Name: determination of the optimal chromosomal location(s) for a dna element in escherichia coli using a novel transposon-mediated approach
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Yazarlar Novo Nordisk Vakfı, Lundbeck Vakfı ve Danimarka Ulusal Araştırma Vakfı (DNRF120) Merkezi aracılığıyla gelen hibe tarafından bakteriyel stres tepkisi ve sebat (BASP) için finanse edilmiştir.

1. Transposon Kütüphane koleksiyonu Not: kromozom DARS2 odağı mini Tn 10 klonlanmış-transposon, NKBOR (pNKBOR üzerinde) 21 sonuçlanan, temel NKBOR:: DARS2 (pJFM1). pNKBOR online 22 elde edilebilir. pNKBOR çoğaltma 23 için başlatıcı protein π gerektirir bir intihar R6K tabanlı vektörü olduğu. Plazmid pJFM1 bu nedenle pir geni kromozom bir kopyasını içeren bir ...

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Burada kullanılan metodoloji ile ilgili genetik bir öğenin en uygun genomik konumunu zor bir soruyu cevaplamak için state-of--art teknikleri yararlanır. (Transposon tarafından aracılı) genetik öğesi rasgele yerleştirilmesi için incelenen genetik öğesinin konumu en iyi seçmek için birbirlerine karşı rekabet yapılabilir klonlar binlerce hızlı ve kolay toplama sağlar (Yani fittest klon).
Burada, DARS2 mini-Tn10eklenmiş-transposon, NKBOR dayalı. T...

Genetik herhangi bir öğe işlev genom konumunda etkilenebilir. Bakterilerde bu esas olarak müdahalelerden komşu genler, yerel DNA topoloji ve/veya çoğaltma ilişkili gen dozaj transkripsiyon tarafından neden olur. Özellikle, DNA ikileşmesi ve segregasyon süreçleri, en azından bölümünde kontrol edilir, kromozom bölgeleri kodlamayan1ve bu bölgeler düzgün genomik konumu/içeriğine bağlıdır. E.coliiçinde kardeşi için gerekli DIF site örnekler kromozom çözüm2; Kromozom segregasyon3için gereken KOPS serilerini; ve veri, DARS1ve DARS2 bölgeleri, uygun kromozom çoğaltma denetimi (aşağıda; için gerekli 4). biz izin rasgele tehcir, seçim ve genetik verilen herhangi bir öğe, en uygun genetik bağlamında belirlenmesi için örneği burada kodlamayan DARS2 bölgesinin çalışma odasına bir yöntem mevcut.
E. coli, DnaA başlatıcı protein DNA dizisi tek çoğaltma kökenoriCaçılış ve helikaz DnaB5,6,7alımı sorumludur. AAA+ (Yani, çeşitli aktiviteler ile ilişkili ATPases) kime ait DnaA proteinler ve ATP ve ADP ile benzer yüksek bağlayabilirsiniz benzeşim5. DnaAATP düzeyini doruklarına başlatma8nerede DnaAATP oluşturan bir multimer üzerinde oriC DNA çift yönlü açılış9tetikler. Başlatma sonra oriC bağlama hemimethylated için SeqA protein içeren bir mekanizma geçici olarak kullanım dışı haciz nedeniyle yeniden başlatma için yapılır oriC10,11. Haciz sırasında en az iki mekanizma ile DnaAATP düzeyi azalır: DnaA (RIDA)12,13 ve veridüzenleme inactivation-bağımlı DnaAATP hidroliz (DDAH)14 ,15. RIDA ve DDAH DnaAATP dönüştürmeye DnaAADPtanıtmak. Önce yeni bir tur başlatma DnaAADP DnaAATP belirli DnaA yeniden etkinleştirme dizileri (DARS) yeniden aktif: DARS1 ve DARS216,17. Kromozom veri, DARS1ve DARS2 bölgeleri kodlamayan ve hareket DnaAATPmodüle için bir refakatçi benzeri şekilde /DnaAADP şekilde. Çoğaltma, kökeni dışında yer alan bu bölgeler, karmaşık bir DnaA Meclisi etkinleştirmek için de inactivation (veri; 14) ya da harekete geçirmek (DARS1 ve DARS2; 17), DnaA. DARS2 hücrede silme kitle katlama zaman ama sonuçları zaman uyumsuz çoğaltma başlatma15,16,18değiştirmez. Ancak, DARS2-eksik hücrelerin bir fitness var aksi halde isogenic bir wildtype için her iki sürekli büyüme rekabet zengin Orta veya fare bağırsak18kolonizasyon kurulması sırasında karşılaştırıldığında maliyet. Bu eşzamansız/kökenli konsantrasyon bile küçük değişiklikler bakteriyel fitness üzerinde olumsuz bir etkisi gösterir. E. coli, kromozom simetri (Yani, iki neredeyse eşit uzunlukta çoğaltma kol)19korumak için seçici bir basınç vardır. Veri, DARS1ve DARS2 bölgeleri aynı göreli uzaklık var oriC içinde tüm E. coli suşları18, kromozom boyutu büyük değişimler rağmen sıralı.
Burada, kromozom pozisyon(lar) için işlevini en iyi tanımlanması için bir örnek olarak E. coli DARS2 bölge kullanırız. DARS2 NKBOR transposon ve sonuç NKBOR içine eklenen:: daha sonra rastgele MG1655 genom eklenenDARS2 transposon ΔDARS2. Böylece hücre koleksiyonu üretilen, kromozom üzerinde farklı bir yerde her sahip DARS2 yerleştirilir. Nerede koleksiyondaki tüm hücreleri havuza alınmış ve birbirlerine karşı sırasında LB sürekli büyüme tahmini 700 nesiller için yarıştı, vitro rekabet deney, gerçekleştirildi. Rekabet denemenin sonucu izlenen/Southern blot, kolay gen yürüyüş ve bütün-genom sıralama (WGS; kullanarak tespit edildi Şekil 1). Kolay gen yürüyerek çözüldü son nokta klonlar hücre döngüsü parametreleri değerlendirmek için Akış Sitometresi tarafından karakterize. Bir akış sitometrik analizinde çok sayıda hücre için hücre boyutu, DNA içeriği ve inisiyasyon senkronizasyonu ölçülebilir. Akış Sitometresi sırasında bir akış tek hücre sonra aynı anda verilmiş Floresans DNA içeriği, bir ölçü birimi toplamak photomultipliers tarafından sağlanan kayıtlı lekeli DNA heyecanlandırmak için uygun dalga boyu bir ışık huzmesi geçer. hücreleri DNA için lekeli. İleri dağınık ışık hücre kitle20ölçüsüdür.
Burada mevcut vitro rekabet deney kromozom pozisyon ve genomik genetik öğesi bağlamında önemi ile ilgili soruları gidermek için kullanılır. Tarafsız ve kolay yöntemdir.

