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

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.

Figure 1: Overview of Protocol I: replication of the PA14NR set. Day 1: Replicate frozen mutant cultures from "master copy" of PA14NR Set into LB Agar media and grow mutants overnight at 37 °C. Day 2: Transfer mutant growth from LB Agar media to Deep Well Blocks containing LB liquid broth, grow overnight at 37 °C with shaking at 950 rpm. Day 3: Mix overnight LB cultures with glycerol, then transfer to 96-well destination plates for long-term storage. Place 96-well plates flat in -80 °C freezer. Please click here to view a larger version of this figure.

Figure 2: Recommended setup. Sterility and smooth workflow should be maintained through the use of appropriate precautions. Please click here to view a larger version of this figure.

1. Protocol I: Replication of the PA14NR Set

NOTE: Replication of the library can be achieved by dividing the PA14NR Set in four subsets of sixteen plates each that can be processed in four consecutive weeks. Generation of 1 to 6 copies adheres to the weekly workflow outlined in Table 1, while generation of more than 6 copies follows the weekly workflow outlined in Table 2. To generate 12 copies of the PA14NR Set, inoculate the same PA14NR subset into liquid LB media on Day 2 and again on Day 3 (from the same set of agar plates replicated on Day 1), and transfer overnight mutant cultures into copy plates on Day 3 and Day 4 respectively.

Table 1: Schedule for replication of 1 – 6 copies of the PA14NR Set. Replication of a small number of copies may adhere to a weekly workflow.

Table 2: Schedule for replication of up to 12 copies of the PA14NR Set. Replication of a larger number of copies will require layering within a weekly workflow.

