We would like to thank Lisa Philpotts of the MGH Treadwell Virtual Library for her guidance in the database search. This work was supported by the Cystic Fibrosis Foundation (YONKER16G0 and HURLEY16G0) and NIH NIAID (BPH and ADE: R01 A1095338).

CAUTION: Utilize standard BSL-2 safety measures when handling P. aeruginosa, a human pathogen. If you are an immunocompromised individual or have any medical condition that increases your susceptibility to bacterial infection, take special caution when working with P. aeruginosa. Consult the biosafety office in your institution and obtain approval from your physician before working with the the PA14 NR Set or mutant libraries of bacterial pathogens.
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The P. aeruginosa PA14NR Set is a valuable resource for the scientific community. According to the March 2017 dataset from Clarivate Analytics&#39; Essential Science Indicators database, Liberati et al. (2006)37, which describes the construction of the PA14NR Set, is ranked in the top 1% of microbiology publications. Google Scholar reports over 600 citations of the Liberati et al. (2006) original manuscript as of August 2017. The library has played an important ...

Pseudomonas aeruginosa is a phenotypically and genotypically diverse and adaptable Gram-negative bacterium present in soil, water, and most human environments, as well as skin microflora. Compared to many bacterial species, P. aeruginosa has a relatively large genome of 5.5-7 Mbp with high G+C content (65-67%). Furthermore, a significant proportion of its genes are involved in metabolic adaptability and are part of regulatory networks, allowing for great flexibility in response to environmental stress1. P. aeruginosa expresses a plethora of virulence factors, exhibits proclivity to form biofilms, possesses the ability to coordinate responses through multiple quorum sensing pathways, and displays a notable capacity to develop antibiotic resistance and tolerance2,3,4,5,6,7,8. These attributes present significant challenges for treating infections caused by P. aeruginosa.
Chronic P. aeruginosa infections can occur in numerous disease states. Cystic fibrosis (CF), a genetic disease caused by mutation of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene, results in inspissated, infected secretions within the airway, progressive bronchiectasis and, ultimately, death from respiratory failure9. By adulthood, the majority of patients with CF are chronically infected with P. aeruginosa, which plays a key role in the morbidity and mortality associated with this disease10. Additionally, patients with severe burn injuries11, tracheostomies12, joint replacements13, or indwelling catheters14 are at risk for P. aeruginosa infection related to the bacteria&#39;s ability to form biofilms and escape host inflammatory responses15. Further, colonization occurs without competition after a multi-antibiotic resistant or tolerant population is selected through broad-spectrum, sequential antimicrobial treatment12,16,17,18. Better understanding the pathogenesis of P. aeruginosa will have significant implications for numerous disease states.
Several P. aeruginosa clinical isolates, including strains PAO1, PA103, PA14 and PAK, have been extensively studied to investigate different features of P. aeruginosa pathogenesis. Strain PA14 is a clinical isolate that belongs to one of the most common clonal groups worldwide19,20 and has not been extensively passaged in the laboratory. PA14is highly virulent in vertebrate models of infection, with a notable endotoxin profile21, pili structure22, pathogenicity islands23, type III secretion system (TTSS), cytotoxicity towards mammalian cells24 and profiles in antibiotic resistance and persistence25. Furthermore, PA14 is also highly virulent in numerous host-pathogen model systems, including plant leaf infiltration models26,27,Caenorhabditis elegans infection models28,29, insect models30,31, as well as mouse pneumonia models32,33 and skin burn models34.
Genome-wide mutant libraries are collections of isogenic mutants in nonessential genes that constitute very powerful tools to understand the biology of an organism by allowing analysis of gene function on a genomic scale. Two near-saturation transposon insertion mutant libraries constructed in P. aeruginosa are currently available for distribution. The insertion sites of the transposons have been determined for both libraries. These so-called nonredundant libraries facilitate genome-wide studies of bacterial strains by considerably decreasing the time and cost involved in screening uncharacterized random transposon mutants. The P. aeruginosa PAO1 transposon mutant library, constructed in the MPAO1 isolate of strain PAO1 using transposons