Generating Transposon Insertion Libraries in Gram-Negative Bacteria for High-Throughput Sequencing

Summary

We describe a method to generate saturating transposon mutant libraries in Gram-negative bacteria and subsequent preparation of DNA amplicon libraries for high-throughput sequencing. As an example, we focus on the ESKAPE pathogen, Acinetobacter baumannii, but this protocol is amenable to a wide range of Gram-negative organisms.

Abstract

Transposon sequencing (Tn-seq) is a powerful method that combines transposon mutagenesis and massive parallel sequencing to identify genes and pathways that contribute to bacterial fitness under a wide range of environmental conditions. Tn-seq applications are extensive and have not only enabled examination of genotype-phenotype relationships at an organism level but also at the population, community and systems levels. Gram-negative bacteria are highly associated with antimicrobial resistance phenotypes, which has increased incidents of antibiotic treatment failure. Antimicrobial resistance is defined as bacterial growth in the presence of otherwise lethal antibiotics. The “last-line” antimicrobial colistin is used to treat Gram-negative bacterial infections. However, several Gram-negative pathogens, including Acinetobacter baumannii can develop colistin resistance through a range of molecular mechanisms, some of which were characterized using Tn-seq. Furthermore, signal transduction pathways that regulate colistin resistance vary within Gram-negative bacteria. Here we propose an efficient method of transposon mutagenesis in A. baumannii that streamlines generation of a saturating transposon insertion library and amplicon library construction by eliminating the need for restriction enzymes, adapter ligation, and gel purification. The methods described herein will enable in-depth analysis of molecular determinants that contribute to A. baumannii fitness when challenged with colistin. The protocol is also applicable to other Gram-negative ESKAPE pathogens, which are primarily associated with drug resistant hospital-acquired infections.

Introduction

The discovery of antibiotics is undoubtedly one of the most impactful health-related events of the 20^th century. Not only do antibiotics quickly resolve serious bacterial infections, they also play a pivotal role in modern medicine. Major surgeries, transplants and advances in neonatal medicine and chemotherapy leave patients susceptible to life threatening infections and these therapies would not be possible without antibiotics^1,2. However, rapid development and spread of antibiotic resistance among human pathogens has significantly decreased the efficacy of all clinically important classes of antibiotics³. Many bacterial infections that were once easily cleared with antibiotics treatment, no longer respond to classic treatment protocols, causing a serious threat to global public health¹. Antimicrobial resistance (AMR) is where bacterial cells grow in otherwise lethal concentrations of antibiotics, regardless of the treatment duration^4,5. There is an urgent need to understand molecular and biochemical factors that regulate AMR, which will help guide alternative antimicrobial development. Specifically, ESKAPE pathogens are problematic in clinical settings and associated with extensive AMR. These include Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter spp. While several mechanisms contribute to AMR in ESKAPE pathogens, the latter four organisms are Gram-negative.

Gram-negative bacteria assemble a defining outer membrane that protects them from adverse environmental conditions. The outer membrane serves as a permeability barrier to restrict entry of toxic molecules, such as antibiotics, into the cell. Unlike other biological membranes, the outer membrane is asymmetrical. The outer leaflet is enriched with surface-exposed lipopolysaccharide, while the inner leaflet is a mixture of phospholipids⁶. Lipopolysaccharide molecules are anchored to the outer membrane by a conserved lipid A moiety embedded within the lipid bilayer⁷. The canonical lipid A domain of Escherichia coli lipopolysaccharide is required for the growth of most Gram-negative bacteria and is synthesized by a nine-step enzymatic pathway that is one of the most fundamental and conserved pathways in Gram-negative organisms^6,7,8.

