Name: high-throughput parallel sequencing to measure fitness of leptospira interrogans transposon insertion mutants during golden syrian hamster infection
Uploaded: 2017-12-18T12:30:00
Description: Watch this Scientific Journal Video about High-throughput Parallel Sequencing to Measure Fitness of Leptospira interrogans Transposon Insertion Mutants During Golden Syrian Hamster Infection at JoVE.c

Эта работа была поддержана заслуги дел ветеранов (в D.A.H.) и национального института здравоохранения предоставлять R01 Ай 034431 (D.A.H.).

Предупреждение: Патогенные штаммы Leptospira spp. должны быть обработаны согласно процедурам включения 2-го уровня биобезопасности (BSL-2). Необходимо носить соответствующие средства индивидуальной защиты (СИЗ). Класс II биобезопасности кабинета должны использоваться для всех манипуляций ...

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W., Camilli, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+fitness+and+genetic+interactions+determined+by+Tn-seq,+a+high-throughput+massively+parallel+sequencing+method+for+microorganisms.">Genome-wide fitness and genetic interactions determined by Tn-seq, a high-throughput massively parallel sequencing method for microorganisms.</a> Curr Protoc Microbiol. 36, 1E.3.1-1E.3.24 (2015).</li><li>Troy, E. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Understanding+barriers+to+Borrelia+burgdorferi+dissemination+during+infection+using+massively+parallel+sequencing.">Understanding barriers to Borrelia burgdorferi dissemination during infection using massively parallel sequencing.</a> Infect Immun. 81 (7), 2347-2357 (2013).</li><li>Wunder, E. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+PCR+reveals+rapid+dissemination+of+Leptospira+interrogans+after+intraperitoneal+and+conjunctival+inoculation+of+hamsters.">Real-time PCR reveals rapid dissemination of Leptospira interrogans after intraperitoneal and conjunctival inoculation of hamsters.</a> Infect Immun. 84 (7), 2105-2115 (2016).</li><li>Lourdault, K., Aviat, F., Picardeau, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+quantitative+real-time+PCR+for+studying+the+dissemination+of+Leptospira+interrogans+in+the+guinea+pig+infection+model+of+leptospirosis.">Use of quantitative real-time PCR for studying the dissemination of Leptospira interrogans in the guinea pig infection model of leptospirosis.</a> J Med Microbiol. 58, 648-655 (2009).</li><li>Coutinho, M. L., 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=Kinetics+of+Leptospira+interrogans+infection+in+hamsters+after+intradermal+and+subcutaneous+challenge.">Kinetics of Leptospira interrogans infection in hamsters after intradermal and subcutaneous challenge.</a> PLoS Negl Trop Dis. 8 (11), e3307 (2014).</li><li>Athanazio, D. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rattus+norvegicus+as+a+model+for+persistent+renal+colonization+by+pathogenic+Leptospira+interrogans.">Rattus norvegicus as a model for persistent renal colonization by pathogenic Leptospira interrogans.</a> Acta Trop. 105 (2), 176-180 (2008).</li><li>Ratet, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+imaging+of+bioluminescent+Leptospira+interrogans+in+mice+reveals+renal+colonization+as+a+stealth+escape+from+the+blood+defenses+and+antibiotics.">Live imaging of bioluminescent Leptospira interrogans in mice reveals renal colonization as a stealth escape from the blood defenses and antibiotics.</a> PLoS Negl Trop Dis. 8 (12), e3359 (2014).</li><li>Gutierrez, M. G., Yoder-Himes, D. R., Warawa, J. 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Хотя результаты от нашего эксперимента для хомяка, внутрибрюшинно оспорены с 42 л interrogans мутантов представлены17, мы ожидаем, что большие бассейны мутантов может проверяться Tn-далее Потому что низкая частота трансконъюгантов (100-200 трансконъюгантов/спаривания), нескольк...