ISphoA/hah and ISlacZ/hah35, is curated by the Manoil lab, University of Washington. The library consists of a sequence-verified collection of 9,437 transposon mutants that provides wide genome coverage and includes two mutants for most genes36. Information about the P. aeruginosa PAO1 transposon mutant library is available at the public, internet-accessible Manoil lab website at http://www.gs.washington.edu/labs/manoil/libraryindex.htm. The P. aeruginosa strain PA14 nonredundant transposon insertion mutant library (PA14NR Set) constructed in strain PA14 using transposons MAR2xT7 and TnphoA37 is currently distributed by the Department of Pediatrics at Massachusetts General Hospital. The PA14NR Set comprises a collection of more than 5,800 mutants with single transposon insertions in nonessential genes37. Details on the construction of the PA14NR Set are described in the public, internet-accessible site http://pa14.mgh.harvard.edu/cgi-bin/pa14/home.cgi?section=NR_LIB, which also contains a variety of online search tools to facilitate the use of the PA14NR Set.
The original PA14NR Set comprised 5,459 mutants, selected from a comprehensive library of approximately 34,000 random transposon insertion mutants, that correspond to 4,596 predicted PA14 genes representing 77% of all predicted PA14 genes37. Since the construction of the library in 2006 new mutants were added, and presently the PA14NR Set includes more than 5,800 mutants38 that represent approximately 4,600 PA14 genes. The majority of the PA14 transposon mutants were generated in the wild type background37. Details concerning each member of the mutant library, including genetic background, are available either through searching the online database, or by downloading the Nonredundant Library spreadsheet, both features available on the PA14 website (http://pa14.mgh.harvard.edu/cgi-bin/pa14/home.cgi). The majority of mutants were created using the MAR2xT7 (MrT7) transposon, with a small set created using the TnPhoA (phoA) transposon37. Each transposon has an antibiotic resistance cassette, which allows for mutant selection using gentamicin (MrT7) or kanamycin (phoA). The PA14NR set of mutants is stored in sixty-three 96-well plates and includes two additional 96-well control plates, which consist of wild type PA14 inoculated and uninoculated wells intercalated in a preset pattern. The 96-well plate format paired with the online search tools greatly facilitates the custom development of screening assays that allow users to easily identify genes associated with mutant phenotypes. The online search tools also facilitate the search and selection of additional relevant mutants required for further studies.
The PA14 and PAO1 transposon mutant libraries are very important global resources for the scientific community, and they complement each other in validating the function of unknown genes and pathways of this bacterial pathogen. Coincidentally, since the construction of the PAO1 and PA14 transposon mutation libraries, full-genome DNA sequencing analysis of many P. aeruginosa isolates has shown that PAO1 and PA14 belong to different major subclades of the P. aeruginosa phylogeny7,39,40,41. Because clinical P. aeruginosa isolates are found distributed throughout the phylogeny, the fact that PAO1 and PA14 belong to different P. aeruginosa subgroups enhances the value of the two transposon mutation libraries for comparative studies.
Publications describing the construction and screening of bacterial mutant libraries, including P. aeruginosa libraries35,37,42, are readily available in the literature. However, to the best of our knowledge, no published protocols describing detailed procedures and techniques used for replication, maintenance, and validation of bacterial mutant libraries are available.
The methodology outlined in this publication describes a set of three protocols that facilitate the use and maintenance of the PA14NR Set. The first protocol describes replication of the library as recommended to recipients of the PA14NR Set. The second protocol includes guidelines for streaking, growing, and storing individual mutants identified using the PA14NR Set. The third protocol describes quality control techniques, including PCR amplification of fragments from transposon mutants and subsequent sequencing to confirm mutant identity. This set of protocols may also be adapted for the replication and maintenance of other bacterial mutant libraries or collections. The replication of bacterial mutant libraries or collections is highly advised to preserve the integrity of the &#34;master copy&#34; (original copy received). Replication of several copies of the PA14NR Set for routine laboratory use minimizes the probability of interwell contamination of the master copy.