Polymyxins are cationic antimicrobial peptides that target the lipid A domain of lipopolysaccharide to perturb the outer membrane and lyse the cell. The electrostatic interaction between positively charged residues of polymyxins and the negatively charged lipid A phosphate groups disrupt the bacterial cell membrane ultimately leading to cell death^{9,10,11,12,13}. Colistin (polymyxin E) is a last-resort antimicrobial used to treat infections caused by multidrug resistant Gram-negative nosocomial pathogens, such as Acinetobacter baumannii^14,15,16. First discovered in 1947, polymyxins are produced by the soil bacteria, Paenibacillus polymyxa^17,18,19. Polymyxins were prescribed to treat Gram-negative infections for years before their clinical use was limited due to reports of significant nephro- and neurotoxicity^20,21.

A. baumannii is a nosocomial Gram-negative pathogen that has dramatically increased the morbidity and mortality of patient outcomes over recent decades²². What was once regarded as a low-threat pathogen, now poses a significant risk for hospital-acquired infection throughout the world due to its incredible ability to acquire AMR and high risk of epidemic^23,24. A. baumannii accounts for more than 10% of nosocomial infections in the United States. Disease manifests as pneumonia, bacteremia, urinary tract infections, skin and soft tissue infections, meningitis, and endocarditis²⁵. Treatment options for A. baumannii infections have dwindled due to resistance against almost all antibiotic classes, including β-lactams, fluoroquinolones, tetracycline, and aminoglycosides^23,24. The prevalence of multidrug resistant, extensively drug-resistant and pan-drug resistant A. baumannii isolates has led to a resurgence in colistin treatment, which was thought to be one of the few remaining therapeutic options still effective against multidrug resistant A. baumannii. However, increased colistin resistance among A. baumannii isolates has further amplified its threat to the global public health^{10,11,12,13,27,30,31}.

Recent advances in high-throughput sequencing technologies, such as transposon sequencing (Tn-seq), have provided important tools to advance our understanding of bacterial fitness in vitro and in vivo. Tn-seq is a powerful tool that can be leveraged to study genotype-phenotype interactions in bacteria. Tn-seq is broadly applicable across bacterial pathogens, where it combines traditional transposon mutagenesis with massive parallel sequencing to rapidly map insertion sites, which can be used to link DNA mutations to phenotypic variants on a genome-wide scale^32,33,34,35. While transposon mutagenesis methods have been previously described, the general steps are similar³³. First, an insertion library is generated using transposon mutagenesis, where each bacterial cell within a population is restricted to a single transposon insertion within the genomic DNA (gDNA). Following mutagenesis, individual mutants are pooled. gDNA is extracted from the insertion mutant pool and the transposon junctions are amplified and subjected to high-throughput sequencing. The reads represent insertion sites, which can be mapped to the genome. Transposon insertions that reduce fitness quickly fall out of the population, while beneficial insertions are enriched. Tn-seq has been instrumental to advance our understanding of how genes impact bacterial fitness in stress³³.

The Himar1 mariner transposon system encoded in pJNW684 was specifically constructed and optimized for the purpose of transposon mutagenesis. It includes a mariner-family transposon flanking the kanamycin resistance gene, which is used for the selection of transposon insertion mutants in A. baumannii. It also encodes an A. baumannii specific promoter that drives expression of the transposase encoding gene³⁶. The mariner-based transposon also contains two translational terminators downstream of the kanamycin resistance gene, which prevents read-through downstream of the insertion³⁷. pJNW684 also carries a RP4/oriT/oriR6K-conditional origin of replication which requires the λpir gene contributed by the donor strain to replicate³⁸. In absence of the λpir gene, the pJNW684 vector carrying the transposition machinery will not be able to replicate in the A. baumannii recipient strain^10,36,38. Therefore, during bacterial conjugation, only the transposon is inserted into the recipient genome without background insertion of the plasmid, which carries the transposase gene. This is significant because the loss of transposase activity along with the plasmid results in single, stable transposition event that prevents the transposon from moving to different locations once it inserts into the recipient genome.

pJNW648 has also been tested for activity in another Gram-negative organism, E. coli. Successful assembly of a saturating Tn-seq library in E. coli strain W3110 indicated the system is amenable to perform mutagenesis in a wide range of pathogens, including Enterobacteriaceae. Furthermore, the A. baumannii specific promoter that drives transposase expression can quickly be exchanged with a species-specific promoter. Lastly, the kanamycin resistance gene can be exchanged for other resistance cassettes, depending on the AMR phenotype of the organism being studied.

