This work was supported by funding from the National Institute of Health (Grant AI146829 to J.M.B.) and is gratefully acknowledged.

1. Bacterial strain preparation
<ol>
	<li>Streak the &#8220;donor&#8221; strain (E. coli MFD DAP-/pJNW684, Table of Materials) for isolated colonies on Luria-Bertani agar supplemented with 600 &#956;M diaminopimelic acid (DAP), 100 mg/L of ampicillin and 25 mg/L of kanamycin. Incubate overnight at 37 &#176;C. Using a single isolated colony, inoculate 50 mL of Luria broth (LB) supplemented with 600 &#956;M DAP, 100 mg/L of ampicillin and 25 mg/L of kanamycin in a 250 mL Erlenmeyer flask and label it as &#8220;donor&#8221;.</li>
	<li>Streak the &#8220;recipient&#8221; strain (A. baumannii strain ATCC 17978, Table of Materials) for isolated colonies on Luria-Bertani agar. Incubate overnight at 37 &#176;C. Using a single isolated colony, inoculate 50 mL of LB in a 250 mL Erlenmeyer flask and label it as &#8220;recipient&#8221;.</li>
	<li>Incubate both cultures (&#8220;donor&#8221; and &#8220;recipient&#8221;) overnight at 37 &#176;C with shaking.</li>
</ol>
2. Bacterial mating
<ol>
	<li>Transfer overnight cultures to 50 mL conical tubes.</li>
	<li>Pellet both recipient and donor cultures using centrifugation at 5,000 x g for 7 min.</li>
	<li>Discard the supernatant and resuspend the &#8220;donor&#8221; strain pellet in 35 mL of LB supplemented with DAP to wash away residual antibiotics.</li>
	<li>Pellet the &#8220;donor&#8221; strain cells using centrifugation at 5,000 x g for 7 min.</li>
	<li>Discard the supernatant and resuspend the &#8220;donor&#8221; strain pellet in 4.5 mL of LB supplemented with DAP. Use a 10 mL serological pipette.</li>
	<li>Transfer the resuspended &#8220;donor&#8221; strain into &#8220;recipient&#8221; strain tube, which contains the pelleted &#8220;recipient&#8221; 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.</li>
	<li>Distribute the mating suspension as individual 100 &#956;L droplets on LB agar plates supplemented with DAP (5-7 droplets per plate) (Figure 1A).</li>
	<li>Incubate plates at room temperature for 30 min.</li>
	<li>Without disturbing the droplets, carefully transfer plates to a 37 &#176;C incubator and allow cultures to mate for 1 h. 
	NOTE: Incubation periods exceeding 1 h risks generation of sister mutants.</li>
	<li>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.</li>
	<li>Combine harvested cells in a 50 mL conical tube.</li>
	<li>Pellet the mated cells using centrifugation at 5,000 x g for 7 min.</li>
	<li>Discard the supernatant and resuspend cells in 50 mL of LB to remove residual DAP.</li>
	<li>Pellet the mating using centrifugation at 5,000 x g for 7 min.</li>
	<li>Repeat the wash step (steps 2.13 and 2.14).</li>
	<li>Using a 10 mL serological pipette, resuspend the pellet in 10 mL of LB supplemented with 25% glycerol.</li>
	<li>Using washed cells, make five serial dilutions in LB broth (1:10, 1:100, 1:1000, 1:10,000, 1:100,000).</li>
	<li>Spread 100 &#956;L 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.</li>
	<li>Incubate plates at 37 &#176;C overnight.</li>
	<li>Aliquot the remaining mating in 1 mL aliquots and store at -80 &#176;C.</li>
</ol>
3. Determine the appropriate dilution of transposon library
<ol>
	<li>Record colony-forming units (CFU) from overnight plates.</li>
	<li>Image a plate with countable colonies for each different plate condition (Figure 2A). 
	NOTE: Both &#8220;donor&#8221; and &#8220;recipient&#8221; strains should grow on agar plates supplemented with DAP, so most plated dilutions will yield a lawn. Only the &#8220;recipient&#8221; strain can grow on agar plates. Neither the &#8220;donor&#8221; nor the &#8220;recipient&#8221; 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.</li>
	<li>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).</li>
</ol>
4. Generation of final bacterial mutant library
<ol>
	<li>Thaw an aliquot of the frozen mating on ice.</li>
	<li>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 &#181;L 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.</li>
	<li>Use sterile glass beads to spread 150 &#181;L 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.</li>
	<li>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.</li>
	<li>Incubate plates at 37 &#176;C for 14 h. 
	NOTE: The incubation time is optimized to prevent overgrowth (colonies touching). Minimizing growth by reducing the incubation time is suggested.</li>