<table>
	<tbody>
		<tr>
			<td>Materials for Library Replication</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile 96-well Tissue-culture treated, case of 50</td>
			<td>Corning Life Sciences</td>
			<td>353072</td>
			<td>via Fisher Scientific</td>
		</tr>
		<tr>
			<td>Sterile 96 Well Clear V-Bottom 2000&#956;L Deep Well Plates, case of 25</td>
			<td>Corning Life Sciences</td>
			<td>3960</td>
			<td>via Fisher Scientific</td>
		</tr>
		<tr>
			<td>Nunc OmniTray (rectangular plates), case of 60</td>
			<td>Thermo Scientific Rochester</td>
			<td>242811</td>
			<td>via Fisher Scientific</td>
		</tr>
		<tr>
			<td>Rectangular Ice Pan, Midi (4L)</td>
			<td>Corning Life Sciences</td>
			<td>432104</td>
			<td>via Fisher Scientific</td>
		</tr>
		<tr>
			<td>Secure-Gard Cone Mask, case of 300</td>
			<td>Cardinal Health</td>
			<td>AT7509</td>
			<td>via Fisher Scientific</td>
		</tr>
		<tr>
			<td>AluminaSeal, pack of 100</td>
			<td>Diversified Biotech</td>
			<td>ALUM-100</td>
			<td>via Fisher Scientific</td>
		</tr>
		<tr>
			<td>Breathe-Easy membrane, pack of 100</td>
			<td>Diversified Biotech</td>
			<td>BEM-1</td>
			<td>via Sigma-Aldrich</td>
		</tr>
		<tr>
			<td>Sterile, individually wrapped, 50mL Solution Trough/Reagent Reservoir, case of 100</td>
			<td>Sorenson</td>
			<td>S50100</td>
			<td>via Westnet Incorporated</td>
		</tr>
		<tr>
			<td>Plate roller</td>
			<td>VWR</td>
			<td>60941-118</td>
			<td>via VWR</td>
		</tr>
		<tr>
			<td>Cryo Laser Labels - CRYOLAZRTAG 2.64&#34; x 0.277&#34;, pack of 16 sheets</td>
			<td>GA International</td>
			<td>RCL-11T1-WH</td>
			<td>via Labtag.com (template for printing also available from Labtag.com)</td>
		</tr>
		<tr>
			<td>96-well replicator</td>
			<td>V &#38; P Scientific, Inc.</td>
			<td>Custom 407C, 3.18mm pin diameter, 57mm long</td>
			<td>via V &#38; P Scientific, Inc.</td>
		</tr>
		<tr>
			<td>Multitron Pro, 3mm Shaking incubator</td>
			<td>Infors HT</td>
			<td>l10003P</td>
			<td>via Infors HT</td>
		</tr>
		<tr>
			<td>Picus 12 Channel 50-1200&#956;L Electronic Pipette</td>
			<td>Sartorius</td>
			<td>735491PR</td>
			<td>via Sartorius</td>
		</tr>
		<tr>
			<td>Filter Tips 50-1200&#956;L, pack of 960</td>
			<td>Biohit</td>
			<td>14-559-512</td>
			<td>via Fisher Scientific; use electronic multichannel-compatible tips</td>
		</tr>
		<tr>
			<td>Dry Ice</td>
			<td>User-specific vendor</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Materials for Individual Mutant Storage</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fisherbrand Premium Microcentrifuge Tubes: 1.5mL</td>
			<td>Fisher Scientific</td>
			<td>05-408-130</td>
			<td>via Fisher Scientific</td>
		</tr>
		<tr>
			<td>Pipettes (P1000, P200, P20, P2)</td>
			<td>Gilson</td>
			<td>F167370</td>
			<td>via Gilson</td>
		</tr>
		<tr>
			<td>Materials for Quality Control PCR</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fisherbrand Premium Microcentrifuge Tubes: 1.5mL</td>
			<td>Fisher Scientific</td>
			<td>05-408-130</td>
			<td>via Fisher Scientific</td>
		</tr>
		<tr>
			<td>NanoDrop</td>
			<td>Thermo Scientific</td>
			<td>ND-2000</td>
			<td>via ThermoFisher</td>
		</tr>
		<tr>
			<td>PCR Thermocycler</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Omnistrips PCR Tubes with domed lids</td>
			<td>Thermo Scientific</td>
			<td>AB0404</td>
