elastin-like polypeptides and their peptide or protein fusions and discusses basic characterization techniques to assess the thermal behavior of pure elastin-like polypeptide products.

Non-chromatographic Purification of Recombinant Elastin-like Polypeptides and their Fusions with Peptides and Proteins from Escherichia coli

Elastin-like polypeptides are stimulus-responsive biopolymers with applications ranging from recombinant protein purification to drug delivery. This protocol describes the purification and characterization of elastin-like polypeptides and their peptide or protein fusions from Escherichia coli using their lower critical solution temperature phase transition behavior as a simple alternative to chromatography.

Peptidler ve proteinler ile rekombinant Elastin gibi Polipeptitlerin ve Fusions olmayan kromatografik saflaştırma<em&gt; Escherichia coli</em

Elastin-like polypeptides are repetitive biopolymers that exhibit a lower critical solution temperature phase transition behavior, existing as soluble ...

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and the inherent instability of many membrane proteins once solubilized in detergents. A protocol is described that combines ligation independent cloning of membrane proteins as GFP fusions with expression in Escherichia coli detected by GFP fluorescence. This enables the construction and expression screening of multiple membrane protein/variants to identify candidates suitable for further investment of time and effort. The GFP reporter is used in a primary screen of expression by visualizing GFP fluorescence following SDS polyacrylamide gel electrophoresis (SDS-PAGE). Membrane proteins that show both a high expression level with minimum degradation as indicated by the absence of free GFP, are selected for a secondary screen. These constructs are scaled and a total membrane fraction prepared and solubilized in four different detergents. Following ultracentrifugation to remove detergent-insoluble material, lysates are analyzed by fluorescence detection size exclusion chromatography (FSEC). Monitoring the size exclusion profile by GFP fluorescence provides information about the mono-dispersity and integrity of the membrane proteins in different detergents. Protein: detergent combinations that elute with a symmetrical peak with little or no free GFP and minimum aggregation are candidates for subsequent purification. Using the above methodology, the heterologous expression in E. coli of SED (shape, elongation, division, and sporulation) proteins from 47 different species of bacteria was analyzed. These proteins typically have ten transmembrane domains and are essential for cell division. The results show that the production of the SEDs orthologues in E. coli was highly variable with respect to the expression levels and integrity of the GFP fusion proteins. The experiment identified a subset for further investigation.

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

A streamlined approach to screening for the expression of recombinant membrane proteins in Escherichia coli based on fusion to green fluorescent protein is presented.

Membran proteinlerinin Yeşil floresan proteini temelli Ekspresyon elemesinde<em&gt; Escherichia coli</em

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and ...

Gap Junctional Intercellular Communication: A Functional Biomarker to Assess Adverse Effects of Toxicants and Toxins, and Health Benefits of Natural Products

This protocol describes a scalpel loading-fluorescent dye transfer (SL-DT) technique that measures intercellular communication through gap junction channels, which is a major intercellular process by which tissue homeostasis is maintained. Interruption of gap junctional intercellular communication (GJIC) by toxicants, toxins, drugs, etc. has been linked to numerous adverse health effects. Many genetic-based human diseases have been linked to mutations in gap junction genes. The SL-DT technique is a simple functional assay for the simultaneous assessment of GJIC in a large population of cells. The assay involves pre-loading cells with a fluorescent dye by briefly perturbing the cell membrane with a scalpel blade through a population of cells. The fluorescent dye is then allowed to traverse through gap junction channels to neighboring cells for a designated time. The assay is then terminated by the addition of formalin to the cells. The spread of the fluorescent dye through a population of cells is assessed with an epifluorescence microscope and the images are analyzed with any number of morphometric software packages that are available, including free software packages found on the public domain. This assay has also been adapted for in vivo studies using tissue slices from various organs from treated animals. Overall, the SL-DT assay can serve a broad range of in vitro pharmacological and toxicological needs, and can be potentially adapted for high throughput set-up systems with automated fluorescence microscopy imaging and analysis to elucidate more samples in a shorter time.

This protocol describes a scalpel loading-fluorescent dye transfer technique that measures intercellular communication through gap junction channels. Gap junctional intercellular communication is a major cellular process by which tissue homeostasis is maintained and disruption of this cell signaling has adverse health effects.

Gap Junctional Hücreler arası iletişim: Doğal Ürünler Toksikantların ve Toksinler Yan Etkileri değerlendirmek için Fonksiyonel Biyomarker ve Sağlık Faydaları

This protocol describes a scalpel loading-fluorescent dye transfer (SL-DT) technique that measures intercellular communication through gap junction ...