Name: construction of synthetic phage displayed fab library with tailored diversity
Uploaded: 2018-05-01T14:30:00
Description: Watch this Scientific Journal Video about Construction of Synthetic Phage Displayed Fab Library with Tailored Diversity at JoVE.com

Forfatterne værdsætter Dr. Frederic Fellouse fra Sidhu lab for kritiske kommentarer på Kunkels metode baseret syntetisk Fab phage bibliotek konstruktion. Forfatterne forstår Mrs Alevtina Pavlenco og andre medlemmer fra Sidhu lab for værdifuld hjælp til at forberede højeffektiv electro-kompetente E. coli celler og høj kvalitet dU-ssDNA. Dette arbejde blev støttet af National Natural Science Foundation of China (Grant No.: 81572698, 31771006) til DW og ved ShanghaiTech Universitet (Grant No.: F-0301-13-005) til laboratoriet af antistof Engineering.

Bemærk: Filteret sterile tips skal bruges i hele når der beskæftiger sig med phage for at undgå kontaminering pipette pistol og omegn. Aseptisk område eller hætte skal bruges når håndtering med bakterier og phage eksperimenter. Phage eksperiment område skal renses op ved hjælp af 2% sodium dodecyl sulfat (SDS) efterfulgt af 70% ethanol til phage kontaminering. For at gøre serielle fortyndinger i denne protokol, skal nye tips bruges til hver fortynding.
1. E. Coli SS320 Electr...

<ol>
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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anatomy+of+the+antibody+molecule.">Anatomy of the antibody molecule.</a> Mol Immunol. 31 (3), 169-217 (1994).</li><li>Virnekas, 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=Trinucleotide+phosphoramidites:+ideal+reagents+for+the+synthesis+of+mixed+oligonucleotides+for+random+mutagenesis.">Trinucleotide phosphoramidites: ideal reagents for the synthesis of mixed oligonucleotides for random mutagenesis.</a> Nucleic Acids Res. 22 (25), 5600-5607 (1994).</li><li>Fellouse, F. A., Sidhu, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Making and Using Antibodies: A Practical Handbook. , 157-180 (2006).</li><li>Fantini, 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=Assessment+of+antibody+library+diversity+through+next+generation+sequencing+and+technical+error+compensation.">Assessment of antibody library diversity through next generation sequencing and technical error compensation.</a> PLoS One. 12 (5), e0177574 (2017).</li><li>Glanville, J., 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=Deep+sequencing+in+library+selection+projects:+what+insight+does+it+bring?.">Deep sequencing in library selection projects: what insight does it bring?.</a> Curr Opin Struct Biol. 33, 146-160 (2015).</li><li>Perelson, A. S., Oster, G. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Theoretical+studies+of+clonal+selection:+minimal+antibody+repertoire+size+and+reliability+of+self-non-self+discrimination.">Theoretical studies of clonal selection: minimal antibody repertoire size and reliability of self-non-self discrimination.</a> J Theor Biol. 81 (4), 645-670 (1979).</li><li>Miersch, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scalable+high+throughput+selection+from+phage-displayed+synthetic+antibody+libraries.">Scalable high throughput selection from phage-displayed synthetic antibody libraries.</a> J Vis Exp. 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For at konstruere høj diversitet, phage-vises Fab biblioteker, kvalitetskontrol check point er nødvendige for at overvåge forskellige stadier af byggeprocessen, herunder kompetenceudvikling af electro-kompetente celler, kvaliteten af dU-ssDNA skabelon, effektiviteten af CCC-dsDNA syntese, titer efter elektroporation, Fab foldning og aminosyre mangfoldighed af CdR af Sekvensanalyse af Fab-phage kloner.
Højt udbytte og renheden af dU-ssDNA er afgørende for høj mutagenese sats. Vores erfari...