			<td>via Fisher Scientific</td>
		</tr>
		<tr>
			<td>ART Barrier low-retention pipette tips (10 uL, 100 uL, 1000 uL)</td>
			<td>Molecular BioProducts, Inc.</td>
			<td>Z676543 (10 uL), Z676713 (100 uL), Z676802 (1000 uL)</td>
			<td>via Sigma-Aldrich</td>
		</tr>
		<tr>
			<td>Pipettes (P1000, P200, P20, P2)</td>
			<td>Gilson</td>
			<td>F167370</td>
			<td>via Gilson</td>
		</tr>
		<tr>
			<td>Fisherbrand Premium Microcentrifuge Tubes: 1.5mL</td>
			<td>Fisher Scientific</td>
			<td>05-408-130</td>
			<td>via Fisher Scientific</td>
		</tr>
		<tr>
			<td>MasterPure DNA Purification Kit</td>
			<td>Epicentre</td>
			<td>MCD85201</td>
			<td>via Epicentre Technologies Corp</td>
		</tr>
		<tr>
			<td>GeneRuler 1 kb Plus DNA Ladder, ready-to-use</td>
			<td>Thermo Scientific</td>
			<td>SM1333</td>
			<td>via ThermoFisher</td>
		</tr>
		<tr>
			<td>RediLoad Loading Buffer</td>
			<td>Invitrogen</td>
			<td>750026</td>
			<td>via ThermoFisher</td>
		</tr>
		<tr>
			<td>Chemicals</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Chemicals for Library and Individual Mutant Storage</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol MB Grade, 1L</td>
			<td>Sigma Aldrich</td>
			<td>G5516</td>
			<td>via Sigma-Aldrich</td>
		</tr>
		<tr>
			<td>LB Broth</td>
			<td></td>
			<td></td>
			<td>Per 1L dH2O: 10g tryptone, 5g yeast extract, 5g NaCl, 1ml 1N NaOH (Current Protocols in Molecular Biology.&#160; Wiley, 1994.)</td>
		</tr>
		<tr>
			<td>Tryptone</td>
			<td>Sigma Aldrich</td>
			<td>T7293</td>
			<td>via Sigma-Aldrich</td>
		</tr>
		<tr>
			<td>Yeast Extract</td>
			<td>Sigma Aldrich</td>
			<td>Y1625</td>
			<td>via Sigma-Aldrich</td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Sigma Aldrich</td>
			<td>S7653</td>
			<td>via Sigma-Aldrich</td>
		</tr>
		<tr>
			<td>Sodium Hydroxide</td>
			<td>Sigma Aldrich</td>
			<td>S8045</td>
			<td>via Sigma-Aldrich</td>
		</tr>
		<tr>
			<td>LB&#160; agar</td>
			<td></td>
			<td></td>
			<td>See preparation above, add 15g Bacto Agar</td>
		</tr>
		<tr>
			<td>Bacto Agar</td>
			<td>Sigma Aldrich</td>
			<td>A5306</td>
			<td>via Sigma-Aldrich</td>
		</tr>
		<tr>
			<td>Gentamicin sulfate, 10g</td>
			<td>BioReagent</td>
			<td>1405-41-0</td>
			<td>via Sigma-Aldrich</td>
		</tr>
		<tr>
			<td>Kanamycin sulfate</td>
			<td>Gibco</td>
			<td>11815024</td>
			<td>via ThermoFisher</td>
		</tr>
		<tr>
			<td>Ethanol, 190 proof</td>
			<td>Decon</td>
			<td>04-355-221</td>
			<td>via Fisher Scientific</td>
		</tr>
		<tr>
			<td>Chemicals for Quality Control PCR</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Primers</td>
			<td>User-preferred vendor</td>
			<td></td>
			<td>See primers listed in Table 3</td>
		</tr>
		<tr>
			<td>Corning cellgro Molecular Biology Grade Water</td>
			<td>Corning</td>
			<td>46000CV</td>
			<td>via Fisher Scientific</td>
		</tr>
		<tr>
			<td>Taq Polymerase Buffer</td>
			<td>Invitrogen</td>
			<td>10342020</td>
			<td>via ThermoFisher</td>
		</tr>
		<tr>
			<td>Taq DNA Polymerase, recombinant</td>
			<td>Invitrogen</td>
			<td>10342020</td>
			<td>via ThermoFisher</td>
		</tr>
		<tr>
			<td>dNTPs</td>
			<td>Invitrogen</td>
			<td>10297018</td>
			<td>via ThermoFisher</td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Sigma</td>
			<td>A9539</td>
			<td>via Sigma-Aldrich</td>
		</tr>
	</tbody>
</table>