1. Day 1: Growth of PA14NR Set Master copy on LB agar plates
   1. Wear gloves, lab coat and mask to handle the PA14NR Set.
   2. Clear bench and wipe surface with 70% ethanol.
   3. Prepare LB agar media⁴³ and sterilize in autoclave for 25 min. Cool media to 55 °C in water bath and add either 15 µg/mL gentamicin, for mutants containing MAR2xT7 transposon insertions, or 200 µg/mL kanamycin, for mutants containing TnphoA insertions.
   4. Pour molten LB agar in rectangular plates using approximately 60 mL of media per plate. Dry plates in a sterile hood for approximately 1 h before use. Make sure there is no water condensation on the surface of the agar. Store plates at 4 °C, if necessary.
   5. Create a sterile field with a Bunsen burner on ethanol-wiped bench top and set up containers with appropriate solutions for replicator pin sterilization (see step 1.1.9). Use open plastic containers (5.25"L x 4.25"W x 1.75"H approximate size) for replicator pin sterilization. Autoclave plastic containers before use.
      NOTE: The heat of the Bunsen burner flame creates a convection current, which heats the space above the flame and lifts any particulates in the air up and away from the cooler air underneath, keeping the work area sterile.
   6. Remove a maximum of four PA14NR Set master plates from the -80 °C freezer (to avoid unnecessary thawing), and place them on dry ice in 4 L ice pan.
   7. Take the 96-well master plate to be replicated from dry ice and place it on the bench to allow brief thawing, which will only take the time necessary for sterilization of the replicator pins (approximately 3-4 min) (see step 1.1.8). Mark the position of the A1 coordinate on the agar plate prior to stamping. Align master plate and agar plate with the A1 coordinate of both plates in the upper left corner.
   8. Sterilize the replicator "pins" by following the steps described below. Tap replicator slightly after each step to remove excess liquid.
      1. Immerse pins in 250 mL of 0.3-0.5% sodium hypochlorite (10% household bleach) for 30 seconds. Minimize contact with sodium hypochlorite solution, as it can lead to damage of pins. Immerse pins in 250 mL sterile ddH₂O or sterile ultrapure water for 10 seconds, then in 250 mL 70% ethanol for 30 seconds, then in 250 mL of 95% ethanol for 2 min.
      2. Flame sterilize replicator pins by holding replicator perpendicularly to the Bunsen burner flame, slowly approaching flame until ethanol ignites, then immediately withdrawing it from flame. Flame will extinguish once all ethanol burns off. Keep a lid or similar container close by to suffocate burning ethanol, if necessary.
         NOTE: Use extreme caution when working with ethanol near a flame. DO NOT hold replicator directly over flame.
      3. Cool pins by pinning onto an unused sterile rectangular plate containing agar LB media for 30 seconds.
   9. Briefly warm aluminum seal from PA14NR Set master plate with your hand before peeling it off. Do this while cooling replicator pins. Remove aluminum seal carefully to prevent the seal from retouching the plate. Use tweezers to peel off any remnants of aluminum seal.
   10. Insert replicator pins into master plate, gently pushing and swinging replicator in all four directions to ensure that pins make contact with 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.
   11. Gently place replicator pins onto surface of the agar plate. Move replicator in a slight circular motion to form mini-lawns of approximately 4-5 mm for each mutant strain. Avoid the possibility that the mini-lawns may overlap, to avoid cross contamination.
   12. Seal PA14NR Set master plate with a new sterile aluminum seal. Do not touch the adhesive side of aluminum seal at any point to avoid contamination. Make sure that each well and the edges of plate are completely sealed by using a plate roller. Return 96-well plate to dry ice.
   13. Repeat procedure for each master plate.
   14. Wipe down all work surfaces with 70% ethanol after handling the PA14NR Set.
   15. Transfer replicated agar plates to 37 °C incubator and incubate overnight.
2. Day 2: Growth of the PA14NR Set Master copy in LB liquid broth
   1. Prepare LB liquid broth⁴³ containing either 15 µg/mL gentamicin, for mutants containing MAR2xT7 transposon insertions, or 200 µg/mL kanamycin, for mutants containing TnphoA insertions.
   2. Clear laminar flow hood of unnecessary equipment and turn hood blower on for a minimum of 10 min before beginning work. Wipe down hood surfaces and any items placed in the hood using 70% ethanol.
   3. Fill 2 mL deep-well blocks with 525 µL of LB liquid broth containing appropriate antibiotics in laminar flow hood using a 50-1200 µL 12-channel electronic pipette. Then, transfer media-filled deep-well blocks to ethanol-wiped bench top. Reuse tips as long as sterile conditions are maintained.
   4. Wear gloves, lab coat, and mask to handle PA14NR Set.
   5. Clear bench and wipe surface with 70% ethanol.
   6. Bring agar plates with overnight grown mutant strains to bench top.
   7. Create a sterile field with a Bunsen burner on ethanol-wiped bench top and set up containers with appropriate solutions for replicator pin sterilization (see next step).
   8. Sterilize the replicator "pins" following the steps described below. Tap replicator slightly after each immersion to remove excess liquid.
      1. Immerse pins in 250 mL of 0.3-0.5% sodium hypochlorite for 30 seconds. Minimize replicator pin contact with sodium hypochlorite solution, as it can lead to damage of pins. Then, immerse pins in 250 mL sterile ddH₂O or ultrapure water for 10 seconds, then in in 250 mL of 70% ethanol for 30 seconds, then in 250 mL of 95% ethanol for 2 min.
      2. Flame sterilize replicator pins, then cool pins by pinning into unused rectangular plate containing agar LB media for 30 seconds.
         NOTE: Use extreme caution when working with ethanol near a flame (see 1.1.8).
   9. Gently place replicator pins onto agar plate containing mutant growth and check that pins are in contact with all 96 mutants on agar plate, then submerge pins into deep wells containing LB liquid broth. Avoid touching the sides of the wells with the pins.
   10. Seal the deep well block with a sterile breathable sealing membrane. Use a plate roller to ensure that each individual well is properly sealed.
   11. Repeat procedure for each agar plate.
   12. Wipe down all work surfaces with 70% ethanol after handling the PA14NR Set.
   13. Grow inoculated liquid cultures for 15 – 16 h at 37°C at 950 rpm using a high speed shaker, if available.
       NOTE: If a high speed shaker is not available, incubation of deep well blocks in shaker at 250-300 rpm is feasible. However, there is a greater chance of small colony variants (SCVs)²⁵ emerging under low oxygenation conditions. Therefore, it is highly advisable to keep incubation times under 15 – 16 h when growing cultures in deep well blocks at lower shaker speeds. Unwanted proliferation of SCVs in mutant wells can alter mutant phenotypes when using the library to perform genetic screens.
       Certain wells in the PA14NR Set contain slow-growing/non-growing mutants, missing clones, or contain uninoculated media. The location of these wells has been recorded and can be found in the Supplemental PA14NR Set Wells Information file included with this publication.
3. Day 3: Transfer of PA14NR Set overnight cultures into destination plates
   1. Clear laminar flow hood and turn hood blower on for a minimum of 10 min before beginning work. Wipe down hood surfaces and any items placed in the hood using 70% ethanol.
   2. Print adhesive waterproof labels (see Supplemental PA14NR Set Labels file for template). Remove 96-well plates from plastic wrap inside a laminar flow hood. Peel-off plate label and place it along the plate's edge closest to 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 label when plate is covered with lid.
   3. Prepare 3.5 L of 60% glycerol (v/v) and sterilize 20 min in autoclave.
   4. Wear gloves, lab coat, and mask to handle the PA14NR Set. Remove deep well blocks from high speed shaker or regular shaker.
   5. Transfer deep-well blocks to sterile laminar flow hood and carefully remove breathable sealing membrane. Replace with aluminum seal.Spin-down the deep-well blocks at very low speed to collect condensation (30 seconds at 50-150 x g, then decelerate quicky by having centrifuge brake on).
   6. Transfer deep-well blocks back to sterile hood and using a 50-1200 µL 12-channel electronic pipette and sterile filtered tips add 525 µL of glycerol/LB liquid broth mix (equal parts LB liquid broth and 60% glycerol solution) to each well. Mix by gently pipetting 300 µL up and down 3 times with electronic pipette. Touch tips to side of well before ejecting tips to prevent dripping. Eject tips and continue with the next row until done with entire deep well block.
   7. Using the 12-channel electronic repetitive pipette and filtered tips, to prevent interwell contamination during aliquoting, pull up 900 µL of mutant culture and dispense 150 µL into each 96-well destination plates to generate 6 copies of the library plate. Prevent drips by touching the wall of the wells with tips before starting transfer of the culture into destination plates. If no repeat pipette is available, use a multichannel pipette to dispense 150 µL into each of the six 96-well plates, using the technique described above to prevent dripping.
   8. Use sterile aluminum seals to cover the plates and use a plate roller to completely seal plate edges and all wells. Make sure not to cover label identifier with the aluminum seal. Do not shake the plates, as culture may splash on sides of the wells or on aluminum seal.
   9. Remove sealed plates from hood and place plates on a flat, even surface in the -80 °C freezer.
   10. Wipe down all work surfaces with 70% ethanol after handling the library.
   11. Perform quality control checks after library replication and after using the library to perform genetic screens (see Protocol III).