</ol>
5. Estimating library density and pooling for storage
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>Pool bacterial suspensions from all plates into 50 mL conical tubes (Figure 1D). This will require multiple 50 mL conical tubes, at least 3.</li>
	<li>Pellet pooled bacterial suspension using centrifugation at 5,000 x g for 7 min.</li>
	<li>Discard the supernatant and resuspend pellet in 5 mL of LB supplemented with 30% glycerol.</li>
	<li>Aliquot 1 mL of the transposon library into cryovials and store at -80 &#176;C.</li>
</ol>
6. Identification of factors that regulate colistin resistance in A. baumannii
<ol>
	<li>Prepare 4 x 250 mL Erlenmeyer flasks with each containing 50 mL LB broth and label as (Figure 2B)
	<ol>
		<li>A. baumannii strain ATCC 17978 Tn-seq library; (-) colistin_1</li>
		<li>A. baumannii strain ATCC 17978 Tn-seq library; (-) colistin_2</li>
		<li>A. baumannii strain ATCC 17978 Tn-seq library; (+) colistin_1</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Add 50 &#181;L of 0.5 mg/L colistin to (+) colistin flasks (6.1.3 and 6.1.4) and 50 &#181;L water to (-) colistin flasks (6.1.1 and 6.1.2).</li>
	<li>Thaw a frozen Tn-seq library aliquot from step 5 on ice.</li>
	<li>Pipette 1 &#181;L of the thawed library into 1 mL of PBS.</li>
	<li>Measure the optical density at 600 nm (OD600) and multiply by 1,000. 
	NOTE: This determines OD600 of 1 &#956;L of the Tn-library.</li>
	<li>Based on the calculation in step 6.5, inoculate each flask containing 50 mL LB to a final OD600 0.001.</li>
	<li>Grow cultures in a shaking incubator at 37 &#176;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.</li>
	<li>Harvest cultures using centrifugation at 5,000 x g at 4 &#176;C for 7 min.</li>
	<li>Remove the supernatant and wash with 50 mL of PBS.</li>
	<li>Pellet using centrifugation at 5,000 x g at 4 &#176;C for 7 min.</li>
	<li>Remove the supernatant and resuspend the pellet in 1 mL of PBS. Aliquot ~ 200 &#181;L into 5 microcentrifuge tubes.</li>
	<li>Pellet using centrifugation at 5,000 x g at 4 &#176;C for 5 min. Remove all of the supernatant using a pipette tip.</li>
	<li>Store pellets at -20 &#176;C or proceed with gDNA extraction.</li>
</ol>
7. gDNA extraction
<ol>
	<li>Thaw one cell pellet on ice.</li>
	<li>Add 0.6 mL lysis buffer (Table of Materials) and vortex to completely resuspend the pellet.</li>
	<li>Incubate at 37 &#176;C for 1 h.</li>
	<li>Add 0.6 mL of phenol/chloroform/isoamyl alcohol to the sample and vortex vigorously.</li>
	<li>Separate phases using centrifugation in a microcentrifuge at max speed at room temp for 5 min.</li>
	<li>Transfer the upper aqueous phase to a new tube. Avoid disturbing the phase interface during transfer.</li>
	<li>Add an equal volume of chloroform to the aqueous phase obtained above and vortex vigorously.</li>
	<li>Separate phases using centrifugation in microcentrifuge at max speed at room temp for 5 min.</li>
	<li>Transfer upper aqueous phase to a new tube. 
	NOTE: Be sure to avoid disturbing the interface during transfer.</li>
	<li>Add 2.5x aqueous phase volume of cold 100 % ethanol and mix gently. Precipitated DNA will be visible.</li>
	<li>Place tube at -80 &#176;C for at least 1 h.</li>
	<li>Pellet DNA using centrifugation at max speed at 4 &#176;C for 30 min.</li>
	<li>Carefully remove supernatant without disturbing the DNA pellet and wash with 150 &#956;L of 70 % ethanol by pipetting.</li>
	<li>Pellet DNA using centrifugation at max speed at 4 &#176;C for 2 min.</li>
	<li>Carefully remove the supernatant.</li>
	<li>Repeat step 7.14 once. Carefully remove all remaining ethanol.</li>
	<li>Dry DNA by incubating at room temp for 5-10 min.</li>
	<li>Resuspend DNA pellet in 100 &#956;L TE buffer by pipetting.</li>
</ol>
8. DNA shearing (Figure 3A)
<ol>
	<li>Dilute gDNA with TE buffer to a concentration of 250 ng/mL in a total volume of 200 &#181;L.</li>
	<li>Place tubes in a water bath sonicator.</li>
	<li>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 &#176;C (Figure 3A).</li>
	<li>Confirm DNA is sheared appropriately by separating 10 &#181;L of unsheared DNA and 10 &#181;L of sheared DNA on a 1% agarose gel. Repeat sonication or optimize as needed (Figure 3B).</li>
</ol>
9. Poly-C tail addition to the 3&#8217; end (Figure 3A)
<ol>
	<li>Setup poly-C reaction according to the Table 1.</li>
	<li>Incubate reaction at 37 &#176;C for 1 h.</li>