replication of the ordered, nonredundant library of pseudomonas aeruginosa strain pa14 transposon insertion mutants

Pseudomonas aeruginosa is a phenotypically and genotypically diverse and adaptable Gram-negative bacterium ubiquitous in human environments. P. aeruginosa is able to form biofilms, develop antibiotic resistance, produce virulence factors, and rapidly evolve in the course of a chronic infection. Thus P. aeruginosa can cause both acute and chronic, difficult to treat infections, resulting in significant morbidity in certain patient populations. P. aeruginosa strain PA14 is a human clinical isolate with a conserved genome structure that infects a variety of mammalian and nonvertebrate hosts making PA14 an attractive strain for studying this pathogen. In 2006, a nonredundant transposon insertion mutant library containing 5,459 mutants corresponding to 4,596 predicted PA14 genes was generated. Since then, distribution of the PA14 library has allowed the research community to better understand the function of individual genes and complex pathways of P. aeruginosa. Maintenance of library integrity through the replication process requires proper handling and precise techniques. To that end, this manuscript presents protocols that describe in detail the steps involved in library replication, library quality control and proper storage of individual mutants.

Twelve new copies of the PA14NR Set were replicated using Protocol I, and a quality control assessment of the new copies generated was conducted using Protocol III.
PA14NR Set mutant plates along with Control Plates, which consist of wild type PA14 inoculated and uninoculated wells intercalated in a preset pattern (Figure 4A), were replicated following methology described in Protocol I. Control plat...

Watch this Scientific Journal Video about Replication of the Ordered, Nonredundant Library of Pseudomonas aeruginosa strain PA14 Transposon Insertion Mutants at JoVE.com

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

Pseudomonas aeruginosa infection causes significant morbidity in vulnerable hosts. The nonredundant transposon insertion mutant library of P. aeruginosa strain PA14, designated as PA14NR Set, facilitates analysis of gene functionality in numerous processes. Presented here is a protocol to generate high-quality copies of the PA14NR Set mutant library.

