Name: a non-coding small rna micc contributes to virulence in outer membrane proteins in salmonella enteritidis
Uploaded: 2021-01-27T14:00:00
Description: Watch this Scientific Journal Video about A Non-Coding Small RNA MicC Contributes to Virulence in Outer Membrane Proteins in Salmonella Enteritidis at JoVE.com

Denne undersøgelse blev støttet af tilskud fra Chinese National Science Foundation (nr. 31972651 og 31101826), Jiangsu High Education Science Foundation (nr. 14KJB230002), State Key Laboratory of Veterinary Biotechnology (nr. SKLVBF201509), Nature Science Foundation Grant of Yangzhou (nr. YZ2014019), et projekt finansieret af det prioriterede akademiske program udvikling af Jiangsu Higher Education Institutions (PAPD).

Alle forsøgene blev udført i overensstemmelse med Det Nationale Forskningsråds vejledning for pleje og brug af forsøgsdyr. Dyrepleje- og brugsudvalget ved Yangzhou University godkendte alle forsøg og procedurer, der blev anvendt på dyrene (SYXK2016-0020).
1. Bakteriestammer, plasmider og kulturbetingelser
<ol><li>Brug de bakterier og plasmider, der er anført i tabel 1.</li>
	<li>Kulturbakterier i LB-bouillon eller på LB-agarplader ved 37 °C under tilstedeværelse af...