	<li>Purify poly-C reaction with 40 &#181;L of size-selection paramagnetic beads (Figure 3A) by following the steps below.
	<ol>
		<li>Add 40 &#181;L of size-selection paramagnetic beads to each sample. Vortex ~ 5 s or pipette up and down.</li>
		<li>Incubate samples at room temperature for 5 min.</li>
		<li>Briefly, centrifuge tubes to collect liquid in the bottom of the tube (~ 2 s).</li>
		<li>Transfer tubes to a magnetic rack and incubate at room temperature for ~ 2 min until the solution is clear.</li>
		<li>With tubes on magnetic rack, carefully remove the supernatant.</li>
		<li>With tubes on magnetic rack, add 200 &#181;L of freshly prepared 80% ethanol (do not disturb beads).</li>
		<li>Incubate samples for at least 30 s until solution is clear.</li>
		<li>With tubes on magnetic rack, carefully remove the supernatant.</li>
		<li>Repeat the wash step (steps 9.3.6 - 9.3.8).</li>
		<li>Collect liquid in the bottom of the tube using a brief centrifugation step (~ 2 s).</li>
		<li>Transfer tubes to the magnetic rack and remove any remaining liquid.</li>
		<li>Incubate at room temperature for 2-5 min to dry samples. Do not over dry.</li>
		<li>Remove tubes from magnetic rack and add 25 &#181;L of water to each. Vortex for ~ 5 s or pipette up and down.</li>
		<li>Briefly, centrifuge tubes to collect liquid in the bottom of the tube (~ 2 s).</li>
		<li>Transfer tubes to the magnetic rack and allow to sit for ~ 2 min until the solution is clear.</li>
		<li>With tubes on the magnetic rack, remove liquid without disturbing the beads and transfer to a new tube (~ 23 &#181;L of DNA).</li>
	</ol>
	</li>
</ol>
10. Transposon junction amplification (Figure 3A)
<ol>
	<li>Setup PCR 1 as described in Table 1 for the first nested PCR (Table 2).</li>
	<li>Perform PCR 1 using conditions described in Table 1.</li>
	<li>Purify PCR products with 40 &#956;L of size-selection paramagnetic beads (steps 9.3.1 &#8211; 9.3-12). Elute in 50 &#956;L of water (steps 9.3.13 &#8211; 9.3.16). At this point the samples can be stored at -20 &#176;C.</li>
	<li>Prepare Streptavidin-coupled paramagnetic beads:
	<ol>
		<li>Resuspend Streptavidin beads by shaking vigorously.</li>
		<li>Add 32 &#956;L of beads per sample to a fresh microcentrifuge tube. 
		NOTE: Beads for 6 + samples can be prepared in a single tube.</li>
		<li>Transfer the tube to a magnetic rack. Incubate for ~ 2 min until solution is clear.</li>
		<li>With tube on magnetic rack, remove supernatant.</li>
		<li>Remove the tube from magnetic rack. Wash beads by resuspending in 1 mL 1x B&#38;W buffer by pipetting up and down.</li>
		<li>Transfer the tube to magnetic rack. Incubate for ~ 2 min until solution is clear.</li>
		<li>With the tube on magnetic rack, remove the supernatant.</li>
		<li>Repeat wash step with 1 mL 1x&#160;B&#38;W buffer (steps 10.4.5 &#8211; 10.4.7) twice more for a total of 3 washes.</li>
		<li>Remove tube from magnetic rack and resuspend beads in 52 &#956;L of 2x&#160;B&#38;W buffer per sample.</li>
	</ol>
	</li>
	<li>Combine 50 &#956;L of the prepared beads with 50 &#956;L of purified PCR1 (from step 10.3). Pipette to mix.</li>
	<li>Rotate at room temperature for 30 min.</li>
	<li>Wash away unbound DNA:
	<ol>
		<li>Briefly, centrifuge to collect liquid at the bottom of the tube (~ 2 s).</li>
		<li>Transfer the tube to magnetic rack. Incubate for ~ 2 min until the solution is clear.</li>
		<li>With the tube on magnetic rack, remove the supernatant.</li>
		<li>Remove the tube from magnetic rack, wash beads by resuspending in 100 &#956;L 1x B&#38;W buffer and pipetting up and down.</li>
		<li>Transfer tube to magnetic rack. Incubate for ~ 2 min until the solution is clear.</li>
		<li>With tube on magnetic rack, remove supernatant.</li>
		<li>Repeat wash steps with 100 &#956;L LoTE (steps 10.7.4 &#8211; 10.7.6) twice more for a total of 3 washes.</li>
	</ol>
	</li>
	<li>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).</li>
	<li>Perform PCR 2 using conditions described in Table 1.</li>
	<li>Purify with 40 &#956;L of size-selection paramagnetic beads (steps 9.3.1 &#8211; 9.3.12). Elute in 17 &#956;L of water (steps 9.3.13 &#8211; 9.3.16), collect ~ 15 &#956;L.</li>
	<li>Quantify DNA concentration using a fluorometer. The final concentrations should be ~ 50 to 250 ng/&#956;l.</li>
	<li>Assess DNA quality using chip-based capillary electrophoresis (Figure 4A).</li>
</ol>