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			<td height="20" style="width:369px;height:20px;">Autoclaved Mili-Q water</td>
			<td style="width:164px;"></td>
			<td style="width:119px;">None</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="width:369px;height:20px;">Electroporation Cuvettes, 0.1 cm</td>
			<td style="width:164px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">P41050</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="width:369px;height:20px;">Bio-Rad MicroPulser Electroporation System</td>
			<td style="width:164px;">Bio-Rad</td>
			<td style="width:119px;">165-2100</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="width:369px;height:42px;">LB Broth</td>
			<td style="width:164px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">12780029&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="width:369px;height:20px;">LB Agar, powder (Lennox L agar)</td>
			<td style="width:164px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">22700025</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="width:369px;height:20px;">Glycerol</td>
			<td style="width:164px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">17904</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:369px;height:21px;">Fisherbrand&#160;Plastic Petri Dishes</td>
			<td style="width:164px;">Fisher Scientific</td>
			<td style="width:119px;">S33580A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:369px;height:21px;">Falcon 50mL Conical Centrifuge Tubes</td>
			<td style="width:164px;">Fisher Scientific</td>
			<td style="width:119px;">14-432-22</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:369px;height:21px;">Falcon 15mL Conical Centrifuge Tubes</td>
			<td style="width:164px;">Fisher Scientific</td>
			<td style="width:119px;">14-959-53A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:369px;height:21px;">Nunc&#160;CryoTubes</td>
			<td style="width:164px;">Sigma-Aldrich</td>
			<td style="width:119px;">V7634&#160;</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="width:369px;height:42px;">Phusion High-Fidelity DNA Polymerase (2 U/&#181;L)</td>
			<td style="width:164px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">F530S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:369px;height:21px;">dATP, [&#945;-32P]- 3000Ci/mmol 10mCi/ml, 250 &#181;Ci</td>
			<td style="width:164px;">PerkinElmer</td>
			<td style="width:119px;">BLU012H250UC</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="width:369px;height:42px;">DECAprime II DNA Labeling Kit</td>
			<td style="width:164px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">AM1455</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="width:369px;height:20px;">Spectrophotometer&#160; SF/MBV/03.32</td>
			<td style="width:164px;">Pharmacia</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:369px;height:21px;">Hermle Centrifuge&#160;&#160;&#160; SF/MBV/03.46</td>
			<td style="width:164px;">Hermle</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:369px;height:21px;">Ole Dich Centrifuge&#160; SF/MBV/03.29</td>
			<td style="width:164px;">Ole Dich</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:369px;height:21px;">Eppendorftubes 1.5 mL</td>
			<td style="width:164px;">Sigma-Aldrich</td>
			<td style="width:119px;">T9661</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:369px;height:21px;">Eppendorftubes 2.0 mL</td>
			<td style="width:164px;">Sigma-Aldrich</td>
			<td style="width:119px;">T2795</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium Chloride</td>
			<td>Merck</td>
			<td>6404</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:369px;height:21px;">96% Ethanol</td>
			<td style="width:164px;">Sigma-Aldrich</td>
			<td style="width:119px;">16368</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:369px;height:21px;">Trizma HCl</td>
			<td style="width:164px;">Sigma-Aldrich</td>
			<td style="width:119px;">T-3253</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:369px;height:21px;">Phenol Ultra Pure</td>
			<td>BRL</td>
			<td>5509UA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:369px;height:21px;">Chloroform</td>
			<td>Merck</td>
			<td>2445</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:369px;height:21px;">Ribonuclease A type II A</td>
			<td style="width:164px;">Sigma-Aldrich</td>
			<td>R5000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:369px;height:21px;">Sodiumdodecylsulphate (SDS)</td>
			<td>Merck</td>
			<td>13760</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:369px;height:21px;">Lysozyme</td>
			<td style="width:164px;">Sigma-Aldrich</td>
			<td>L 6876</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:369px;height:21px;">Isopropanol</td>