Идентификация генов вирулентности для некоторых бактерий, таких как Leptospira spp., затруднено из-за ограниченного числа генетических инструментов, доступных. Один из часто используемых подходов является создание коллекции мутантов, случайных transposon мутагенеза, следуют идентификации включение сайта в каждом мутант и вирулентности тестирование отдельных transposon мутантов в животной модели. Этот подход является длительным, дорогостоящим и требует большое количество животных.
При случайных мутагенеза была впервые разработана для патогена Лептоспире interrogans, генов, участвующих в вирулентности были определены путем тестирования отдельных мутантов в животной модели1. Мутанты были отобраны на основе таких критериев, как их потенциальной роли в сигнализации подвижности или их предсказал внешней мембраны или поверхности местоположение. Как и большинство leptospiral гены кодируют гипотетических белки неизвестной функции2, выбрав мутантов основанный на эти критерии ограничения возможность обнаружения генов вирулентности Роман leptospiral.
Совсем недавно бассейны л interrogans transposon мутантов были экранированы для инфективности в хомяк и мыши модели3. Каждое животное было оспорено с бассейном до 10 мутантов. Инфективности мутант был забит как положительным, если он был обнаружен методом ПЦР культур, полученных из крови и почек. PCR тестирования был трудоемким, потому что он требует индивидуальной реакции PCR для каждого мутант в бассейне. Потому что частота каждого мутант в культурах количественно не оценивалась, подход был предвзято к идентификации весьма ослабленный мутантов.
Мы описываем transposon виртуализации (Tn-Seq) технику, как стратегия более эффективно экран для генов вирулентности. TN-seq состоит из создания библиотеки мутантов transposon мутагенеза, следуют массивной параллельной последовательности4,5,6. Кратко transposon мутантов пуле, привитых животных и впоследствии оправился от различных органов (вывода бассейны). ДНК из пулов вывода извлечены и переваривается с энзимами ограничения или стриженый sonication. Два раунда ПЦР ориентации развязок transposon вставки сайтов выполняются. Этот шаг позволяет адаптеров, необходимые для последовательности. Результате продукты PCR анализируются высокопроизводительного секвенирования для выявления transposon вставки сайт каждого мутант бассейна наряду с их относительного изобилия, который сравнивается с первоначальный состав пула мутант.
Основным преимуществом этого подхода является возможность одновременно экран большое количество мутантов с небольшое количество животных. TN-Seq не требует предварительных знаний о transposon вставки сайты, что увеличивает шансы выявления новых Leptospira-специфических генов, участвующих в вирулентности с меньше времени и повышения эффективности. Поскольку бремя leptospiral в тканях является относительно высоким в грызун модели восприимчивы к смертельной инфекции (обычно 104 108 бактерий/g ткани)7,8,9 , также как и хозяева водохранилище 10,11, тканей могут быть проанализированы непосредственно, без необходимости культуры, снижения отклонений вследствие роста в пробирке .
В большинство бактериальных патогенов, описанные на сегодняшний день исследований, Tn-Seq высокая частота инсерционному мутагенезу допускается инфекции с большими бассейнами, содержащие мутантов вместе с нескольких близко расположенных transposon вставок в каждый ген4 ,12,,1314. TN-Seq была также разработана для бактерий, для которого мутагенеза частота является намного ниже6. С Leptospiraбиблиотека transposon мутантов могут создаваться путем введения transposon на мобилизационный плазмиды путем конъюгации, как описано в Slamti et al15. Однако частота transposon мутагенеза л interrogans является низким. Когда Himar1 transposon была введена на сопряженных плазмида, частота transconjugant сообщается только 8,5 x 10-8 для каждого получателя ячейки с Лай штамм L. interrogans16 и, вероятно, будет Аналогичным образом бедных с большинства других штаммов L. interrogans. В части, на основе, которая разработана для Borrelia burgdorferi, в котором частота transposon инсерционному мутагенезу является также низкая6протокол, описанных здесь.
Для нашего эксперимента с протоколом17мы провели transposon мутагенеза с л interrogans серовар штамм Manilae L495 из-за успеха других групп в Изолирующие вставки мутантов transposon в штамм наряду с его низкой LD50 (летальная доза) для вирулентности1. Мы экранированный 42 мутантов, Tn-Seq и определили несколько мутантов кандидатов дефектных в вирулентности, включая два с вставками в гене Аденилатциклаза кандидата. Индивидуальные испытания двух мутантов в хомяков подтвердил, что они являются несовершенными в вирулентности17.