The authors have nothing to disclose.

A. baumannii is an emerging threat to global public health due to the rapid acquisition of AMR against &#8220;last-line&#8221; therapeutics, such as colistin10,11,12,23,24,30,31. In recent decades, Tn-seq has played a critical role in elucidating genotype-phenotype interactions acro...

The discovery of antibiotics is undoubtedly one of the most impactful health-related events of the 20th 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 antibiotics1,2. 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 duration4,5. 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 organisms6,7,8.
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 death9,10,11,12,13. Colistin (polymyxin E) is a last-resort antimicrobial used to treat infections caused by multidrug resistant Gram-negative nosocomial pathogens, such as Acinetobacter baumannii14,15,16. First discovered in 1947, polymyxins are produced by the soil bacteria, Paenibacillus polymyxa17,18,19. Polymyxins were prescribed to treat Gram-negative infections for years before their clinical use was limited due to reports of significant nephro- and neurotoxicity20,21.
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 epidemic23,24. 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 &#946;-lactams, fluoroquinolones, tetracycline, and aminoglycosides23,24. 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 health10,11,12,13,27,30,31.
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 scale32,33,34,35. 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 &#955;pir gene contributed by the donor strain to replicate38. In absence of the &#955;pir gene, the pJNW684 vector carrying the transposition machinery will not be able to replicate in the A. baumannii recipient strain10,36,38. 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 population11,12,30,31. 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 transposons40,41. While several Tn-seq protocols generate 20,000 - 100,000 mutants35,42,43,44,45,46, 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.