mAbs har bredt programmer lige fra grundforskning til sygdom diagnostik og terapi. I 2016, er mere end 60 mAbs blevet godkendt af USA 's Food and Drug Administration (USFDA) til klinisk behandling af autoimmune sygdomme, cancer og infektionssygdomme1,2.
I 1975 rapporterede Kohler og Milstein en teknik til kontinuerlig produktion af antistoffer af en enkelt klonede specificitet fra en cellulær kilde omtales som 'hybridomer' og denne teknik er efterfølgende blevet en hjørnesten i medicin og industri3 ,4. Generation af mAbs ved denne metode kræver forskellige foranstaltninger herunder antigen produktion, mus immunisering, udvinding af B-lymfocytter, fusion af B-celler med myelomatose celler til at danne udødelige hybridom celler, klon udvalg, og til terapeutisk anvendelse, menneskeliggørelse er forpligtet til at undgå menneskelig anti-mus antistof (HAMA)4,5. Men for denne teknologi, antigener herunder toksiner, patogener og meget velbevarede proteiner er relativt ineffektive i udløser en i vivo immunrespons for mAb produktion5.
I 1978 rapporterede Hutchison et al. brugen af en oligonukleotid til direkte mutagenese af rester i en enkelt-strenget bacteriophage virus6. I 1985 rapporterede Smith, at udenlandske gen fragmenter kan være smeltet i rammen med det gen, der koder phage frakke protein III og kan således vises på phage overflade uden akkord dens smitteevne7. Disse pionérarbejde lagt et fundament for den efterfølgende opførelse af phage-vises antistof biblioteker i immun, naiv, og syntetisk danner med formater af single-kæde variable fragment (scFv) og antigen-bindende fragment (Fab) for terapeutisk mAb udvikling8,9. Fra et teknisk synspunkt phage display-baserede antistof udvikling tilbyder en supplerende tilgang til hybridom-baserede mAb udvikling, der kan bidrage til at omgå de begrænsninger, nogle antigener kan udgøre og menneskeliggørelse proces, der hybridom-afledte antistoffer kræver ofte5. I 2016, 6 phage display-afledte mAbs er blevet godkendt på markedet herunder Humira, en af de mest succesfulde mAbs anvendes til behandling af reumatoid arthritis, og mange phage display-afledte antistof kandidater er i øjeblikket på forskellige stadier af kliniske undersøgelsen10.
For immunforsvaret og naive phage antistof biblioteker, mangfoldighed af komplementaritet-bestemmende områder (CdR) i lette og tunge kæde er afledt fra naturlige immun repertoire (dvs.fra B-celler). Derimod er mangfoldighed af CDR i syntetisk phage antistof biblioteker helt kunstig. Syntetisk tilgange til biblioteket konstruktion giver præcis kontrol over design af sekvens mangfoldighed og muligheder for Mekanistiske undersøgelser af antistof struktur og funktion11,12. Desuden kan rammer for syntetisk biblioteker optimeres før bibliotek opbygning at lette nedstrøms, omfattende industriel udvikling11,12.
I 1985, Kunkel rapporterede en enkeltstrenget DNA (ssDNA) skabelon-baseret mutagenese tilgang at indføre site-directed mutationer i M13 bacteriophage effektivt13. Denne tilgang blev efterfølgende anvendes bredt til opførelse af phage-vises biblioteker. Kemisk syntetiseret DNA oligonukleotider designet til at indføre mangfoldighed i Fab CdR er indarbejdet i et phagemid med et antistof rygraden skabelon. I denne proces, phagemid er udtrykt som en uracil-holdige ssDNA (dU-ssDNA) og oligonukleotider udglødet på en CDR og udvidet til at syntetisere dobbelt-strenget DNA (dsDNA) T7 DNA polymerase og T4 DNA ligase. Endelig kan genererede ds-DNA føres ind Escherichia coli af elektroporation.
Høj mangfoldighed, phage-vises bibliotek opbygning, høj spænding elektroporation af en to-komponent blanding af electro-kompetente celler og kovalent bør lukkede cirkulære dsDNA (CCC-dsDNA) forberedes omhyggeligt. Sidhu et al. ændrede forberedelse af electro-kompetente celler og DNA fra traditionelle metoder og stærkt forbedret bibliotek mangfoldighed14.
I denne protokol beskriver vi en metode til konstruktion af syntetiske phage-vises Fab biblioteker med EDD 109-1010 fås med en enkelt elektroporation. Figur 1 viser en oversigt over biblioteket konstruktion herunder: 1) højeffektiv electro-kompetente celle forberedelse; 2) udvinding af dU-ssDNA; 3) Kunkel metode baseret oligonukleotid-directed mutagenese; 4) elektroporation og beregning af bibliotek størrelse; 5) protein A/L-baserede ELISA for foldning og funktionelle mangfoldighed evaluering; og 6) DNA Sekvensanalyse af forskellighed. Alle reagenser, stammer og udstyr er angivet i materialets tabel. Tabel 1 viser opsætningen reagens.