<ol>
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H., Romby, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+RNAs+in+bacteria+and+archaea:+who+they+are,+what+they+do,+and+how+they+do+it.">Small RNAs in bacteria and archaea: who they are, what they do, and how they do it.</a> Advances In Genetics. 90, 133-208 (2015).</li><li>Vogel, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rough+guide+to+the+non-coding+RNA+world+of+Salmonella.">A rough guide to the non-coding RNA world of Salmonella.</a> Molecular Microbiology. 71 (1), 1-11 (2009).</li><li>Dutta, T., Srivastava, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+RNA-mediated+regulation+in+bacteria:+A+growing+palette+of+diverse+mechanisms.">Small RNA-mediated regulation in bacteria: A growing palette of diverse mechanisms.</a> Gene. 656, 60-72 (2018).</li><li>Waters, L. S., Storz, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulatory+RNAs+in+bacteria.">Regulatory RNAs in bacteria.</a> Cell. 136 (4), 615-628 (2009).</li><li>Chen, S., Zhang, A., Blyn, L. B., Storz, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicC,+a+second+small-RNA+regulator+of+Omp+protein+expression+in+Escherichia+coli.">MicC, a second small-RNA regulator of Omp protein expression in Escherichia coli.</a> Journal of Bacteriology. 186 (20), 6689-6697 (2004).</li><li>Pfeiffer, V., Papenfort, K., Lucchini, S., Hinton, J. C., Vogel, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coding+sequence+targeting+by+MicC+RNA+reveals+bacterial+mRNA+silencing+downstream+of+translational+initiation.">Coding sequence targeting by MicC RNA reveals bacterial mRNA silencing downstream of translational initiation.</a> Nature Structural &amp; Molecular Biology. 16 (8), 840-846 (2009).</li><li>Dam, S., Pag&#232;s, J. M., Masi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dual+Regulation+of+the+Small+RNA+MicC+and+the+Quiescent+Porin+OmpN+in+Response+to+Antibiotic+Stress+in+Escherichia+coli.">Dual Regulation of the Small RNA MicC and the Quiescent Porin OmpN in Response to Antibiotic Stress in Escherichia coli.</a> Antibiotics (Basel). 6 (4), 33 (2017).</li><li>Hao, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Porin+Deficiency+in+Carbapenem-Resistant+Enterobacter+aerogenes+Strains.">Porin Deficiency in Carbapenem-Resistant Enterobacter aerogenes Strains.</a> Microbial Drug Resistance. 24 (9), 1277-1283 (2018).</li><li>Wroblewska, Z., Olejniczak, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hfq+assists+small+RNAs+in+binding+to+the+coding+sequence+of+ompD+mRNA+and+in+rearranging+its+structure.">Hfq assists small RNAs in binding to the coding sequence of ompD mRNA and in rearranging its structure.</a> RNA. 22 (7), 979-994 (2016).</li><li>Santiviago, C. 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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=Analysis+of+relative+gene+expression+data+using+real-time+quantitative+PCR+and+the+2(-Delta+Delta+C(T))+Method.">Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method.</a> Methods. 25 (4), 402-408 (2001).</li><li>Vander Velden, A. W., B&#228;umler, A. J., Tsolis, R. M., Heffron, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+fimbrial+adhesins+are+required+for+full+virulence+of+Salmonella+typhimurium+in+mice.">Multiple fimbrial adhesins are required for full virulence of Salmonella typhimurium in mice.</a> Infection and Immunity. 66 (6), 2803-2808 (1998).</li><li>Prescott, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Salmonella+enterica+serovar+enteritidis+in+humans+and+animals:+Epidemiology,+pathogenesis,+and+control.">Salmonella enterica serovar enteritidis in humans and animals: Epidemiology, pathogenesis, and control.</a> Canadian Veterinary Journal La Revue Veterinaire Canadienne. 40 (10), 736 (1999).</li><li>Balasubramanian, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+global+burden+and+epidemiology+of+invasive+non-typhoidal.">The global burden and epidemiology of invasive non-typhoidal.</a> Hum Vaccin Immunother. 15 (6), 1421-1426 (2019).</li><li>De Buck, J., Van Immerseel, F., Haesebrouck, F., Ducatelle, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Colonization+of+the+chicken+reproductive+tract+and+egg+contamination+by+Salmonella.">Colonization of the chicken reproductive tract and egg contamination by Salmonella.</a> Journal of General and Applied Microbiology. 97 (2), 233-245 (2004).</li><li>Padalon-Brauch, 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=Small+RNAs+encoded+within+genetic+islands+of+Salmonella+typhimurium+show+host-induced+expression+and+role+in+virulence.">Small RNAs encoded within genetic islands of Salmonella typhimurium show host-induced expression and role in virulence.</a> Nucleic Acids Research. 36 (6), 1913-1927 (2008).</li><li>Santiviago, C. 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=Analysis+of+pools+of+targeted+Salmonella+deletion+mutants+identifies+novel+genes+affecting+fitness+during+competitive+infection+in+mice.">Analysis of pools of targeted Salmonella deletion mutants identifies novel genes affecting fitness during competitive infection in mice.</a> PLoS Pathogens. 5 (7), 1000477 (2009).</li><li>Gong, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Salmonella+small+non-coding+RNA+facilitates+bacterial+invasion+and+intracellular+replication+by+modulating+the+expression+of+virulence+factors.">A Salmonella small non-coding RNA facilitates bacterial invasion and intracellular replication by modulating the expression of virulence factors.</a> PLoS Pathogens. 7 (9), 1002120 (2011).</li><li>H&#233;brard, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=sRNAs+and+the+virulence+of+Salmonella+enterica+serovar+Typhimurium.">sRNAs and the virulence of Salmonella enterica serovar Typhimurium.</a> RNA Biology. 9 (4), 437-445 (2012).</li><li>Vogel, J., Papenfort, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+non-coding+RNAs+and+the+bacterial+outer+membrane.">Small non-coding RNAs and the bacterial outer membrane.</a> Current Opinion in Microbiology. 9 (6), 605-611 (2006).</li><li>Papenfort, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SigmaE-dependent+small+RNAs+of+Salmonella+respond+to+membrane+stress+by+accelerating+global+omp+mRNA+decay.">SigmaE-dependent small RNAs of Salmonella respond to membrane stress by accelerating global omp mRNA decay.</a> Molecular Microbiology. 62 (6), 1674-1688 (2006).</li><li>Udekwu, K. I., 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=Hfq-dependent+regulation+of+OmpA+synthesis+is+mediated+by+an+antisense+RNA.">Hfq-dependent regulation of OmpA synthesis is mediated by an antisense RNA.</a> Genes and Development. 19 (19), 2355-2366 (2005).</li><li>Papenfort, K., Vogel, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+target+regulation+by+small+noncoding+RNAs+rewires+gene+expression+at+the+post-transcriptional+level.">Multiple target regulation by small noncoding RNAs rewires gene expression at the post-transcriptional level.</a> Research in Microbiology. 160 (4), 278-287 (2009).</li><li>Lease, R. A., Cusick, M. E., Belfort, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Riboregulation+in+Escherichia+coli:+DsrA+RNA+acts+by+RNA:RNA+interactions+at+multiple+loci.">Riboregulation in Escherichia coli: DsrA RNA acts by RNA:RNA interactions at multiple loci.</a> Proceedings of the National Academy of Sciences of the United States of America. 95 (21), 12456-12461 (1998).</li><li>Sharma, C. M., Darfeuille, F., Plantinga, T. 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S. Enteritidis er et vigtigt fakultativt intracellulært patogen, der kan inficere unge kyllinger og producerer symptomer fra enteritis til systemisk infektion og død17,18. Derudover forårsager S. Enteritidis latente infektioner hos voksne kyllinger, og kroniske bærere forurener fjerkræprodukter, hvilket resulterer i fødevarebårne infektioner hos mennesker19. Den patogene mekanisme af S. Enteritidis skal undersøges...