<table><tbody><tr><td>10 mM ddCTP, 2&amp;rsquo;,3&amp;rsquo;-Dideoxycytidine-5&amp;rsquo;-Triphosphate</td><td>Affymetrix</td><td>77112</td><td></td></tr><tr><td>100 mM dCTP 2&amp;rsquo;-Deoxycytidine-5&amp;rsquo;-Triphosphate</td><td>Invitrogen</td><td>10217-016</td><td></td></tr><tr><td>100bp DNA Ladder Molecular Weight Marker</td><td>Promega</td><td>PR-G2101</td><td></td></tr><tr><td>100mm x 15mm Petri Dishes</td><td>Corning</td><td>351029</td><td></td></tr><tr><td>150mm x 15mm Petri Dishes</td><td>Corning</td><td>351058</td><td></td></tr><tr><td>1X B&amp;amp;W</td><td>N/A</td><td>N/A</td><td>Dilute 2X B&amp;amp;W by half to get 1X B&amp;amp;W.</td></tr><tr><td>2,6-Diaminopimelic acid</td><td>Alfa Aesar</td><td>B2239103</td><td>used at 600 &amp;micro;M</td></tr><tr><td>2X B&amp;amp;W</td><td>N/A</td><td>N/A</td><td>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.</td></tr><tr><td>50mL Conical Sterile Polypropylene Centrifuge Tubes</td><td>Fisher Scientific</td><td>12-565-271</td><td></td></tr><tr><td>9.5 mM dCTP/0.5 mM ddCTP</td><td>N/A</td><td>N/A</td><td>9.5 ml 100 mM dCTP; 5 ml 10 mM ddCTP; 85.5 ml water. Store at -20&amp;deg;C.</td></tr><tr><td>AccuPrimeTM Pfx DNA Polymerase</td><td>Invitrogen</td><td>12344</td><td></td></tr><tr><td>Acinetobacter baumannii ATCC 17978</td><td>ATCC</td><td>N/A</td><td>Amp&lt;sup&gt;S&lt;/sup&gt;, Kan&lt;sup&gt;S&lt;/sup&gt;</td></tr><tr><td>Ampicillin (100 mg/L)</td><td>Fisher Scientific</td><td>BP1760</td><td>used at 100 mg/L</td></tr><tr><td>AMPure XP PCR purification system</td><td>BECKMAN COULTER</td><td>A63881</td><td></td></tr><tr><td>BioAnalyzer</td><td>Agilent</td><td>G2939B</td><td></td></tr><tr><td>Bioanalyzer High Sensitivity DNA Analysis</td><td>Agilent</td><td>5067-4626</td><td></td></tr><tr><td>Deoxynucleotide Solution Mix (dNTP)</td><td>New England Biolabs (NEB)</td><td>N0447L</td><td></td></tr><tr><td>DynaMag-2 Magnetic rack</td><td>Invitrogen</td><td>12321D</td><td></td></tr><tr><td>E.coli MFD Dap-</td><td>N/A</td><td>N/A</td><td>DAP Auxotroph, requires 600 mM exogenously added DAP to grow. Contains RP4 machinery for plasmid transfer. Carrier for JNW68 (36).</td></tr><tr><td>Ethanol</td><td>Fisher Scientific</td><td>A4094</td><td></td></tr><tr><td>Externally Threaded Cryogenic Vials</td><td>Corning</td><td>09-761-71</td><td></td></tr><tr><td>Glass beads</td><td>Corning</td><td>72684</td><td></td></tr><tr><td>Glycerol</td><td>Fisher Scientific</td><td>G33</td><td></td></tr><tr><td>Inoculating loops</td><td>Fisher Scientific</td><td>22-363-602</td><td>Scraping tool</td></tr><tr><td>Kanamycin</td><td>Fisher Scientific</td><td>BP906</td><td>used at 25 mg/L</td></tr><tr><td>LB agar, Miller</td><td>Fisher Scientific</td><td>BP1425</td><td></td></tr><tr><td>LB broth, Miller</td><td>Fisher Scientific</td><td>BP1426</td><td></td></tr><tr><td>LoTE</td><td>N/A</td><td>N/A</td><td>Add 3 mM Tris-HCl, 0.2 mM EDTA (pH 7.5) in water. Used with Streptavidin beads. Solutions keep at room temperature.</td></tr><tr><td>Lysis buffer</td><td>N/A</td><td>N/A</td><td>9.34 mL TE buffer; 600 ml of 10% SDS; 60 ml of proteinase K (20 mg/mL)</td></tr><tr><td>Phenol/Chloroform/Isoamyl Alcohol (25:24:1 Mixture, pH 6.7/8.0, Liq.)</td><td>Fisher Scientific</td><td>BP1752I</td><td></td></tr><tr><td>Phosphate Buffered Saline, 10X Solution</td><td>Fisher Scientific</td><td>BP39920</td><td>Diluted to 1X</td></tr><tr><td>Qubit 4 Fluorometer</td><td>Thermo Fisher</td><td>Q33238</td><td></td></tr><tr><td>Qubit Assay Tubes</td><td>Thermo Fisher</td><td>Q32856</td><td></td></tr><tr><td>Qubit dsDNA HS Assay Kit</td><td>Thermo Fisher</td><td>Q32851</td><td></td></tr><tr><td>Sonicator with refridgerated waterbath</td><td>Qsonica Sonicators</td><td>Q2000FCE</td><td></td></tr><tr><td>Streptavidin Magnetic Beads</td><td>New England Biolabs (NEB)</td><td>S1420S</td><td></td></tr><tr><td>TE buffer</td><td>N/A</td><td>N/A</td><td>10 mM Tris-HCl (pH 8.0); 1 mM EDTA (pH 8.0)</td></tr><tr><td>Terminal Deoxynucleotidyl Transferase (rTdt)</td><td>Promega</td><td>PR-M1875</td><td></td></tr></tbody></table>

generating transposon insertion libraries in gram-negative bacteria for high-throughput sequencing

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 &#8220;last-line&#8221; 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.

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 &#955;pir+ donor strain to the A. baumannii recipient strain. This is an efficient and inexpensive method for generati...

Watch this Scientific Journal Video about Generating Transposon Insertion Libraries in Gram-Negative Bacteria for High-Throughput Sequencing at JoVE.com

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

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.

Transposon sequencing (Tn-seq) is a powerful method that combines transposon mutagenesis and massive parallel sequencing to identify genes and pathways ...

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Universidad de Cartagena UDEC

Research

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Biology

10.0K Views.  University of Texas at Arlington. 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.