			<td style="width:164px;">Sigma-Aldrich</td>
			<td>405-7</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:369px;height:21px;">0.5M Na-EDTA pH 8.0</td>
			<td>BRL</td>
			<td>5575 UA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:369px;height:21px;">Kanamycin sulfate</td>
			<td style="width:164px;">Sigma-Aldrich</td>
			<td style="width:119px;">10106801001</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="width:369px;height:42px;">PvuI (10 U/&#181;L)</td>
			<td style="width:164px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">ER0621</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="width:369px;height:42px;">UltraPure Agarose</td>
			<td style="width:164px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">16500500</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="width:369px;height:42px;">DNA Gel Loading Dye (6X)</td>
			<td style="width:164px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">R0611</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:369px;height:21px;">Tris-Borate-EDTA buffer</td>
			<td style="width:164px;">Sigma-Aldrich</td>
			<td style="width:119px;">T4415</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:369px;height:21px;">Ethidium bromide</td>
			<td style="width:164px;">Sigma-Aldrich</td>
			<td style="width:119px;">E7637</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:369px;height:21px;">Hydrochloric acid</td>
			<td style="width:164px;">Sigma-Aldrich</td>
			<td style="width:119px;">433160</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:369px;height:21px;">Sodium Hydroxide</td>
			<td style="width:164px;">Sigma-Aldrich</td>
			<td style="width:119px;">71687</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:369px;height:21px;">Whatman&#160;3MM papers</td>
			<td style="width:164px;">Sigma-Aldrich</td>
			<td style="width:119px;">WHA3030931</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:369px;height:21px;">SSC Buffer 20&#215; Concentrate</td>
			<td style="width:164px;">Sigma-Aldrich</td>
			<td style="width:119px;">S6639</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:369px;height:21px;">Amersham Hybond-N+</td>
			<td style="width:164px;">GE Healthcare</td>
			<td style="width:119px;">RPN119B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:369px;height:21px;">Ficoll&#160;400</td>
			<td style="width:164px;">Sigma-Aldrich</td>
			<td style="width:119px;">F8016</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:369px;height:21px;">Polyvinylpyrrolidone</td>
			<td style="width:164px;">Sigma-Aldrich</td>
			<td style="width:119px;">PVP40</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:369px;height:21px;">Bovine Serum Albumin - Fraction V</td>
			<td style="width:164px;">Sigma-Aldrich</td>
			<td style="width:119px;">85040C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:369px;height:21px;">Deoxyribonucleic acid sodium salt from salmon testes</td>
			<td style="width:164px;">Sigma-Aldrich</td>
			<td style="width:119px;">D1626</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:369px;height:21px;">Carestream Kodak &#160;BioMax &#160;light film</td>
			<td style="width:164px;">Sigma-Aldrich</td>
			<td style="width:119px;">Z373494</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:369px;height:21px;">GenElute &#160;Gel Extraction Kit</td>
			<td style="width:164px;">Sigma-Aldrich</td>
			<td style="width:119px;">NA1111&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:369px;height:21px;">GenElute &#160;PCR Clean-Up Kit</td>
			<td style="width:164px;">Sigma-Aldrich</td>
			<td style="width:119px;">NA1020</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:369px;height:21px;">T100&#160; Thermal Cycler</td>
			<td style="width:164px;">Bio-Rad</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:369px;height:21px;">SmartSpec Plus Spectrophotometer</td>
			<td style="width:164px;">Bio-Rad</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:369px;height:21px;">Rifampicin</td>
			<td style="width:164px;">Serva</td>
			<td style="width:119px;">34514.01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:369px;height:21px;">Cephalexin</td>
			<td style="width:164px;">Sigma-Aldrich</td>
			<td style="width:119px;">C4895</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:369px;height:21px;">Mithramycin</td>
			<td style="width:164px;">Serva</td>
			<td>29803.02</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:369px;height:21px;">Magnesium chloride hexahydrate</td>
			<td style="width:164px;">Sigma-Aldrich</td>
			<td style="width:119px;">246964</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:369px;height:21px;">Apogee A10 instrument</td>
			<td style="width:164px;">Apogee</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