Ikke-kodende små RNA'er (sRNA'er) er 40-400 nukleotider lange, som generelt ikke koder for proteiner, men kan transkriberes uafhængigt i bakteriekromosomer 1,2,3. De fleste sRNA'er er kodet i de intergene regioner (IGR'er) mellem genkodende regioner og interagerer med mål-mRNA'er gennem baseparringshandlinger og regulerer målgenekspression på posttranskriptionsniveau 4,5. De spiller vigtige reguleringsroller i stofmetabolisme, proteinsyntese i ydre membran, quorum sensing og virulens genekspression5.
MicC er et 109-nukleotid lille RNA-transkript til stede i Escherichia coli og Salmonella enterica serovar Typhimurium, som kunne regulere flere ydre membranproteinekspressioner såsom OmpC, OmpD, OmpN, Omp35 og Omp36 6,7,8,9. MicC regulerer ekspressionen af OmpC ved at hæmme ribosombinding til ompC mRNA-lederen in vitro, og det kræver Hfq RNA-chaperon for dets funktion i Escherichia coli6. I Salmonella Typhimurium tavsgør MicC ompD mRNA via en ≤12-bp RNA duplex inden for kodningssekvensen (kodoner 23-26) og destabiliserer derefter endonukleolytisk mRNA7. Denne reguleringsproces assisteres af chaperonprotein Hfq10. OmpC er et rigeligt ydre membranprotein, der blev anset for at være vigtigt i miljøer, hvor næringsstof- og toksinkoncentrationer var høje, såsom i tarmen6. OmpD porin er det mest rigelige ydre membranprotein i Salmonella Typhimurium og repræsenterer ca. 1% af det samlede celleprotein11. OmpD er involveret i overholdelse af humane makrofager og intestinale epitelceller12. MicC undertrykker også udtrykket af både OmpC og OmpD poriner. Det menes, at MicC kan regulere virulens. For at udforske nye målgener reguleret af MicC og studere virulensreguleringsfunktionen af micC, klonede vi micC-genet i Salmonella Enteritidis (SE) stammen 50336 og konstruerede derefter mutanten 50336ΔmicC og den komplementerede mutant 50336ΔmicC / p micC. Nye målgener blev screenet af qRT-PCR. Virulensen af 50336ΔmicC blev påvist af mus og kyllingeinfektioner.

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	<tbody>
		<tr>
			<td>dextrose</td>
			<td>Sangon Biotech</td>
			<td>A610219</td>
			<td>for broth preparation</td>
		</tr>
		<tr>
			<td>DNA purification kit</td>
			<td>TIANGEN</td>
			<td>DP214</td>
			<td>for DNA purification</td>
		</tr>
		<tr>
			<td>Ex Taq</td>
			<td>TaKaRa</td>
			<td>RR01A</td>
			<td>PCR</td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>Sinopharm Chemical Reagent</td>
			<td>10017608</td>
			<td>for broth preparation</td>
		</tr>
		<tr>
			<td>K2HPO4</td>
			<td>Sinopharm Chemical Reagent</td>
			<td>20032116</td>
			<td>for broth preparation</td>
		</tr>
		<tr>
			<td>L-Arabinose</td>
			<td>Sangon Biotech</td>
			<td>A610071</td>
			<td>&#955;-Red recombination</td>
		</tr>
		<tr>
			<td>Mini Plasmid Kit</td>
			<td>TIANGEN</td>
			<td>DP106</td>
			<td>plasmid extraction</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sinopharm Chemical Reagent</td>
			<td>10019308</td>
			<td>for broth preparation</td>
		</tr>
		<tr>
			<td>(NH4)2SO4</td>
			<td>Sinopharm Chemical Reagent</td>
			<td>10002917</td>
			<td>for broth preparation</td>
		</tr>
		<tr>
			<td>PrimeScriptRRT reagent Kit with gDNA Eraser&#160;</td>
			<td>TaKaRa</td>
			<td>RR047</td>
			<td>qRT-PCR</td>
		</tr>
		<tr>
			<td>SYBRR Premix Ex Taq II</td>
			<td>TaKaRa</td>
			<td>RR820</td>
			<td>qRT-PCR</td>
		</tr>
		<tr>
			<td>T4 DNA Ligase</td>
			<td>NEB</td>
			<td>M0202</td>
			<td>Ligation</td>
		</tr>
		<tr>
			<td>TRIzol&#160;</td>
			<td>Invitrogen</td>
			<td>15596018</td>
			<td>RNA isolation</td>
		</tr>
		<tr>
			<td>Tryptone</td>
			<td>Oxoid</td>
			<td>LP0042</td>
			<td>for broth preparation</td>
		</tr>
		<tr>
			<td>Yeast extract</td>
			<td>Oxoid</td>
			<td>LP0021</td>
			<td>for broth preparation</td>
		</tr>
		<tr>
			<td>centrifuge</td>
			<td>Eppendorf</td>
			<td>5418</td>
			<td>centrifugation</td>
		</tr>
		<tr>
			<td>Electrophoresis apparatus</td>
			<td>Bio-Rad</td>
			<td>164-5050</td>
			<td>Electrophoresis</td>
		</tr>
		<tr>
			<td>&#160;Electroporation System</td>
			<td>Bio-Rad</td>
			<td>165-2100</td>
			<td>for bacterial transformation</td>
		</tr>
		<tr>
			<td>Spectrophotometer</td>
			<td>BioTek</td>
			<td>Epoch</td>
			<td>Absorbance detection</td>
		</tr>
		<tr>
			<td>Real-Time PCR system</td>
			<td>Applied Biosystems</td>
			<td>7500 system</td>
			<td>qRT-PCR</td>
		</tr>
	</tbody>
</table>