One factor that contributes to colistin resistance in A. baumannii is administration of insufficient doses, where bacteria are exposed to selective pressure at non-lethal levels³⁹. Several reports showed that subinhibitory antimicrobial concentrations can induce regulated responses that alter cell physiology to reduce susceptibility of the entire bacterial population^11,12,30,31. Using Tn-seq, we discovered factors that regulate colistin resistance in A. baumannii strain ATCC 17978 after exposure to inhibitory¹⁰ and subinhibitory concentrations of colistin. This example details a Tn-seq method that streamlines the construction and enrichment of a saturated transposon mutant library using the mariner-based family of transposons^40,41. While several Tn-seq protocols generate 20,000 - 100,000 mutants^{35,42,43,44,45,46}, the protocol described herein can rapidly generate a transposon library of 400,000 + mutants, which roughly equates to a transposon insertion every 10-base pairs in A. baumannii¹⁰. Furthermore, the library size can be scaled up without significant additional effort. This method also eliminates the requirement for restriction endonucleases, adapter ligation and gel purification, which can reduce final library diversity.

Protocol

1. Bacterial strain preparation

1. Streak the “donor” strain (E. coli MFD DAP^-/pJNW684, Table of Materials) for isolated colonies on Luria-Bertani agar supplemented with 600 μM diaminopimelic acid (DAP), 100 mg/L of ampicillin and 25 mg/L of kanamycin. Incubate overnight at 37 °C. Using a single isolated colony, inoculate 50 mL of Luria broth (LB) supplemented with 600 μM DAP, 100 mg/L of ampicillin and 25 mg/L of kanamycin in a 250 mL Erlenmeyer flask and label it as “donor”.
2. Streak the “recipient” strain (A. baumannii strain ATCC 17978, Table of Materials) for isolated colonies on Luria-Bertani agar. Incubate overnight at 37 °C. Using a single isolated colony, inoculate 50 mL of LB in a 250 mL Erlenmeyer flask and label it as “recipient”.
3. Incubate both cultures (“donor” and “recipient”) overnight at 37 °C with shaking.

2. Bacterial mating

1. Transfer overnight cultures to 50 mL conical tubes.
2. Pellet both recipient and donor cultures using centrifugation at 5,000 x g for 7 min.
3. Discard the supernatant and resuspend the “donor” strain pellet in 35 mL of LB supplemented with DAP to wash away residual antibiotics.
4. Pellet the “donor” strain cells using centrifugation at 5,000 x g for 7 min.
5. Discard the supernatant and resuspend the “donor” strain pellet in 4.5 mL of LB supplemented with DAP. Use a 10 mL serological pipette.
6. Transfer the resuspended “donor” strain into “recipient” strain tube, which contains the pelleted “recipient” cells. Use the same 10 mL serological pipette from step 2.5 to mix the cultures. Immediately move to the next step.
   NOTE: The total volume of the final suspension should be 5 mL.
7. Distribute the mating suspension as individual 100 μL droplets on LB agar plates supplemented with DAP (5-7 droplets per plate) (Figure 1A).
8. Incubate plates at room temperature for 30 min.
9. Without disturbing the droplets, carefully transfer plates to a 37 °C incubator and allow cultures to mate for 1 h.
   NOTE: Incubation periods exceeding 1 h risks generation of sister mutants.
10. Following incubation, add 1.5 mL of LB onto each plate and harvest by resuspending bacteria from the plates. Use a 1 mL micropipette for resuspension. Final volume should be approximately 12 - 15 mL.
11. Combine harvested cells in a 50 mL conical tube.
12. Pellet the mated cells using centrifugation at 5,000 x g for 7 min.
13. Discard the supernatant and resuspend cells in 50 mL of LB to remove residual DAP.
14. Pellet the mating using centrifugation at 5,000 x g for 7 min.
15. Repeat the wash step (steps 2.13 and 2.14).
16. Using a 10 mL serological pipette, resuspend the pellet in 10 mL of LB supplemented with 25% glycerol.
17. Using washed cells, make five serial dilutions in LB broth (1:10, 1:100, 1:1000, 1:10,000, 1:100,000).
18. Spread 100 μL of each dilution on 4 different plates using sterile glass beads: Luria-Bertani agar supplemented with kanamycin, agar supplemented with ampicillin, agar supplemented with DAP and agar only.
19. Incubate plates at 37 °C overnight.
20. Aliquot the remaining mating in 1 mL aliquots and store at -80 °C.