2. Protocol II: Handling and Storage of Individual Mutants from the PA14NR Set

1. Day 1: Streak mutant of interest
   1. Identify the location of the mutant of interest through the PA14NR Set link http://pa14.mgh.harvard.edu/cgi-bin/pa14/search.cgi?searchType=SEARCH_PLATE_POSITIONS or using the Nonredundant Library.xls file downloaded from the link http://pa14.mgh.harvard.edu/cgi-bin/pa14/downloads.cgi). Make a note of the antibiotic needed to select every particular mutant (gentamicin or kanamycin).
   2. Prepare LB agar media, sterilize in autoclave for 20-25 min, and cool to 55 °C in water bath. Add 15 µg/mL gentamicin for mutants containing MAR2xT7 transposon insertions, and 200 µg/mL kanamycin for mutants containing TnphoA transposon insertions. Pour LB agar media on plates (round or rectangular plates are adequate). Dry plates in sterile hood for approximately 30 min to 1 h before use.
   3. Autoclave all plasticware and non-sterile supplies before use.
   4. Wear gloves, lab coat, and mask to handle the PA14NR Set. Clear bench and wipe surface with 70% ethanol before working with the PA14NR Set.
   5. Create a sterile field with a Bunsen burner.
   6. Remove PA14NR Set 96-well plate with mutant of interest from -80 °C freezer and place it on dry ice, take dry ice container to the bench, and briefly place 96-well plate on top of bench to allow slight thawing (approximately 1-2 min).
      NOTE: Keep a record of all PA14NR Set 96-well plates accessed to streak individual mutants, as more access to library plates is correlated with a greater risk for interwell contamination.
   7. Warm aluminum seal with hand before peeling it off, being careful to prevent the seal from retouching the plate. Use tweezers to peel off any remnants of aluminum seal.
   8. Locate the mutant of interest on the 96-well plate. Use either sterile wooden stick or sterile pipette tip to pick a small amount of frozen culture from the individual well containing the mutant of interest.
   9. Streak frozen culture on agar plate for single mutant colonies as follows: Gently spread the bacteria over a section of the plate to create streak 1, using a fresh, sterile wooden stick or pipette tip, drag through streak 1 and spread the bacteria over a second section of the plate, to create streak 2. Using a third sterile wooden stick or pipette tip, drag through streak 2 and spread the bacteria over the last section of the plate, to create streak 3.
   10. Seal source plate with a new sterile aluminum seal. Do not touch adhesive side of aluminum seal at any point to avoid contamination. Make sure that each well and edges of plate are completely sealed by using a plate roller. Return 96-well plate to dry ice and then the -80 °C freezer.
   11. Incubate agar plate in 37 °C incubator overnight.
   12. Wipe down all work surfaces with 70% ethanol after handling the library.
2. Day 2: Growth of mutant of interest in LB liquid broth
   1. Prepare liquid LB broth containing 15 µg/mL gentamicin or 200 µg/mL kanamycin, as per transposon insertion.
   2. Wear gloves, lab coat, and mask to handle P. aeruginosa.
   3. Clear bench and wipe surface with 70% ethanol. Create a sterile field with a Bunsen burner.
   4. Transfer 3 – 5 mL of LB broth with appropriate antibiotic into sterile culture tube with cap.
   5. Using a sterile applicator or a sterile pipette tip, pick a single colony of mutant strain and inoculate it into LB media.
   6. Incubate LB liquid cultures at 37 °C in a shaker at 225 – 250 rpm overnight.
   7. Wipe down all work surfaces with 70% ethanol after handling P. aeruginosa.
3. Day 3: Store mutant of interest in -80 °C freezer
   1. Label cryovial with mutant name, antibiotic added to LB broth and date of storage.
   2. Prepare 500 mL of 50% Glycerol (v/v) and sterilize in autoclave.
   3. Wear gloves, lab coat, and mask to handle P. aeruginosa.
   4. Clear bench and/or laminar flood hood and wipe surface with 70% ethanol.
   5. Remove tube containing mutant culture from shaker.
   6. Prepare a small container with dry ice.
   7. Use a laminar flow hood or create a sterile field on ethanol-wiped bench top with Bunsen burner.
   8. Add equal amounts of bacterial culture and 50% glycerol to the labeled cryovial using sterile conditions, and gently mix with pipette (final volume 1 – 2 mL/vial depending on the size of the cryovials used). Place cryovial on dry ice to quick-freeze.
   9. Place cryovial in labeled box in -80 °C freezer.
   10. Wipe down all work surfaces with 70% ethanol after handling P. aeruginosa.