<table>
	<tbody>
		<tr>
			<td>Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1.0 M H3PO4</td>
			<td>Fisher</td>
			<td>AC29570</td>
			<td></td>
		</tr>
		<tr>
			<td>1.0 M Tris, pH 8.0</td>
			<td>Invitrogen</td>
			<td>15568-025</td>
			<td></td>
		</tr>
		<tr>
			<td>10 mM ATP</td>
			<td>Invitrogen</td>
			<td>18330-019</td>
			<td></td>
		</tr>
		<tr>
			<td>100 mM dithiothreitol</td>
			<td>Fisher</td>
			<td>BP172</td>
			<td></td>
		</tr>
		<tr>
			<td>100 mM dNTP mix</td>
			<td>GE Healthcare</td>
			<td>28-4065-60</td>
			<td>solution containing 25 mM each of dATP, dCTP, dGTP and dTTP.</td>
		</tr>
		<tr>
			<td>3,3&#8217;,5,5&#8217;-tetramethylbenzidine (TMB)</td>
			<td>Kirkegaard &#38; Perry Laboratories Inc</td>
			<td>50-76-02</td>
			<td></td>
		</tr>
		<tr>
			<td>50X TAE</td>
			<td>Invitrogen</td>
			<td>24710030</td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Fisher</td>
			<td>BP160</td>
			<td></td>
		</tr>
		<tr>
			<td>Carbenicillin, carb</td>
			<td>Sigma</td>
			<td>C1389</td>
			<td>100 mg/mL in water,&#160; 0.22 &#956;m filter-sterilize, work concentration: 100 &#956;g/mL.</td>
		</tr>
		<tr>
			<td>Chloramphenicol, cmp</td>
			<td>Sigma</td>
			<td>C0378</td>
			<td>100 mg/mL in ethanol, 0.22 &#956;m filter-sterilize, work concentration: 10 &#956;g/mL.</td>
		</tr>
		<tr>
			<td>EDTA 0.5 M, pH 8.0</td>
			<td>Invitrogen</td>
			<td>AM9620G</td>
			<td></td>
		</tr>
		<tr>
			<td>Granulated agar</td>
			<td>VWR</td>
			<td>J637-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>H2O2 peroxidase substrate</td>
			<td>Kirkegaard &#38; Perry Laboratories Inc</td>
			<td>50-65-02</td>
			<td></td>
		</tr>
		<tr>
			<td>K2HPO4</td>
			<td>Sigma</td>
			<td>795488</td>
			<td></td>
		</tr>
		<tr>
			<td>Kanamycin, kan</td>
			<td>Fisher</td>
			<td>AC61129</td>
			<td>50 mg/mL in water,&#160; 0.22 &#956;m filter-sterilize, work concentration: 50 &#956;g/mL.</td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>Sigma</td>
			<td>P2222</td>
			<td></td>
		</tr>
		<tr>
			<td>Na2HPO4</td>
			<td>Sigma</td>
			<td>94046</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Alfa Aesar</td>
			<td>U19C015</td>
			<td></td>
		</tr>
		<tr>
			<td>Nanodrop</td>
			<td>Fisher</td>
			<td>ND2000C</td>
			<td></td>
		</tr>
		<tr>
			<td>NaOH</td>
			<td>Fisher</td>
			<td>SS256</td>
			<td>! CAUTION NaOH causes burns.</td>
		</tr>
		<tr>
			<td>NON-Fat Powdered Milk</td>
			<td>Sangon Biotech</td>
			<td>A600669</td>
			<td></td>
		</tr>
		<tr>
			<td>PEG-8000</td>
			<td>Fisher</td>
			<td>BP233</td>
			<td></td>
		</tr>
		<tr>
			<td>Protein A-HRP conjugate</td>
			<td>Invitrogen</td>
			<td>101123</td>
			<td></td>
		</tr>
		<tr>
			<td>QIAprep Spin M13 Kit</td>
			<td>Qiagen</td>
			<td>22704</td>
			<td></td>
		</tr>
		<tr>
			<td>QIAquick Gel Extraction Kit</td>
			<td>Qiagen</td>
			<td>28706</td>
			<td></td>
		</tr>
		<tr>
			<td>QIAquick PCR Purification Kit</td>
			<td>Qiagen</td>
			<td>28104</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Protein L</td>
			<td>Fisher</td>
			<td>77679</td>
			<td></td>
		</tr>
		<tr>
			<td>T4 DNA polymerase</td>
			<td>New England Biolabs</td>
			<td>M0203S</td>