3. Determine the appropriate dilution of transposon library

1. Record colony-forming units (CFU) from overnight plates.
2. Image a plate with countable colonies for each different plate condition (Figure 2A).
   NOTE: Both “donor” and “recipient” strains should grow on agar plates supplemented with DAP, so most plated dilutions will yield a lawn. Only the “recipient” strain can grow on agar plates. Neither the “donor” nor the “recipient” strain can grow on agar plates supplemented with ampicillin, so there should be none/minimal growth. Only target strain cells encoding the transposon insertion can grow on agar plates supplemented with kanamycin. Colonies should vary in size, indicating transposon insertions in genes that contribute to fitness on agar supplemented with kanamycin.
3. Calculate the number of transposon mutants in the frozen mating by counting the number of colonies on LB agar plates supplemented with kanamycin.
   NOTE: For the A. baumannii genome (approximately 4 Mbps), the goal was to obtain about 400,000 colonies in order to generate a high-resolution mutant library (approximately one transposon insertion/10 base pairs). However, this number should be optimized based on genome size of the target species).

4. Generation of final bacterial mutant library

1. Thaw an aliquot of the frozen mating on ice.
2. Plate mating on 150 mm Luria-Bertani agar plates supplemented with kanamycin based on CFUs 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 as 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 adds 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.

5. 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 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.

6. Identification of factors that regulate colistin resistance in A. baumannii

1. Prepare 4 x 250 mL Erlenmeyer flasks with each containing 50 mL LB broth and label as (Figure 2B)
   1. A. baumannii strain ATCC 17978 Tn-seq library; (-) colistin_1
   2. A. baumannii strain ATCC 17978 Tn-seq library; (-) colistin_2
   3. A. baumannii strain ATCC 17978 Tn-seq library; (+) colistin_1
   4. A. baumannii strain ATCC 17978 Tn-seq library; (+) colistin_2
      NOTE: In the challenge growth described here, each condition, (-) colistin control and (+) colistin challenge, is being tested in duplicate. Therefore, the setup requires 4 x 250 mL Erlenmeyer flasks, two per condition.
2. Add 50 µL of 0.5 mg/L colistin to (+) colistin flasks (6.1.3 and 6.1.4) and 50 µL water to (-) colistin flasks (6.1.1 and 6.1.2).
3. Thaw a frozen Tn-seq library aliquot from step 5 on ice.
4. Pipette 1 µL of the thawed library into 1 mL of PBS.
5. Measure the optical density at 600 nm (OD₆₀₀) and multiply by 1,000.
   NOTE: This determines OD₆₀₀ of 1 μL of the Tn-library.
6. Based on the calculation in step 6.5, inoculate each flask containing 50 mL LB to a final OD₆₀₀ 0.001.
7. Grow cultures in a shaking incubator at 37 °C to OD₆₀₀ 0.5.
   NOTE: It is important that cultures remain in logarithmic growth phase, so if your strain is at a different OD₆₀₀ during exponential growth, the OD₆₀₀ needs to be adjusted to ensure the culture replicates as many times as possible within logarithmic growth phase. If the culture only replicates 3 times, the power to detect fitness defects in mutants is capped at 3-fold differences, in theory. Since different bacteria have different doubling times, it is important to seed the cultures at a fixed OD₆₀₀ to normalize the starting inoculum. This ensures consistent representation of the entire library in all cultures.
8. Harvest cultures using centrifugation at 5,000 x g at 4 °C for 7 min.
9. Remove the supernatant and wash with 50 mL of PBS.
10. Pellet using centrifugation at 5,000 x g at 4 °C for 7 min.
11. Remove the supernatant and resuspend the pellet in 1 mL of PBS. Aliquot ~ 200 µL into 5 microcentrifuge tubes.
12. Pellet using centrifugation at 5,000 x g at 4 °C for 5 min. Remove all of the supernatant using a pipette tip.
13. Store pellets at -20 °C or proceed with gDNA extraction.