determination of the optimal chromosomal location(s) for a dna element in escherichia coli using a novel transposon-mediated approach

Belirli bir DNA öğeyi en iyi kromozom pozisyon(lar)/rasgele ekleme transposon-aracılı fitness seçime göre takip tarafından belirlendi. Bakterilerde genetik bir öğe işlev genetik içeriğine etkisini değerlendirmek zor olabilir. Topolojik etkileri, komşu genler ve/veya çoğaltma ilişkili gen dozaj, transkripsiyon müdahalelerden de dahil olmak üzere çeşitli mekanizmalar belirli bir genetik öğeyi işlevini etkileyebilir. Burada, kromozom Escherichia coli ve basit büyüme rekabet kullanarak en uygun konumları deneme Seç rasgele entegrasyonu, bir DNA öğesi izin veren bir yöntem açıklanmaktadır. Yöntem rasgele ekleme, büyüme avantajı, deneysel ihtiyaçlarına kolayca ayarlanabilir bir yordam tarafından fittest clone(s) bir seçim ile birleştiğinde iyi tarif bir transposon tabanlı sistem yararlanır. Fittest clone(s) doğası karmaşık bir çok klonal nüfusun bütün-genom sıralama veya seçili klonlar hızlı tanımlanması için yürüyüş kolay gen tarafından belirlenebilir. Burada, kodlamayan DNA bölge E. colikromozom çoğaltması başlatma kontrol, DARS2, örnek olarak kullanılmıştır. DARS2 fonksiyonu çoğaltma ilişkili gen dozaj tarafından etkilendiği bilinmektedir; daha yakın DARS2 DNA ikileşmesi, kökeni daha etkin hale gelir alır. DARS2 rasgele bir DARS2kromozom eklenen-zorlanma silinmiş. Bireysel eklemeler içeren sonuç klonlar havuza alınmış ve birbirlerine karşı yüzlerce nesiller için yarıştı. Son olarak, fittest klonlar ile karakterize ve yakın özgün DARS2 konumuna eklenen DARS2 içerecek şekilde bulundu.

Southern blot DARS2 kromozom transposon kütüphanede boyunca rasgele dağıtılan doğrulamak için yapılır (t = 0) ve fittest klonlar zamanla kalıcı. Southern blot ilk transposon havuzundan çıkarılan DNA üzerinde gerçekleştirildi (t = 0) ve rekabet (şekil 3) 700 nesiller dışarı tahmin edilen her 100. Burada, her zaman noktasından toplam hücresel DNA'sı sindirilmiş ile Pvuben restriksiyon enzimi, transposon NKBOR kesmek i?...

Watch this Scientific Journal Video about Determination of the Optimal Chromosomal Locations for a DNA Element in Escherichia coli Using a Novel Transposon-mediated Approach at JoVE.com

Determination of the Optimal Chromosomal Locations for a DNA Element in Escherichia coli Using a Novel Transposon-mediated Approach

Burada, bir transposon-aracılı rasgele ekleme kodlamayan DNA öğesinin gücünü en iyi kromozom konumunu gidermek için kullanıldı.