<table>
	<tbody>
		<tr>
			<td>Kanamycin sulfate from Streptomyces kanamyceticus</td>
			<td>Sigma-Aldrich</td>
			<td>K4000</td>
			<td></td>
		</tr>
		<tr>
			<td>2,6-diaminopimelic acid</td>
			<td>Sigma-Aldrich</td>
			<td>D1377</td>
			<td></td>
		</tr>
		<tr>
			<td>Spectinomycin dihydrochloride pentahydrate</td>
			<td>Sigma-Aldrich</td>
			<td>S4014</td>
			<td></td>
		</tr>
		<tr>
			<td>Axio Lab A1 microscope with a darkfieldcondenser</td>
			<td>Zeiss</td>
			<td>490950-001-000</td>
			<td></td>
		</tr>
		<tr>
			<td>DNeasy blood and tissue kit</td>
			<td>Qiagen</td>
			<td>69504/69506</td>
			<td></td>
		</tr>
		<tr>
			<td>MinElute PCR Purification</td>
			<td>Qiagen</td>
			<td>28004/28006</td>
			<td></td>
		</tr>
		<tr>
			<td>QIAquick PCR purification kit</td>
			<td>Qiagen</td>
			<td>28104/28106</td>
			<td></td>
		</tr>
		<tr>
			<td>Model 505 Sonic Dismembrator</td>
			<td>Fisher Scientific</td>
			<td>FB-505</td>
			<td></td>
		</tr>
		<tr>
			<td>2.5&#34; Cup horn</td>
			<td>Fisher Scientific</td>
			<td>FB-4625</td>
			<td></td>
		</tr>
		<tr>
			<td>Bead Ruptor 24</td>
			<td>Omni International</td>
			<td>19-010</td>
			<td>Step 3.1.2.4</td>
		</tr>
		<tr>
			<td>Terminal deoxynucleotidyl transferase</td>
			<td>Promega</td>
			<td>M828C</td>
			<td></td>
		</tr>
		<tr>
			<td>Master mix Phusion</td>
			<td>Thermo Scientific</td>
			<td>F531</td>
			<td>Preparation of genomic libraries, step 3.4.</td>
		</tr>
		<tr>
			<td>DreamTaq Master Mix</td>
			<td>Thermo Scientific</td>
			<td>K9011/K9012</td>
			<td>Identification of the transposon insertion site, step 1.2.</td>
		</tr>
		<tr>
			<td>dCTP</td>
			<td>Thermo Scientific</td>
			<td>R0151</td>
			<td></td>
		</tr>
		<tr>
			<td>ddCTP</td>
			<td>Affymetrix/ USBProducts</td>
			<td>77112</td>
			<td></td>
		</tr>
		<tr>
			<td>T100 Thermal cycler</td>
			<td>BioRad</td>
			<td>1861096</td>
			<td></td>
		</tr>
		<tr>
			<td>Qubit 2.0 fluorometer</td>
			<td>Invitrogen</td>
			<td>Q32866</td>
			<td>step 3.6.</td>
		</tr>
		<tr>
			<td>Qubit dsDNA HS assay kit</td>
			<td>Invitrogen</td>
			<td>Q32851/Q32854</td>
			<td>step 3.6.</td>
		</tr>
		<tr>
			<td>Qubit assay tubes</td>
			<td>life technologies</td>
			<td>Q32856</td>
			<td>step 3.6.</td>
		</tr>
		<tr>
			<td>PBS pH 7.2 (1X)</td>
			<td>Gibco</td>
			<td>20012-027 20012-050</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable scalpel No10</td>
			<td>Feather</td>
			<td>2975#10</td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic K2 EDTA 2 ml tubes</td>
			<td>BD vacutainer</td>
			<td>367841</td>
			<td></td>
		</tr>
		<tr>
			<td>syringe U-100 with 26G x &#189;&#8221; needle</td>
			<td>BD vacutainer</td>
			<td>329652</td>
			<td>IP challenge, step 2.2.1.</td>
		</tr>
		<tr>
			<td>3 mL Luer-Lok tip syringe</td>
			<td>BD vacutainer</td>
			<td>309657</td>
			<td>Cardiac puncture, step 2.4.2.</td>
		</tr>
		<tr>
			<td>25G X 5/8&#8221; needle</td>
			<td>BD vacutainer</td>
			<td>305901</td>
			<td>Cardiac puncture, step 2.4.2.</td>
		</tr>
		<tr>
			<td>25 mm fritted glass base with stopper</td>
			<td>EMD Millipore</td>
			<td>XX1002502</td>
			<td>Filtration unit system, step 1.1.7.</td>
		</tr>
		<tr>
			<td>25 mm aluminum spring clamp</td>
			<td>EMD Millipore</td>
			<td>XX1002503</td>
			<td>Filtration unit system, step 1.1.7.</td>
		</tr>
		<tr>
			<td>15 ml borosilcate glass funnel</td>
			<td>EMD Millipore</td>
			<td>XX1002514</td>
			<td>Filtration unit system, step 1.1.7.</td>
		</tr>
		<tr>
			<td>125 ml side-arm Erlenmeyer flask</td>
			<td>EMD Millipore</td>
			<td>XX1002505</td>
			<td>Filtration unit system, step 1.1.7.</td>
		</tr>
		<tr>
			<td>Acetate-cellulose filter VVPP (pore size 0.1 mm; diameter 25 mm)</td>
			<td>EMD Millipore</td>
			<td>VVLP02500</td>
			<td></td>
		</tr>
	</tbody>
</table>