a non-coding small rna micc contributes to virulence in outer membrane proteins in salmonella enteritidis

Et ikke-kodende lille RNA (sRNA) er en ny faktor til regulering af genekspression på posttranskriptionelt niveau. En slags sRNA MicC, kendt i Escherichia coli og Salmonella Typhimurium, kunne undertrykke ekspressionen af ydre membranproteiner. For yderligere at undersøge reguleringsfunktionen af micC i Salmonella Enteritidis klonede vi micC-genet i Salmonella Enteritidis-stammen 50336 og konstruerede derefter mutanten 50336Δ micC af λ Red-baseret rekombinationssystem og den komplementerede mutant 50336Δ micC/p micC, der bærer rekombinant plasmid pBR322, der udtrykker micC. qRT-PCR-resultater viste, at transkription af ompD i 50336Δ micC var 1,3 gange højere end i vildtypestammen, mens transkriptionen af ompA og ompC i 50336ΔmicC var 2,2 gange højere end dem i vildtypestammen. Disse indikerede, at micC undertrykker ekspressionen af ompA og ompC. I den følgende undersøgelse blev patogeniciteten af 50336ΔmicC påvist af både inficerende 6 uger gamle Balb / c-mus og 1 dag gamle kyllinger. Resultatet viste, at LD 50 af vildtypestammen 50336, mutanterne 50336Δ micC og 50336Δ micC/pmicC for 6 uger gamle Balb/c mus var henholdsvis 12,59 CFU, 5,01 CFU og 19,95 CFU. LD50 af stammerne til 1 dag gamle kyllinger var henholdsvis 1,13 x 109 CFU, 1,55 x 10 8 CFU og 2,54 x 108 CFU. Det indikerede, at sletning af micC øgede virulensen af S. Enteritidis hos mus og kyllinger ved at regulere ekspression af ydre membranproteiner.

Konstruktion af den mutante 50336Δ micC og suppleret stamme 50336Δ micC /pmicC MicC-genklonresultatet indikerede, at dette gen var sammensat af 109 bp, der viste 100% identitet med S. Typhimurium. Baseret på sekvensdataene blev deletionsmutanten 50336ΔmicC og den komplementerede mutant 50336ΔmicC/pmicC konstrueret med succes....

Watch this Scientific Journal Video about A Non-Coding Small RNA MicC Contributes to Virulence in Outer Membrane Proteins in Salmonella Enteritidis at JoVE.com

A Non-Coding Small RNA MicC Contributes to Virulence in Outer Membrane Proteins in Salmonella Enteritidis

Et λ-rødmedieret rekombinationssystem blev brugt til at skabe en deletionsmutant af en lille ikke-kodende RNA-micC.

En ikke-kodende lille RNA-mikrofon bidrager til virulens i ydre membranproteiner i Salmonella enteritidis

A non-coding small RNA (sRNA) is a new factor to regulate gene expression at the post-transcriptional level. A kind of sRNA MicC, known in Escherichia ...