7. gDNA extraction

1. Thaw one cell pellet on ice.
2. Add 0.6 mL lysis buffer (Table of Materials) and vortex to completely resuspend the pellet.
3. Incubate at 37 °C for 1 h.
4. Add 0.6 mL of phenol/chloroform/isoamyl alcohol to the sample and vortex vigorously.
5. Separate phases using centrifugation in a microcentrifuge at max speed at room temp for 5 min.
6. Transfer the upper aqueous phase to a new tube. Avoid disturbing the phase interface during transfer.
7. Add an equal volume of chloroform to the aqueous phase obtained above and vortex vigorously.
8. Separate phases using centrifugation in microcentrifuge at max speed at room temp for 5 min.
9. Transfer upper aqueous phase to a new tube.
   NOTE: Be sure to avoid disturbing the interface during transfer.
10. Add 2.5x aqueous phase volume of cold 100 % ethanol and mix gently. Precipitated DNA will be visible.
11. Place tube at -80 °C for at least 1 h.
12. Pellet DNA using centrifugation at max speed at 4 °C for 30 min.
13. Carefully remove supernatant without disturbing the DNA pellet and wash with 150 μL of 70 % ethanol by pipetting.
14. Pellet DNA using centrifugation at max speed at 4 °C for 2 min.
15. Carefully remove the supernatant.
16. Repeat step 7.14 once. Carefully remove all remaining ethanol.
17. Dry DNA by incubating at room temp for 5-10 min.
18. Resuspend DNA pellet in 100 μL TE buffer by pipetting.

8. DNA shearing (Figure 3A)

1. Dilute gDNA with TE buffer to a concentration of 250 ng/mL in a total volume of 200 µL.
2. Place tubes in a water bath sonicator.
3. Sonicate DNA to yield fragments of approximately 300 nucleotides. Power: 60 %, Total Time: 20 min, Cycles: 10 s ON and 10 s OFF at 4 °C (Figure 3A).
4. Confirm DNA is sheared appropriately by separating 10 µL of unsheared DNA and 10 µL of sheared DNA on a 1% agarose gel. Repeat sonication or optimize as needed (Figure 3B).