			<td></td>
		</tr>
		<tr>
			<td>T4 polynucleotide kinase</td>
			<td>New England Biolabs</td>
			<td>M0201S</td>
			<td></td>
		</tr>
		<tr>
			<td>T7 DNA polymerase</td>
			<td>New England Biolabs</td>
			<td>M0274S</td>
			<td></td>
		</tr>
		<tr>
			<td>Tetracycline, tet</td>
			<td>Sigma</td>
			<td>T7660</td>
			<td>50 mg/mL in water, 0. 22 &#956;m filter-sterilize, work concentration: 10 &#956;g/mL.</td>
		</tr>
		<tr>
			<td>Tryptone</td>
			<td>Fisher</td>
			<td>0123-07-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>Sigma</td>
			<td>P2287</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrapure glycerol</td>
			<td>Invitrogen</td>
			<td>15514-011</td>
			<td></td>
		</tr>
		<tr>
			<td>Uridine</td>
			<td>Sigma</td>
			<td>U3750</td>
			<td>25 mg/mL in ethanol, work concentration: 0.25 &#956;g/mL.</td>
		</tr>
		<tr>
			<td>Yeast extract</td>
			<td>VWR</td>
			<td>DF0127-08</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company </td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Strains</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>E.coli CJ236</td>
			<td>New England Biolabs</td>
			<td>E4141</td>
			<td>Genotype: dut-&#160; ung-&#160; thi-1 relA1 spoT1 mcrA/pCJ105(F&#39; camr). Used for preparation of dU-ssDNA.</td>
		</tr>
		<tr>
			<td>E.coli SS320</td>
			<td>Lucigen</td>
			<td>60512</td>
			<td>Genotype: [F&#39;proAB+lacIq lacZ&#916;M15 Tn10 (tetr)] hsdR mcrB araD139 &#916;(araABC-leu)7679 &#916;lacX74 galUgalK rpsL thi. Optimized for high-efficiency electroporation and filamentous bacteriophage production.</td>
		</tr>
		<tr>
			<td>M13KO7</td>
			<td>New England Biolabs</td>
			<td>N0315S</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company </td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>0.2-cm gap electroporation cuvette</td>
			<td>BTX</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>96-well 2mL Deep-well plates</td>
			<td>Fisher</td>
			<td>278743</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well Maxisorp immunoplates</td>
			<td>Nunc</td>
			<td>151759</td>
			<td></td>
		</tr>
		<tr>
			<td>Baffled flasks</td>
			<td>Corning</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Benchtop centrifuge</td>
			<td>Eppendorf</td>
			<td>5811000096</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge bottles</td>
			<td>Nalgene</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ECM-630 electroporator</td>
			<td>BTX</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Magnetic stir bars</td>
			<td>Nalgene</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Thermo Fisher centrifuge</td>
			<td>Fisher</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>High speed shaker</td>
			<td>TAITEK</td>
			<td>MBR-034P</td>
			<td></td>
		</tr>
		<tr>
			<td>Microplate shaker</td>
			<td>QILINBEIER</td>
			<td>QB-9002</td>
			<td></td>
		</tr>
		<tr>
			<td>Liquid handler for 96 and 384 wells</td>
			<td>RAININ</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Mutil-channel pipette</td>
			<td>RAININ</td>
			<td>E4XLS</td>
			<td></td>
		</tr>
		<tr>
			<td>Amicon concentrator</td>
			<td>Merck</td>
			<td>UFC803096</td>
			<td></td>
		</tr>
	</tbody>
</table>

