Isolation of Gram-Negative Bacteria with Genomic Transposon Insertions

Samenvatting

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 ....

Resultaten

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

Materialen

Lijst van materialen gebruikt in dit artikel

Naam	Bedrijf	Catalogusnummer	Opmerkingen
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