9. Poly-C tail addition to the 3’ end (Figure 3A)

1. Setup poly-C reaction according to the Table 1.
2. Incubate reaction at 37 °C for 1 h.
3. Purify poly-C reaction with 40 µL of size-selection paramagnetic beads (Figure 3A) by following the steps below.
   1. Add 40 µL of size-selection paramagnetic beads to each sample. Vortex ~ 5 s or pipette up and down.
   2. Incubate samples at room temperature for 5 min.
   3. Briefly, centrifuge tubes to collect liquid in the bottom of the tube (~ 2 s).
   4. Transfer tubes to a magnetic rack and incubate at room temperature for ~ 2 min until the solution is clear.
   5. With tubes on magnetic rack, carefully remove the supernatant.
   6. With tubes on magnetic rack, add 200 µL of freshly prepared 80% ethanol (do not disturb beads).
   7. Incubate samples for at least 30 s until solution is clear.
   8. With tubes on magnetic rack, carefully remove the supernatant.
   9. Repeat the wash step (steps 9.3.6 - 9.3.8).
   10. Collect liquid in the bottom of the tube using a brief centrifugation step (~ 2 s).
   11. Transfer tubes to the magnetic rack and remove any remaining liquid.
   12. Incubate at room temperature for 2-5 min to dry samples. Do not over dry.
   13. Remove tubes from magnetic rack and add 25 µL of water to each. Vortex for ~ 5 s or pipette up and down.
   14. Briefly, centrifuge tubes to collect liquid in the bottom of the tube (~ 2 s).
   15. Transfer tubes to the magnetic rack and allow to sit for ~ 2 min until the solution is clear.
   16. With tubes on the magnetic rack, remove liquid without disturbing the beads and transfer to a new tube (~ 23 µL of DNA).

10. Transposon junction amplification (Figure 3A)

1. Setup PCR 1 as described in Table 1 for the first nested PCR (Table 2).
2. Perform PCR 1 using conditions described in Table 1.
3. Purify PCR products with 40 μL of size-selection paramagnetic beads (steps 9.3.1 – 9.3-12). Elute in 50 μL of water (steps 9.3.13 – 9.3.16). At this point the samples can be stored at -20 °C.
4. Prepare Streptavidin-coupled paramagnetic beads:
   1. Resuspend Streptavidin beads by shaking vigorously.
   2. Add 32 μL of beads per sample to a fresh microcentrifuge tube.
      NOTE: Beads for 6 + samples can be prepared in a single tube.
   3. Transfer the tube to a magnetic rack. Incubate for ~ 2 min until solution is clear.
   4. With tube on magnetic rack, remove supernatant.
   5. Remove the tube from magnetic rack. Wash beads by resuspending in 1 mL 1x B&W buffer by pipetting up and down.
   6. Transfer the tube to magnetic rack. Incubate for ~ 2 min until solution is clear.
   7. With the tube on magnetic rack, remove the supernatant.
   8. Repeat wash step with 1 mL 1x B&W buffer (steps 10.4.5 – 10.4.7) twice more for a total of 3 washes.
   9. Remove tube from magnetic rack and resuspend beads in 52 μL of 2x B&W buffer per sample.
5. Combine 50 μL of the prepared beads with 50 μL of purified PCR1 (from step 10.3). Pipette to mix.
6. Rotate at room temperature for 30 min.
7. Wash away unbound DNA:
   1. Briefly, centrifuge to collect liquid at the bottom of the tube (~ 2 s).
   2. Transfer the tube to magnetic rack. Incubate for ~ 2 min until the solution is clear.
   3. With the tube on magnetic rack, remove the supernatant.
   4. Remove the tube from magnetic rack, wash beads by resuspending in 100 μL 1x B&W buffer and pipetting up and down.
   5. Transfer tube to magnetic rack. Incubate for ~ 2 min until the solution is clear.
   6. With tube on magnetic rack, remove supernatant.
   7. Repeat wash steps with 100 μL LoTE (steps 10.7.4 – 10.7.6) twice more for a total of 3 washes.
8. Setup PCR 2 as described in Table 1 to amplify transposon junctions and add a single barcode to each sample (Table 2 and Table 3).
9. Perform PCR 2 using conditions described in Table 1.
10. Purify with 40 μL of size-selection paramagnetic beads (steps 9.3.1 – 9.3.12). Elute in 17 μL of water (steps 9.3.13 – 9.3.16), collect ~ 15 μL.
11. Quantify DNA concentration using a fluorometer. The final concentrations should be ~ 50 to 250 ng/μl.
12. Assess DNA quality using chip-based capillary electrophoresis (Figure 4A).