En iyi kromozom Location(s) belirlenmesi için yeni bir Transposon-aracılı yaklaşımın kullanarak Escherichia coli DNA öğesinde

The optimal chromosomal position(s) of a given DNA element was/were determined by transposon-mediated random insertion followed by fitness selection. In ...

Research

JoVE Journal

Genetics

Determination of the Optimal Chromosomal Location(s) for a DNA Element in Escherichia coli Using a Novel Transposon-mediated Approach

Analysis of the Ambient Particulate Matter-induced Chromosomal Aberrations Using an In Vitro System

Analysis of the Ambient Particulate Matter-induced Chromosomal Aberrations Using an In Vitro System

Bir Kullanarak Ortam Partikül Madde kaynaklı kromozomal anormallikler Analizi<em&gt; İn Vitro</em&gt; Sistem

Exposure to particulate matter (PM) is a major world health concern, which may damage various cellular components, including the nuclear genetic material. ...

Detection of Copy Number Alterations Using Single Cell Sequencing

Tek Hücre sıralama kullanarak Kopya sayısı Değişmeler Algılama

Detection of genomic changes at single cell resolution is important for characterizing genetic heterogeneity and evolution in normal tissues, cancers, and ...

Genome-wide Determination of Mammalian Replication Timing by DNA Content Measurement

DNA İçerik Ölçüm tarafından Memeli Çoğaltma Zamanlama genom Tayini

Replication of the genome occurs during S phase of the cell cycle in a highly regulated process that ensures the fidelity of DNA duplication. Each genomic ...

Robust DNA Isolation and High-throughput Sequencing Library Construction for Herbarium Specimens

Sağlam DNA izolasyon ve yüksek işlem hacmi sıralama kitaplığı inşaat sulak numuneler için

Herbaria are an invaluable source of plant material that can be used in a variety of biological studies. The use of herbarium specimens is associated with ...

A Novel Saturation Mutagenesis Approach: Single Step Characterization of Regulatory Protein Binding Sites in RNA Using Phosphorothioates

Bir roman doygunluk Mutagenesis yaklaşım: Tek adım karakterizasyonu düzenleyici protein RNA Phosphorothioates kullanarak siteleri bağlama

Gene regulation plays an important role in development. Numerous DNA- and RNA-binding proteins bind their target sequences with high specificity to ...

Producing Gene Deletions in Escherichia coli by P1 Transduction with Excisable Antibiotic Resistance Cassettes

Producing Gene Deletions in Escherichia coli by P1 Transduction with Excisable Antibiotic Resistance Cassettes

Escherichia coli Excisable antibiyotik direnci kasetleri ile P1 iletim tarafından üreten gen silme

A first approach to study the function of an unknown gene in bacteria is to create a knock-out of this gene. Here, we describe a robust and fast protocol ...

Using In Vitro and In-cell SHAPE to Investigate Small Molecule Induced Pre-mRNA Structural Changes

İn vitro ve hücre içi şeklinde küçük molekül araştırmak için kullanarak Pre-mRNA yapısal değişiklikler indüklenen

In the process of drug development of RNA-targeting small molecules, elucidating the structural changes upon their interactions with target RNA sequences ...

In vivo Application of the REMOTE-control System for the Manipulation of Endogenous Gene Expression

Uzaktan kumandalı sistem in vivo uygulama için işleme endojen gen ekspresyonu

Here we describe a protocol for implementing the REMOTE-control system (Reversible Manipulation of Transcription at Endogenous loci), which allows for ...

Mass Spectrometry-Based Proteomics Analyses Using the OpenProt Database to Unveil Novel Proteins Translated from Non-Canonical Open Reading Frames

Kütle spektrometresi tabanlı proteomik analizleri roman proteinler açıklayacak OpenProt veritabanı kullanarak kanonik olmayan açık okuma çerçevesi tercüme

Genome annotation is central to today's proteomic research as it draws the outlines of the proteomic landscape. Traditional models of open reading frame ...

Use of Frozen Tissue in the Comet Assay for the Evaluation of DNA Damage

DNA Hasarının Değerlendirilmesinde Kuyruklu Yıldız Testinde Donmuş Dokunun Kullanımı

The comet assay is gaining popularity as a means to assess DNA damage in cultured cells and tissues, particularly following exposure to chemicals or other ...