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

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.