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

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

The overall goal of this protocol is to generate high quality copies of the ordered, non-redundant transposon mutant library of Pseudomonas aeruginosa strain PA14 referred to as the PA14NR set. This protocol preserves the integrity of the PA14NR set during replication, generating high quality copies of this library. The main advantage of this protocol is that it provides a detailed description of all steps involved in library replication, quality control, and proper storage of individual mutants.Although this protocol was designed to replicate P14NR set, it can be adapted to replicate other bacterial libraries. Generally, individuals new to this method will struggle because of the risk of contamination during the replication process. Therefore, this protocol provides detailed step-by-step instructions to reduce that risk.Our laboratory distributes the PA14NR set to the global scientific community. And we recommend that the libraries replicated by the end user. To provide guidance, we offer a protocol detailing how to generate high quality copies of the library.Visual demonstration of this protocol is critical, especially the steps designed to prevent cross-contamination. For self-protection, as well as protection of the bacterial cultures, a lab coat, gloves and a mask should always be worn when handling the non-redundant transposon insertion mutant library of P aeruginosa strain PA14, designated PA14NR set. Prepare LB Agar plates by pouring approximately 60 milliliters of molten LB agar into rectangular plates.Dry the plates in a sterile hood for approximately one hour before use. Create a sterile field with a bunsen burner on an ethanol wiped benchtop. And set up containers with appropriate solutions for replicator pin sterilization.Remove a PA14NR set masterplate from the minus 80 degrees Celsius freezer, and place it on dry ice in a 4 liter ice pan. Take the 96 well masterplate to be replicated from the dry ice and place it on the bench to allow brief thawing. Align the masterplate and the agar plate with the A1 coordinate of both plates in the upper left corner.Mark the position of the A1 coordinate on the rectangular agar plate. To sterilize the replicator pins, immerse the pins in 250 milliliters of 0.3 to 0.5%sodium hypochlorite for 30 seconds. Tap the replicator slightly to remove excess liquid and immerse the pins in 250 milliliters of sterile ultrapure water for 10 seconds, followed by 250 milliliters of 70%ethanol for 30 seconds and 250 milliliters of 95%ethanol for two minutes.Flame sterilize the replicator pins by holding the replicator perpendicularly to the bunsen burner flame, slowly approaching the flame until the ethanol ignites and then immediately withdrawing it from the flame. The flame will extinguish once all the ethanol burns off. Cool the pins by pinning on to an unused rectangular plate containing sterile LB agar for 30 seconds.While the replicator pins are cooling, briefly warm the aluminum seal from the PA14NR set masterplate with your hand, and then peel it off carefully, making sure that the seal does not retouch the plate. Use tweezers to peel off any remnants of the aluminum seal. Insert the replicator pins into the masterplate, gently pushing and swinging the replicator in all four directions to ensure that pins make contact with the frozen bacterial cultures in each of the 96 wells.Pay extra attention to wells located in the outer edge of the plate by pushing replicator pins against frozen cultures in the wells located at the edge of the plate. Gently place the replicator pins on to the surface of the agar plates. Move the replicator in a slight circular motion to form many lawns of approximately four to five millimeters for each mutant strain.Seal the PA14NR set masterplate with a new sterile aluminum seal. Use a plate roller to ensure that each well and the edges of the plate are completely sealed. Return the 96 well masterplate to dry ice.After handling the PA14NR set, wipe down all work surfaces with 70%ethanol. Transfer the replicated agar plate to a 37 degrees Celsius incubator and incubate overnight. Prior to starting this procedure, make sure the laminar flow hood is clear of unnecessary equipment and turn the hood blower on for a minimum of 10 minutes.Wipe down the hood surfaces and any items placed in the hood using 70%ethanol. Using a 50 to 1200 microliter 12 channel electronic pipette, fill each well of a 12 milliliter deep well block with 525 microliters of LB liquid broth containing appropriate antibiotics. Then seal the deep well block before removing it from the laminar flow hood.Next, transfer the media filled deep well block to an ethanol wiped benchtop. Create a sterile field with a bunsen burner on the benchtop, and set up containers with appropriate solutions for replicator pin sterilization. Immerse the replicator pins in each solution as demonstrated earlier.Flame sterilize the