Isolation of Gram-Negative Bacteria with Genomic Transposon Insertions

Abstract

Source: Kazi, M. I., et. al., Generating Transposon Insertion Libraries in Gram-Negative Bacteria for High-Throughput Sequencing. J. Vis. Exp. (2020)

This video demonstrates the selection, quantification, and preservation of Gram-negative recipient bacteria carrying transposon insertions based on kanamycin resistance.

Protocol

Generation of the final bacterial mutant library

1. Thaw an aliquot of the frozen mating on ice.
2. Plate mating on 150 mm Luria-Bertani (LB) agar plates supplemented with kanamycin based on colony-forming units (CFU) calculated in step 3.1. Adjust the volume with LB to yield 13,333 colonies per 150 µL before plating.
   NOTE: The CFU count here was determined to be about 10⁵ CFU/mL, so the mating volume was adjusted to obtain 13,333 colonies per plate. This would provide an optimal number of colonies on 30 plates for a high-resolution mutant library without overcrowding the plates.
3. Use sterile glass beads to spread 150 µL of the dilution per plate on 30 x 150 mm Luria-Bertani agar plates supplemented with kanamycin to obtain 400,000 colonies (Figure 1C).
   NOTE: Sterile rods or any kind of sterile spreader tool (i.e., glass beads) may be used to spread the bacteria on plates.
4. Dispose of the used tube containing excess mating.
   NOTE: Freeze/thaw cycles add selective pressures on the bacterial culture, which can skew the Tn-seq experiment results. Use a fresh aliquot each time.
5. Incubate plates at 37 °C for 14 h.
   NOTE: The incubation time is optimized to prevent overgrowth (colonies touching). Minimizing growth by reducing the incubation time is suggested.

Estimating library density and pooling for storage

1. Count CFUs on each plate to estimate the total mutants in the transposon library. Count 20% of at least 3 plates to determine the colony count estimate for the entire group of plates (Figure 2A). Ensure that the colonies are not touching another colony.
2. After calculating the estimated colony yield, add 3-5 mL of LB (or more if needed) to each plate and scrape off the bacteria using a sterile scraping tool.NOTE: Sterile inoculating loops were used to efficiently scrape plates.
3. Pool bacterial suspensions from all plates into 50 mL conical tubes (Figure 1D). This will require multiple 50 mL conical tubes, at least 3.
4. Pellet pooled bacterial suspension using centrifugation at 5,000 x g for 7 min.
5. Discard the supernatant and resuspend the pellet in 5 mL of LB supplemented with 30% glycerol.
6. Aliquot 1 mL of the transposon library into cryovials and store at -80 °C.

Results

Figure 1: Schematic of transposon mutant library construction. (A) Bacterial conjugation. The &#...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
150mm x 15mm Petri Dishes	Corning	351058
50mL Conical Sterile Polypropylene Centrifuge Tubes	Fisher Scientific	12-565-271
Acinetobacter baumannii ATCC 17978	ATCC	N/A	AmpS, KanS
Escherichia coli MFD Dap-	N/A	N/A	DAP Auxotroph, requires 600 mM exogenously added DAP to grow. Contains RP4 machinery for plasmid transfer. Carrier for JNW68 (36).
Externally Threaded Cryogenic Vials	Corning	09-761-71
Glass beads	Corning	72684
Glycerol	Fisher Scientific	G33
Inoculating loops	Fisher Scientific	22-363-602	Scraping tool
Kanamycin	Fisher Scientific	BP906	Used at 25 mg/L
LB agar, Miller	Fisher Scientific	BP1425
LB broth, Miller	Fisher Scientific	BP1426
Phosphate Buffered Saline, 10X Solution	Fisher Scientific	BP39920	Diluted to 1X