high-throughput parallel sequencing to measure fitness of leptospira interrogans transposon insertion mutants during golden syrian hamster infection

В этой рукописи мы описываем transposon виртуализации (Tn-Seq) техника для идентификации и количественной оценки Лептоспире interrogans мутантов, изменены в фитнес во время инфекции Золотой сирийских хомяков. TN-Seq сочетает в себе случайных transposon мутагенеза с силой высокопроизводительного секвенирования технологии. Животных сталкиваются с бассейном transposon мутантов (ввода бассейн), следуют заготовки крови и тканей через несколько дней позже определить и подсчитать количество мутантов в каждом органе (вывода бассейны). Бассейны вывода по сравнению с ввода бассейна оценить фитнес в естественных условиях каждого мутант. Этот подход позволяет скрининг большого пула мутантов в ограниченное количество животных. С незначительными изменениями этот протокол может выполняться с любого животного модель лептоспироза, водохранилище принимающей таких крыс и острой инфекции модели таких хомячков, а также в vitro исследования. TN-Seq обеспечивает мощный инструмент для экрана для мутантов с дефектами фитнес в vivo и in vitro .

Создание библиотеки transposon мутантов в л interrogans путем конъюгации требует фильтрующего блока, как показано на рисунке 1. 100-200 трансконъюгантов мы оправились от каждого спаривания.
Transposon вставки сайт обозначаются в каж...

Watch this Scientific Journal Video about High-throughput Parallel Sequencing to Measure Fitness of Leptospira interrogans Transposon Insertion Mutants During Golden Syrian Hamster Infection at JoVE.c

High-throughput Parallel Sequencing to Measure Fitness of Leptospira interrogans Transposon Insertion Mutants During Golden Syrian Hamster Infection

Здесь мы опишем технику, которая сочетает в себе transposon мутагенеза с высокой пропускной способностью последовательности для идентификации и количественной оценки transposon leptospiral мутантов в тканях после вызова хомяков. Этот протокол может использоваться для экрана мутантов для выживания и распространения в животных и могут также применяться к в vitro исследования.

Высок объём параллельного секвенирования для измерения фитнес Лептоспире interrogans Transposon вставки мутантов во время Золотой сирийского хомяка инфекции

In this manuscript, we describe a transposon sequencing (Tn-Seq) technique to identify and quantify Leptospira interrogans mutants altered in fitness ...