replicator pins. Then cool the pins by pinning into an unused rectangular plate containing LB agar for 30 seconds. Gently place the replicator pins on to the agar plate containing grown mutant strains, and check that the pins are in contact with all 96 mutants on the agar plate.Then submerge the pins into the deep wells containing LB liquid broth for five to 10 seconds. Avoid touching the sides of the wells with the pins. Seal the deep well block with a sterile breathable sealing membrane.Use a plate roller to ensure that each individual well is properly sealed. Wipe down all work surfaces with 70%ethanol after handling the PA14NR set. Grow the inoculated liquid cultures in a high speed shaker at 37 degrees Celsius and 950 rpm for 15 to 16 hours.Begin by clearing the laminar flow hood, and turning the hood blower on for a minimum of 10 minutes. Wipe down all hood surfaces and any items placed in the hood with 70%ethanol. Remove the 96 well plates from the plastic wrap.Peel off the plate label for each plate and place it along the plate's edge closest to the A1 to H1 wells of the destination plate. Slightly lift the lid of the destination plate and place the label on the lower edge to display the label when the plate is covered with a lid. Remove the deep well block from the high speed shaker, and transfer the block to the sterile laminar flow hood.Carefully remove the breathable sealing membrane and replace with an aluminum seal. Spin down the deep well block at very low speed to collect condensation. Since transferring the cultures to the destination plates presents the greatest risk for potential inter-well contamination, it is critical to carefully follow all procedures for maintaining sterility.Return the deep well block to the sterile hood. Using a 50 to 1200 microliter 12 channel electronic pipette and sterile filtered tips, add 525 microliters of glycerol LB liquid broth mix to each well. Mix by gently pipetting 300 microliters up and down three times with an electronic pipette.Touch the tips to the side of the wells, before injecting the tips to prevent dripping. Reload the pipette with new sterile filtered tips, and fill the next row of wells with the glycerol LB liquid broth mix. After all wells have been filled with the glycerol LB liquid broth mix, use the 12 channel electronic pipette and filtered tips to pull up 900 microliters of glycerol mutant culture mix from each row of the deep well block, and dispense 150 microliters into wells of the corresponding row in the destination plates to generate six copies of the library plate.Use the sterile aluminum seal to cover each 96 well destination plates, and use a plate roller to completely seal the plate edges and all wells. Make sure not to cover the label identifier with the aluminum seal. Remove the sealed plates from the hood and place them on a flat even surface in a minus 80 degrees Celsius freezer.Wipe down all work surfaces with 70%ethanol after handling the library. Using the protocol demonstrated in this video, 12 copies of the PA14NR set including control plates which consist of well type PA14 inoculated and un-inoculated wells intercalated in a preset pattern, were replicated. The control plates were visually inspected after replication on LB agar plates to ensure the presence of expected growth patterns.38 mutant strains were randomly selected from one of the newly generated copies of the PA14NR set, and PCR amplification using arbitrary primers was performed to confirm the identity of the transposon mutants. Subsequent sequencing of the DNA fragments confirmed mutant identity for 35 of the 38 mutants tested. Once mastered, replication of the entire library takes roughly 80 hours.While using this protocol, it's important to mind things sterility of the work area at all times. After watching this video, you should have a good understanding of how to generate high quality copies of the P14NR set or other bacterial libraries. Don't forget, working with Pseudomonas can be hazardous and personal protective equipment should always be worn while performing this protocol.This protocol preserves the Pseudomonas aeruginosa PA14 non-redundant mutant library which continues to be an important resource, not only for high throughput screening, but also as a source for individual mutations in most Pseudomonas aeruginosa genes.

replication-ordered-nonredundant-library-pseudomonas-aeruginosa

Research

JoVE Journal

Immunology and Infection

7.9K visualizações.  Massachusetts General Hospital. The overall goal of this protocol is to generate high quality copies of the ordered, non-redundant transposon mutant library of Pseudomonas aeruginosa strain PA14 referred to as the PA14NR set. This protocol preserves the integrity of the PA14NR set during replication, generating high quality copies of this library. The main advantage of this protocol is that it provides a detailed description of all steps involved in library replication, quality control, and proper storage of individual muta...