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

Automated Gel Size Selection to Improve the Quality of Next-generation Sequencing Libraries Prepared from Environmental Water Samples

Next-generation sequencing of environmental samples can be challenging because of the variable DNA quantity and quality in these samples. High quality DNA libraries are needed for optimal results from next-generation sequencing. Environmental samples such as water may have low quality and quantities of DNA as well as contaminants that co-precipitate with DNA. The mechanical and enzymatic processes involved in extraction and library preparation may further damage the DNA. Gel size selection enables purification and recovery of DNA fragments of a defined size for sequencing applications. Nevertheless, this task is one of the most time-consuming steps in the DNA library preparation workflow. The protocol described here enables complete automation of agarose gel loading, electrophoretic analysis, and recovery of targeted DNA fragments. 
In this study, we describe a high-throughput approach to prepare high quality DNA libraries from freshwater samples that can be applied also to other environmental samples. We used an indirect approach to concentrate bacterial cells from environmental freshwater samples; DNA was extracted using a commercially available DNA extraction kit, and DNA libraries were prepared using a commercial transposon-based protocol. DNA fragments of 500 to 800 bp were gel size selected using Ranger Technology, an automated electrophoresis workstation. Sequencing of the size-selected DNA libraries demonstrated significant improvements to read length and quality of the sequencing reads.

This manuscript describes an automated gel size selection approach for purifying DNA fragments for next-generation sequencing. The Ranger Technology provides complete automation of the entire process of agarose gel loading, electrophoretic analysis, and recovery of targeted DNA fragments allowing for high-throughput and high quality next-generation sequencing libraries.

Next-generation sequencing of environmental samples can be challenging because of the variable DNA quantity and quality in these samples. High quality DNA ...

Environment

Determination of the Optimal Chromosomal Location(s) for a DNA Element in Escherichia coli Using a Novel Transposon-mediated Approach

The optimal chromosomal position(s) of a given DNA element was/were determined by transposon-mediated random insertion followed by fitness selection. In bacteria, the impact of the genetic context on the function of a genetic element can be difficult to assess. Several mechanisms, including topological effects, transcriptional interference from neighboring genes, and/or replication-associated gene dosage, may affect the function of a given genetic element. Here, we describe a method that permits the random integration of a DNA element into the chromosome of Escherichia coli and select the most favorable locations using a simple growth competition experiment. The method takes advantage of a well-described transposon-based system of random insertion, coupled with a selection of the fittest clone(s) by growth advantage, a procedure that is easily adjustable to experimental needs. The nature of the fittest clone(s) can be determined by whole-genome sequencing on a complex multi-clonal population or by easy gene walking for the rapid identification of selected clones. Here, the non-coding DNA region DARS2, which controls the initiation of chromosome replication in E. coli, was used as an example. The function of DARS2 is known to be affected by replication-associated gene dosage; the closer DARS2 gets to the origin of DNA replication, the more active it becomes. DARS2 was randomly inserted into the chromosome of a DARS2-deleted strain. The resultant clones containing individual insertions were pooled and competed against one another for hundreds of generations. Finally, the fittest clones were characterized and found to contain DARS2 inserted in close proximity to the original DARS2 location.

Determination of the Optimal Chromosomal Locations for a DNA Element in Escherichia coli Using a Novel Transposon-mediated Approach

Here, the power of a transposon-mediated random insertion of a non-coding DNA element was used to resolve its optimal chromosomal position.

The optimal chromosomal position(s) of a given DNA element was/were determined by transposon-mediated random insertion followed by fitness selection. In ...

Genetics

Generating Transgenic Plants with Single-copy Insertions Using BIBAC-GW Binary Vector

When generating transgenic plants, generally the objective is to have stable expression of a transgene. This requires a single, intact integration of the transgene, as multi-copy integrations are often subjected to gene silencing. The Gateway-compatible binary vector based on bacterial artificial chromosomes (pBIBAC-GW), like other pBIBAC derivatives, allows the insertion of single-copy transgenes with high efficiency. As an improvement to the original pBIBAC, a Gateway cassette has been cloned into pBIBAC-GW, so that the sequences of interest can now be easily incorporated into the vector transfer DNA (T-DNA) by Gateway cloning. Commonly, the transformation with pBIBAC-GW results in an efficiency of 0.2&#8211;0.5%, whereby half of the transgenics carry an intact single-copy integration of the T-DNA. The pBIBAC-GW vectors are available with resistance to Glufosinate-ammonium or DsRed fluorescence in seed coats for selection in plants, and with resistance to kanamycin as a selection in bacteria. Here, a series of protocols is presented that guide the reader through the process of generating transgenic plants using pBIBAC-GW: starting from recombining the sequences of interest into the pBIBAC-GW vector of choice, to plant transformation with Agrobacterium, selection of the transgenics, and testing the plants for intactness and copy number of the inserts using DNA blotting. Attention is given to designing a DNA blotting strategy to recognize single- and multi-copy integrations at single and multiple loci.