3. Protocol III: Quality Control of PA14NR Set

1. Select random set of mutants from newly replicated plates to detect possible interwell contamination (testing of 30-40 mutants is recommended).
   NOTE: In cases where it is necessary to confirm the identity of a mutant being used for characterization of a specific gene, it is recommended to perform PCR amplifications using gene-specific primers designed for the known sequence of the gene that contains the transposon insertion. Although more challenging, the benefits of using arbitrary PCR primers rather than gene-specific PCR primers when amplyfing DNA fragments from transposon mutants include ease of large-scale mutant confirmation and the ability to detect the presence of potential contaminants. For quality control purposes, it is not necessary to obtain high quality PCR sequencing data for all randomly slected mutants, as long as a sufficient number of mutants are screened to assess error rate.
2. Follow "Protocol II" to streak and grow mutant strains.
3. Autoclave all plasticware and non-sterile supplies before use.
4. Isolate genomic DNA by preferred method. For the analysis described in this work, a genomic DNA isolation kit was utilized following manufacturer's protocols. Multiple mutant strains may be analyzed simultaneously.
5. Measure genomic DNA concentration using a microvolume spectrophotometer. Adjust genomic DNA concentration to approximately 100 ng/µL.
6. Use genomic DNA as template to run "PCR1 reaction" as described in Step 1 of Table 3, generating Arb1 PCR fragments (Figure 3). Primers for this step are listed in Table 4.
7. Add 0.5 µL of 10x loading buffer to 5 µL of PCR1 reaction, load into a 1.5-2% agarose gel and run the gel at 80-150 V.
   NOTE: A specific fragment length or a particular fragment range is not expected, and more than one band may be present as the arbitrary primer may bind to multiple locations.
8. Use DNA from PCR1 reaction as template to run "PCR2 reaction" as described in Step 2 of Table 3, generating Arb2 PCR fragments (Figure 3). Primers for this step are listed in Table 4.
9. Add 0.5 µL of 10x loading buffer to 5 µL of PCR2 reaction, load into a 1.5-2% agarose gel, and run the gel at 80-150 V.
   NOTE: A specific fragment length or a particular fragment range is not expected, and more than one band may be present as the arbitrary primer may bind to multiple locations.
10. Send PCR2 reaction along with appropriate transposon-specific primer for sequencing.
11. Analize sequencing results byBLASTing them against the complete PA14 genome using the BLAST link provided on the PA14NR Library website (http://pa14.mgh.harvard.edu/cgi-bin/pa14/blast.cgi) or by directly BLASTing them against the specific gene sequence of the mutant of interest.
    NOTE: Information about selected mutants can be found by searching the PA14NR Set website (search http://pa14.mgh.harvard.edu/cgi-bin/pa14/search.cgi?searchType=SEARCH_PLATE_POSITIONS or download the Nonredundant Library.xls file from the link http://pa14.mgh.harvard.edu/cgi-bin/pa14/downloads.cgi).

Figure 3: PCR amplification and sequencing of transposon insertion mutants. Schematic view of steps involved in PCR amplification and sequencing for verification of mutant identity. Please click here to view a larger version of this figure.

Table 3: PCR reaction set-up and thermocycler conditions used for Arbitrary PCR. Arbitrary PCR reactions are performed sequentially, and fragments generated during PCR1 reaction are used as template in PCR2 reaction. Specific thermocycler settings are used for each set of reactions.

Table 4: List of Primers used in Quality Control Experiments. Primers used for PCR amplification and sequencing of transposon insertion mutants to confirm mutant identity.