Denne protokol kan bruges til inaktivering af kromosomale gener for at isolere mutanter til genfunktionsundersøgelse og bakterier. Det gør den homologe rekombinationsproces enkel, hurtig og yderst effektiv. Ved hjælp af denne teknik kan patogenets virulensrelaterede gener slettes for at opnå den svækkede mutant, som kan anvendes som kandidat til den svækkede vaccine.Begynd med at forstærke chloramphenicolkassetten indeholdende hæmologifragmenter af micC-genet. Design fremadgående og omvendte primere for at forstærke chloramphenicolkassetten fra plasmidet PKD3, herunder 50 baseparhomologiudvidelser fra de fem primære og tre primære ender af micC-genet. Udfør PCR som beskrevet i tekstmanuskriptet ved hjælp af PKD3-plasmidet som skabelon til at forstærke chloramphenicolkassetten.Bestem størrelsen af PCR-produktet med agarosegelelektroforese. Rens derefter produktet med et DNA-gelgendannelsessæt og bestem koncentrationen af DNA ved spektrofotometri. Udfør en anden PCR-reaktion for at eliminere interferensen af yderligere rekombination af PKD3-plasmidet.Fortynd det første PCR-produkt i forholdet 1 til 200, og brug det som skabelon til den sekundære PCR. For at konstruere den første rekombinante stamme 50336 delta micC dot cat, bland 100 mikroliter SE 50336 kompetente celler med fem mikroliter PKD 46 plasmid og inkuber på is i 30 minutter. Varmechok blandingen ved 42 grader Celsius i 90 sekunder, og overfør den hurtigt tilbage på is i to minutter for at omdanne plasmidet til cellerne.Screene positive kolonier ved at dyrke cellerne natten over ved 30 grader Celsius på en ampicillinresistent plade. Den næste dag tilsættes 30 milli mere L arabinose til SE 50336 PKD 46 flydende kultur og inducerer rekombinaseekspression med en times inkubation ved 30 grader Celsius under omrystning. Bland 100 nanogram af det rensede PCR-produkt og 40 mikroliter SE 50336 PKD 46 kompetente celler i en elektrisk stødkop.Udfør derefter elektrisk stødtransformation. Efter elektrotransformation overføres blandingen til en milliliter SOC-medium og en rystekulturinkubator ved 150 o / min og 30 grader Celsius i en time. Spred blandingen på en chloramphenicolresistent LB-plade og kultur ved 37 grader Celsius natten over for at screene for positive kolonier.Dyrk de positive kolonier ved 42 grader Celsius i to timer. Derefter screenes kolonien for følsomhed over for ampicillin og resistens over for chloramphenicol ved 37 grader Celsius natten over for at opnå den første rekombinante stamme uden PKD 46. Efter ekstraktion af 50336 Delta micC dot cat genomisk DNA ved hjælp af PCR og gelelektroforese, elektroplade 100 nanogram plasmid PCP 20 i 40 mikroliter 50336 Delta micC dot cat kompetente celler.Skærm positive transformanter på både ampicillin og chloramphenicol resistente plader ved 30 grader Celsius. Overfør de positive transformanter til ikke-resistent LB flydende medium og dyrk dem natten over ved 42 grader Celsius. Isoler derefter enkeltkolonier på en LB-plade ved 37 grader Celsius.Vælg den koloni, der er følsom over for både ampicillin og chloramphenicol. Denne mutant er micC-deletionsmutanten SE 50336 Delta micC. Kontroller 50336 Delta micC ved at udføre PCR som beskrevet i tekstmanuskriptet.Denne protokol blev brugt til at konstruere deletionsmutanten 50336 Delta micC i den komplimenterede mutant 50336 Delta micC, pmicC. Den første rekombinante 50336 Delta micC dot cat blev valideret af PCR med en forventet båndstørrelse på ca. 1200 basepar PCR-produkter med chloramphenicolindsættelse sammenlignet med 279 basepar i vildtypestammen. I den anden rekombination blev chloramphenicolkassetten elimineret af PCP 20.PCR-resultaterne kombineret med sekventering bekræftede, at den isogene micC-mutant blev konstrueret med succes. Ekspressionen af ompA-, ompC- og ompD-gener i stammer 50336, 50336 Delta micC og 50336 delta micC pmicC blev analyseret ved kvantitativ PCR i realtid. Transskription af ompA og ompC og 50336 delta micC steg ca. 2,2 gange og tredoblet sammenlignet med vildtypestammen.Mens transkription af ompD kun steg lidt. LD 50-analyser viste, at efter at have inficeret seks til otte uger gamle BALB / c-mus med hver af de tre stammer, viste de fleste mus inficeret med 50336 Delta micC slaphed, appetitløshed eller diarré 48 timer efter infektion og døde efter 96 timer. Musene inficeret med vildtypestamme og 50336 delta micC pmicC viste de samme symptomer 72 timer efter infektion og døde efter 120 timer.LD 50'erne af de tre stammer indikerede, at virulensen af den mutante 50336 Delta micC forbedrede 2,5 gange sammenlignet med vildtypen. Denne teknik hjælper forskere med at studere funktionen af bakteriegener og opnå den ønskede konstruerede stamme ved at slette målgener.