The overall goal of this high-throughput transposon sequencing technique is to identify and quantify transposon mutants with in vivo and in vitro fitness defects, and thereby, identify Leptospira genes involved in virulence. This method can help answer key questions in the Leptospira field, such as identifying genes involved in colonization, dissemination, and survival of the bacteria in the host. The main advantage of this technique is that a large number of mutants can be screened with a limited number of animals.Though this method was designed to examine the fitness of Leptospira interrogans mutants in the hamster model with acute leptospirosis, it can also be applied through a chronic infection models, in vitro studies, and other strains of Leptospira. Demonstrating the procedure will be Kritel Lourdault and Karen Evangelista, both post-docs from my laboratory. First, assemble a filtration unit.Place the base on a 125 milliliter side arm Erlenmeyer flask. Position an acetate cellulose filter at the base and clamp a funnel onto the base. Connect the filtration unit to a vacuum system.Next, add five milliliters of Leptospira culture and 0.5 milliliters of E.coli culture into the funnel. Turn on the vacuum to filter the solution. Then place the filter with bacteria-side up onto an EMJH plate, supplemented with DAP.Incubate the plate at 30 degrees Celsius over night. The next day, transfer the filter into a 15 milliliter tube containing one milliliter of EMJH, and vortex the tube for 10 seconds to release the bacteria in the suspension. Then spread the suspension onto five EMJH plates, supplemented with kanamycin using glass beads or a spreader.Wrap the plates in parafilm and incubate them upside down at 30 degrees Celsius until colonies are visible. When colonies are visible, collect and transfer them individually into three milliliters of EMJH containing kanamycin. Then incubate the tubes at 30 degrees Celsius with 150 rpm agitation for seven to 10 days, or to a density of 100 million cells per milliliter.To identify the transposon insertion site, perform nested PCRs. First, prepare the mix for the first PCR. Then, add the samples from culture to the PCR mixes.After running the first reaction, prepare the mix for the second PCR. Add two microliters of PCR product from the first PCR to the complete the reaction mixes. After running the PCR, examine the products on an agarose gel.Proceed by sequencing the products to determine the transposon insertion site. To begin the animal experiment, first grow individual mutants in EMJH, supplemented with kanamycin. Then determine the culture densities using a dark field microscope.Dilute each cultured mutant strain in EMJH to the same density. Then, pool an equal volume of all the mutants to make the input pool. Now challenge the animals as described in the text protocol.The number of animals required depends on the size of the input pool. Be sure to consult a biostatistician to determine the number of animals to inoculate. Allow the infection to develop for four days, then harvest blood and tissues as described in the text protocol.To extract the DNA from the blood sample, use a commercial extraction kit. For tissue, dice 50 to 80 milligrams of tissue into one millimeter cubes. Then, transfer the diced tissues to a screw cap tube and weigh them on a precision balance.Next, add 500 microliters of sterile PBS into the tube. Homogenize it using a disrupter for one minute. Then use a volume corresponding to 25 milligrams of tissue to extract the DNA.Use a commercial kit according to the manufacturer's instructions. Dilute the DNA in 100 microliters of elution buffer. Next, transfer 50 microliters of extracted DNA into a 1.5 milliliter microcentrifuge tube.Then shear the DNA. Place the tube into a sonicator cup horn filled with water at four degrees Celsius. Run the sonicator for three minutes while wearing ear protection.Next, load 2.5 microliters of the sheared DNA into a 2%agarose gel to confirm that most of the DNA fragments are under 600 base pairs. Next, prepare the reaction mix to add cytosine tails to the DNA fragments. Incubate the reaction for an hour at 37 degrees Celsius.Then, inactivate the enzyme by increasing the temperature to 75 degrees Celsius for 20 minutes. When the reaction is done, clean it up using a PCR purification kit and elute the DNA with 12 microliters of elution buffer. Run two PCRs to create the genomic libraries.For the first PCR, prepare mixes with TnkN3 and olj376 primers. Primer TnkN3 is specific to the transposon and primer olj376 is specific to the C-tail. For each sample, combine 22 microliters of mix with three microliters of C-tailed purified DNA.Then, run the PCR as described in the text protocol. For the second PCR, use one microliter of the first PCR product as a template. For primers, use pMargent2 to target the transposon and use the IP primers.The IP primers each have a six base pair barcode and specific sequences recognizable by the next generation sequencing platform. Next run out three microliters of the second PCR products on a 2%agarose gel. The library should show a smear with the majority of the signal between 200 to 600 base pairs, and no amplification should be observable in the control reaction.Next, clean the genomic libraries with a PCR purification kit and elute the DNA with 30 microliters of elution buffer. Then measure the DNA concentration with a fluorometer. Now, calculate the volume of each library needed for 15 to 20 nanograms of DNA.Mix the libraries and determine the DNA molar concentration in the pool, using a publicly available calculator. Now send the pooled genomic libraries out for high-throughput sequencing. Later, process the FASTQ files obtained from sequencing using open source software and analyze the results.Eight hamsters were challenged with one milliliter of pooled transposon mutants and the infection was allowed to develop for four days. After extracting the DNA collected from these hamsters'livers, it was sheared and characterized on an agarose gel. Most of the fragments were between 200 and 600 base pairs.After adding C-tail to the sheared DNA, PCRs were used to prepare genomic libraries. Electrophoresis was performed to confirm the size of the PCR products. The size of most of the PCR products was between 200 and 600 base pairs.After processing the sequencing reads, the output/input ratios were determined using the transposon sequencing approach. As expected, mutants known to be virulent, such as flaA1 and IlgB do not show any change in fitness. In contrast, mutants known to be attenuated, such as loa22 show a decrease in fitness.Mutants with a reduced in vivo fitness level were identified, such as lic12327. Follow up experimentation showed the virulence of these mutants to be attenuated in hamsters. After watching this video, you should have a good understanding of how to generate genomic DNA libraries in order to quantify and identify mutants with altered in vivo fitness.After mutants with in vivo fitness defects are identified, they can be tested individually in hamsters to determine if their virulence is attenuated. Don't forget that Leptospira interrogans can be lethal and must be handled using BSL2 containment. Personal protective equipment including gloves and a lab coat must be worn while handling the bacteria.