Using a pBIBAC-GW binary vector makes generating transgenic plants with intact single-copy insertions, an easy process. Here, a series of protocols is presented that guide the reader through the process of generating transgenic Arabidopsis plants, and testing the plants for intactness and copy number of the inserts.

When generating transgenic plants, generally the objective is to have stable expression of a transgene. This requires a single, intact integration of the ...

Genetic Mapping of Thermotolerance Differences Between Species of Saccharomyces Yeast via Genome-Wide Reciprocal Hemizygosity Analysis

A central goal of modern genetics is to understand how and why organisms in the wild differ in phenotype. To date, the field has advanced largely on the strength of linkage and association mapping methods, which trace the relationship between DNA sequence variants and phenotype across recombinant progeny from matings between individuals of a species. These approaches, although powerful, are not well suited to trait differences between reproductively isolated species. Here we describe a new method for genome-wide dissection of natural trait variation that can be readily applied to incompatible species. Our strategy, RH-seq, is a genome-wide implementation of the reciprocal hemizygote test. We harnessed it to identify the genes responsible for the striking high temperature growth of the yeast Saccharomyces cerevisiae relative to its sister species S. paradoxus. RH-seq utilizes transposon mutagenesis to create a pool of reciprocal hemizygotes, which are then tracked through a high-temperature competition via high-throughput sequencing. Our RH-seq workflow as laid out here provides a rigorous, unbiased way to dissect ancient, complex traits in the budding yeast clade, with the caveat that resource-intensive deep sequencing is needed to ensure genomic coverage for genetic mapping. As sequencing costs drop, this approach holds great promise for future use across eukaryotes.

Genetic Mapping of Thermotolerance Differences Between Species of Saccharomyces Yeast via Genome-Wide Reciprocal Hemizygosity Analysis

Reciprocal hemizygosity via sequencing (RH-seq) is a powerful new method to map the genetic basis of a trait difference between species. Pools of hemizygotes are generated by transposon mutagenesis and their fitness is tracked through competitive growth using high-throughout sequencing. Analysis of the resulting data pinpoints genes underlying the trait.

A central goal of modern genetics is to understand how and why organisms in the wild differ in phenotype. To date, the field has advanced largely on the ...

The Production of C. elegans Transgenes via Recombineering with the galK Selectable Marker

The creation of transgenic animals is widely utilized in C. elegans research including the use of GFP fusion proteins to study the regulation and expression pattern of genes of interest or generation of tandem affinity purification (TAP) tagged versions of specific genes to facilitate their purification. Typically transgenes are generated by placing a promoter upstream of a GFP reporter gene or cDNA of interest, and this often produces a representative expression pattern. However, critical elements of gene regulation, such as control elements in the 3' untranslated region or alternative promoters, could be missed by this approach. Further only a single splice variant can be usually studied by this means. In contrast, the use of worm genomic DNA carried by fosmid DNA clones likely includes most if not all elements involved in gene regulation in vivo which permits the greater ability to capture the genuine expression pattern and timing. To facilitate the generation of transgenes using fosmid DNA, we describe an E. coli based recombineering procedure to insert GFP, a TAP-tag, or other sequences of interest into any location in the gene. The procedure uses the galK gene as the selection marker for both the positive and negative selection steps in recombineering which results in obtaining the desired modification with high efficiency. Further, plasmids containing the galK gene flanked by homology arms to commonly used GFP and TAP fusion genes are available which reduce the cost of oligos by 50% when generating a GFP or TAP fusion protein. These plasmids use the R6K replication origin which precludes the need for extensive PCR product purification. Finally, we also demonstrate a technique to integrate the unc-119 marker on to the fosmid backbone which allows the fosmid to be directly injected or bombarded into worms to generate transgenic animals. This video demonstrates the procedures involved in generating a transgene via recombineering using this method.

The Production of C. elegans Transgenes via Recombineering with the galK Selectable Marker

The ability to produce transgenes for Caenorhabditis elegans using genomic DNA carried by fosmids is particularly attractive as all of the native regulatory elements are retained. Described is a simple and robust procedure for the production of transgenes via recombineering with the galK selectable marker.

The creation of transgenic animals is widely utilized in C. elegans research including the use of GFP fusion proteins to study the regulation and ...