Research

JoVE Journal

Immunology and Infection

A Non-Coding Small RNA MicC Contributes to Virulence in Outer Membrane Proteins in Salmonella Enteritidis

An Allelotyping PCR for Identifying Salmonella enterica serovars Enteritidis, Hadar, Heidelberg, and Typhimurium

An Allelotyping PCR for Identifying Salmonella enterica serovars Enteritidis, Hadar, Heidelberg, and Typhimurium

Current commercial PCRs tests for identifying Salmonella target genes unique to this genus.  However, there are two species, six subspecies, and over ...

Locked Nucleic Acid Flow Cytometry-fluorescence in situ Hybridization (LNA flow-FISH): a Method for Bacterial Small RNA Detection

Locked Nucleic Acid Flow Cytometry-fluorescence in situ Hybridization LNA flow-FISH: a Method for Bacterial Small RNA Detection

Locked Nucleic Acid Flowcytometri-fluorescens In situ Hybridisering (LNA flow-FISH): En metode til bakteriel små RNA Detection

Fluorescence in situ hybridization (FISH) is a powerful technique that is used to detect and localize specific nucleic acid sequences in the cellular ...

Monitoring Changes in Membrane Polarity, Membrane Integrity, and Intracellular Ion Concentrations in Streptococcus pneumoniae Using Fluorescent Dyes

Monitoring Changes in Membrane Polarity, Membrane Integrity, and Intracellular Ion Concentrations in Streptococcus pneumoniae Using Fluorescent Dyes

Overvåge ændringer i Membrane polaritet, Membrane Integritet, og intracellulære Ion Koncentrationerne i<em&gt; Streptococcus pneumoniae</em&gt; Brug fluorescerende farvestoffer

Membrane depolarization and ion fluxes are events that have been studied extensively in biological systems due to their ability to profoundly impact ...

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

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

Brug fluorescerende proteiner til at visualisere og kvantificere<em&gt; Chlamydia</em&gt; Vakuole Vækst Dynamics i levende celler

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

A Protocol to Infect Caenorhabditis elegans with Salmonella typhimurium

A Protocol to Infect Caenorhabditis elegans with Salmonella typhimurium

En protokol til at inficere<em&gt; Caenorhabditis elegans</em&gt; Med<em&gt; Salmonella typhimurium</em

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

High-throughput Assay to Phenotype Salmonella enterica Typhimurium Association, Invasion, and Replication in Macrophages

High-throughput Assay to Phenotype Salmonella enterica Typhimurium Association, Invasion, and Replication in Macrophages

Højkapacitetsforskning Assay til Fænotype<em&gt; Salmonella enterica</em&gt; Typhimurium Association, Invasion, og replikering i makrofager

Salmonella species are zoonotic pathogens and leading causes of food borne illnesses in humans and livestock1. Understanding the mechanisms underlying ...

Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents

Neutrofil Isolering og analyse for at fastslå deres rolle i lymfom Cell Følsomhed til terapeutiske midler

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of ...

Small RNA Transfection in Primary Human Th17 Cells by Next Generation Electroporation

Lille RNA transfektion i primære humane Th17 Celler af Next Generation Elektroporation

CD4+ T cells can differentiate into several subsets of effector T helper cells depending on the surrounding cytokine milieu. Th17 cells can be generated ...

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

Isolering af Salmonella typhimurium-indeholdende Phagosomes fra makrofager

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. ...

Identification of Coding and Non-coding RNA Classes Expressed in Swine Whole Blood

Identifikation af kodning og ikke-kodende RNA klasser udtrykt i svin fuldblod

The advent of innovative and increasingly powerful next generation sequencing techniques has opened new avenues into the ability to examine the underlying ...