Research

JoVE Journal

Immunology and Infection

High-throughput Parallel Sequencing to Measure Fitness of Leptospira interrogans Transposon Insertion Mutants During Golden Syrian Hamster Infection

High-throughput Detection Method for Influenza Virus

Высокая пропускная способность Метод обнаружения для вируса гриппа

Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, ...

Determining Soil-transmitted Helminth Infection Status and Physical Fitness of School-aged Children

Определение передаваемые через почву гельминтами статус-инфекции и физической подготовленности детей школьного возраста

Soil-transmitted helminth (STH) infections are common. Indeed, more than 1 billion people are affected, mainly in the developing world where poverty ...

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Генерация и нескольких фенотипическая Скрининг высокого содержания<em&gt; Coxiella burnetii</em&gt; транспозонов мутанты

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence ...

High-throughput Gene Tagging in Trypanosoma brucei

High-throughput Gene Tagging in Trypanosoma brucei

Высокая пропускная способность гена Tagging в<em&gt; Trypanosoma brucei</em

Improvements in mass spectrometry, sequencing and bioinformatics have generated large datasets of potentially interesting genes. Tagging these proteins ...

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

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

Создание плотного Transposon вставки библиотеки с использованием бактериальной сопряжения в энтеробактерий штаммов таких как Кишечная палочка или шигеллам Флекснера

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 ...

Replication of the Ordered, Nonredundant Library of Pseudomonas aeruginosa strain PA14 Transposon Insertion Mutants

Replication of the Ordered, Nonredundant Library of Pseudomonas aeruginosa strain PA14 Transposon Insertion Mutants

Репликация Ordered, неизбыточной библиотека штамма PA14 Transposon вставки мутантов синегнойной палочки

Pseudomonas aeruginosa is a phenotypically and genotypically diverse and adaptable Gram-negative bacterium ubiquitous in human environments. P. aeruginosa ...

High-throughput Measurement of Plasma Membrane Resealing Efficiency in Mammalian Cells

Высок объём измерение плазматической мембраны запечатывания эффективность в клетках млекопитающих

In their physiological environment, mammalian cells are often subjected to mechanical and biochemical stresses that result in plasma membrane damage. In ...

A High-throughput Assay to Assess and Quantify Neutrophil Extracellular Trap Formation

Высок объём Assay оценить и количественно нейтрофилов внеклеточного ловушку формирования

Neutrophil extracellular traps (NETs) are immunogenic extracellular DNA structures that can be released by neutrophils upon a wide variety of triggers. ...

A High-throughput Shigella-specific Bactericidal Assay

A High-throughput Shigella-specific Bactericidal Assay

Высок объём шигеллезом-конкретные бактерицидной Assay

Serum bactericidal assays (SBAs) measure the functional activity of antibodies and have been used for many decades. SBAs directly measure antibody killing ...

Constructing Mutants in Serotype 1 Streptococcus pneumoniae strain 519/43

Constructing Mutants in Serotype 1 Streptococcus pneumoniae strain 519/43

Конструирование мутантов серотипа 1 Streptococcus pneumoniae штамма 519/43

Streptococcus pneumoniae serotype 1 remains a huge problem in low-and-middle income countries, particularly in sub-Saharan Africa. Despite its importance, ...