Results

The outlined methods describe the generation of a high-density transposon library in A. baumannii strain ATCC 17978 through bacterial conjugation using E. coli MFD DAP^-, which replicates the plasmid pJNW684 (Figure 4B). The detailed protocol uses bi-parental bacterial conjugation for transfer of pJNW684 from the E. coli λpir+ donor strain to the A. baumannii recipient strain. This is an efficient and inexpensive method for generati...

Discussion

A. baumannii is an emerging threat to global public health due to the rapid acquisition of AMR against “last-line” therapeutics, such as colistin^{10,11,12,23,24,30,31}. In recent decades, Tn-seq has played a critical role in elucidating genotype-phenotype interactions acro...

Materials

Name	Company	Catalog Number	Comments
10 mM ddCTP, 2’,3’-Dideoxycytidine-5’-Triphosphate	Affymetrix	77112
100 mM dCTP 2’-Deoxycytidine-5’-Triphosphate	Invitrogen	10217-016
100bp DNA Ladder Molecular Weight Marker	Promega	PR-G2101
100mm x 15mm Petri Dishes	Corning	351029
150mm x 15mm Petri Dishes	Corning	351058
1X B&W	N/A	N/A	Dilute 2X B&W by half to get 1X B&W.
2,6-Diaminopimelic acid	Alfa Aesar	B2239103	used at 600 µM
2X B&W	N/A	N/A	Add 2 M NaCl, 10 mM Tris-HCl, 1 mM EDTA (pH 7.5) in water. Used with Streptavidin beads. Solutions keep at room temperature.
50mL Conical Sterile Polypropylene Centrifuge Tubes	Fisher Scientific	12-565-271
9.5 mM dCTP/0.5 mM ddCTP	N/A	N/A	9.5 ml 100 mM dCTP; 5 ml 10 mM ddCTP; 85.5 ml water. Store at -20°C.
AccuPrimeTM Pfx DNA Polymerase	Invitrogen	12344
Acinetobacter baumannii ATCC 17978	ATCC	N/A	AmpS, KanS
Ampicillin (100 mg/L)	Fisher Scientific	BP1760	used at 100 mg/L
AMPure XP PCR purification system	BECKMAN COULTER	A63881
BioAnalyzer	Agilent	G2939B
Bioanalyzer High Sensitivity DNA Analysis	Agilent	5067-4626
Deoxynucleotide Solution Mix (dNTP)	New England Biolabs (NEB)	N0447L
DynaMag-2 Magnetic rack	Invitrogen	12321D
E.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).
Ethanol	Fisher Scientific	A4094
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
LoTE	N/A	N/A	Add 3 mM Tris-HCl, 0.2 mM EDTA (pH 7.5) in water. Used with Streptavidin beads. Solutions keep at room temperature.
Lysis buffer	N/A	N/A	9.34 mL TE buffer; 600 ml of 10% SDS; 60 ml of proteinase K (20 mg/mL)
Phenol/Chloroform/Isoamyl Alcohol (25:24:1 Mixture, pH 6.7/8.0, Liq.)	Fisher Scientific	BP1752I
Phosphate Buffered Saline, 10X Solution	Fisher Scientific	BP39920	Diluted to 1X
Qubit 4 Fluorometer	Thermo Fisher	Q33238
Qubit Assay Tubes	Thermo Fisher	Q32856
Qubit dsDNA HS Assay Kit	Thermo Fisher	Q32851
Sonicator with refridgerated waterbath	Qsonica Sonicators	Q2000FCE
Streptavidin Magnetic Beads	New England Biolabs (NEB)	S1420S
TE buffer	N/A	N/A	10 mM Tris-HCl (pH 8.0); 1 mM EDTA (pH 8.0)
Terminal Deoxynucleotidyl Transferase (rTdt)	Promega	PR-M1875