construction of synthetic phage displayed fab library with tailored diversity

Behovet for monoklonale antistoffer (papagøjesyge) i grundforskning og medicin stiger årligt. Hybridom teknologi har været den dominerende metode til mAb udvikling siden sin første rapport i 1975. Som en alternativ teknologi er phage display metoder for mAb udvikling i stigende grad attraktivt da Humira, den første phage-afledte antistof og en af de bedst sælgende mAbs, blev godkendt for klinisk behandling af reumatoid arthritis i 2002. Som en ikke-animalsk baseret mAb udvikling teknologi, omgår phage display antigen immunogenicitet, menneskeliggørelse og animalske vedligeholdelse, der kræves fra traditionelle hybridom teknologi baseret antistof udvikling. I denne protokol beskriver vi en metode til konstruktion af syntetiske phage-vises Fab biblioteker med EDD 109-1010 fås med en enkelt elektroporation. Denne protokol består af: 1) højeffektiv electro-kompetente celle forberedelse; 2) udvinding af uracil-holdige enkeltstrenget DNA (dU-ssDNA); 3) Kunkel metode baseret oligonukleotid-directed mutagenese; 4) elektroporation og beregning af bibliotek størrelse; 5) protein A/L-baserede enzym-forbundet immunosorbent assay (ELISA) for foldning og funktionelle mangfoldighed evaluering; og 6) DNA Sekvensanalyse af forskellighed.

Efter flowdiagram af Fab bibliotek konstruktion (Se figur 1), vi forberedte M13KO7 helper phage pre-inficeret E. coli SS320 electro-kompetente celler. Effektiviteten af disse electro-kompetente celler er anslået 2 X 109 cfu/µg når Fab phagemid rygraden for bibliotek opbygning blev brugt (figur 4).
Uracil indarbejdelse effektivitet blev ved sammenl...

Watch this Scientific Journal Video about Construction of Synthetic Phage Displayed Fab Library with Tailored Diversity at JoVE.com

Construction of Synthetic Phage Displayed Fab Library with Tailored Diversity

Denne protokol beskriver en detaljeret procedure for opførelsen af en phage-vises syntetisk antistof bibliotek med skræddersyede mangfoldighed. Syntetisk antistoffer har bredt programmer fra grundforskning til sygdom diagnostik og terapi.

Opførelsen af syntetiske Phage vises Fab bibliotek med skræddersyede mangfoldighed

Demand for monoclonal antibodies (mAbs) in basic research and medicine is increasing yearly. Hybridoma technology has been the dominant method for mAb ...

Research

JoVE Journal

Immunology and Infection

'Bioluminescent' Reporter Phage for the Detection of Category A Bacterial Pathogens

Yersinia pestis and Bacillus anthracis are Category A bacterial pathogens that are the causative agents of the plague and anthrax, respectively 1. ...

Live Cell Imaging of Bacillus subtilis and Streptococcus pneumoniae using Automated Time-lapse Microscopy

Live Cell Imaging of Bacillus subtilis and Streptococcus pneumoniae using Automated Time-lapse Microscopy

During the last few years scientists became increasingly aware that average data obtained from microbial population based experiments are not ...

Separation of Single-stranded DNA, Double-stranded DNA and RNA from an Environmental Viral Community Using Hydroxyapatite Chromatography

Viruses, particularly bacteriophages (phages), are the most numerous biological entities on Earth1,2.  Viruses modulate host cell abundance and diversity, ...

Following Cell-fate in E. coli After Infection by Phage Lambda

Following Cell-fate in E. coli After Infection by Phage Lambda

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the ...

Imaging of HIV-1 Envelope-induced Virological Synapse and Signaling on Synthetic Lipid Bilayers

Scanning af HIV-1 Envelope-induceret Virologisk Synapse og signaler på syntetiske lipiddobbeltlagenes

Human immunodeficiency virus type 1 (HIV-1) infection occurs most efficiently via cell to cell transmission2,10,11. This cell to cell transfer between ...

Scalable High Throughput Selection From  Phage-displayed Synthetic Antibody Libraries

Skalerbar High Throughput Selection Fra fagpræsenterede Syntetiske antistofbiblioteker

The demand for antibodies that fulfill the needs of both basic and clinical research applications is high and will dramatically increase in the future. ...

Phage Phenomics: Physiological Approaches to Characterize Novel Viral Proteins

Fag Phenomics: Fysiologiske tilgange til Karakterisere Novel virale proteiner

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

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Analyse af lymfocyt Ekstravasation Ved anvendelse af en<em&gt; In vitro</em&gt; Model af humant blod-hjernebarrieren

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte ...

Overlapping Peptide Library to Map Qa-1 Epitopes in a Protein

Overlappende peptid Bibliotek kortlægge Qa-1 epitoper i et Protein

Qa-1 (HLA-E in human) belongs to a group of non-classical major histocompatibility complex 1b (MHC-Ib) molecules. Recent data suggest that Qa-1 molecules ...

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

Replikering af bestilt, Nonredundant bibliotek af Pseudomonas aeruginosa stamme PA14 Transposon indsættelse mutanter

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