Creation of a Dense Transposon Insertion Library Using Bacterial Conjugation in Enterobacterial Strains Such As Escherichia Coli or Shigella flexneri

Transposon mutagenesis is a method that allows gene disruption via the random genomic insertion of a piece of DNA called a transposon. The protocol below outlines a method for high efficiency transfer between bacterial strains of a plasmid harboring a transposon containing a kanamycin resistance marker. The plasmid-borne transposase is encoded by a variant tnp gene that inserts the transposon into the genome of the recipient strain with very low insertional bias. This method thus allows the creation of large mutant libraries in which transposons have been inserted into unique genomic positions in a recipient strain of either Escherichia coli or Shigella flexneri bacteria. By using bacterial conjugation, as opposed to other methods such as electroporation or chemical transformation, large libraries with hundreds of thousands of unique clones can be created. This yields high-density insertion libraries, with insertions occurring as frequently as every 4-6 base pairs in non-essential genes. This method is superior to other methods as it allows for an inexpensive, easy to use, and high efficiency method for the creation of a dense transposon insertion library. The transposon library can be used in downstream applications such as transposon sequencing (Tn-Seq), to infer genetic interaction networks, or more simply, in mutational (forward genetic) screens.

Creation of a Dense Transposon Insertion Library Using Bacterial Conjugation in Enterobacterial Strains Such As Escherichia Coli or Shigella flexneri

Presented here is a simple method for creating a high-density transposon insertion library in Escherichia coli or Shigella flexneri using bacterial conjugation. This protocol allows the creation of a collection of hundreds of thousands of unique mutants in bacteria by the random genomic insertion of a transposon.

Transposon mutagenesis is a method that allows gene disruption via the random genomic insertion of a piece of DNA called a transposon. The protocol below ...

Immunology and Infection

High-throughput Parallel Sequencing to Measure Fitness of Leptospira interrogans Transposon Insertion Mutants During Golden Syrian Hamster Infection

In this manuscript, we describe a transposon sequencing (Tn-Seq) technique to identify and quantify Leptospira interrogans mutants altered in fitness during infection of Golden Syrian hamsters. Tn-Seq combines random transposon mutagenesis with the power of high-throughput sequencing technology. Animals are challenged with a pool of transposon mutants (input pool), followed by harvesting of blood and tissues a few days later to identify and quantify the number of mutants in each organ (output pools). The output pools are compared to the input pool to evaluate the in vivo fitness of each mutant. This approach enables screening of a large pool of mutants in a limited number of animals. With minor modifications, this protocol can be performed with any animal model of leptospirosis, reservoir host models such as rats and acute infection models such as hamsters, as well as in vitro studies. Tn-Seq provides a powerful tool to screen for mutants with in vivo and in vitro fitness defects.

High-throughput Parallel Sequencing to Measure Fitness of Leptospira interrogans Transposon Insertion Mutants During Golden Syrian Hamster Infection

We describe here a technique that combines transposon mutagenesis with high-throughput sequencing to identify and quantify transposon leptospiral mutants in tissues after a challenge of hamsters. This protocol can be used to screen mutants for survival and dissemination in animals and can also be applied to in vitro studies.

In this manuscript, we describe a transposon sequencing (Tn-Seq) technique to identify and quantify Leptospira interrogans mutants altered in fitness ...

Genome-wide Gene Deletions in Streptococcus sanguinis by High Throughput PCR

Transposon mutagenesis and single-gene deletion are two methods applied in genome-wide gene knockout in bacteria 1,2. Although transposon mutagenesis is less time consuming, less costly, and does not require completed genome information, there are two weaknesses in this method: (1) the possibility of a disparate mutants in the mixed mutant library that counter-selects mutants with decreased competition; and (2) the possibility of partial gene inactivation whereby genes do not entirely lose their function following the insertion of a transposon. Single-gene deletion analysis may compensate for the drawbacks associated with transposon mutagenesis. To improve the efficiency of genome-wide single gene deletion, we attempt to establish a high-throughput technique for genome-wide single gene deletion using Streptococcus sanguinis as a model organism. Each gene deletion construct in S. sanguinis genome is designed to comprise 1-kb upstream of the targeted gene, the aphA-3 gene, encoding kanamycin resistance protein, and 1-kb downstream of the targeted gene. Three sets of primers F1/R1, F2/R2, and F3/R3, respectively, are designed and synthesized in a 96-well plate format for PCR-amplifications of those three components of each deletion construct. Primers R1 and F3 contain 25-bp sequences that are complementary to regions of the aphA-3 gene at their 5' end. A large scale PCR amplification of the aphA-3 gene is performed once for creating all single-gene deletion constructs. The promoter of aphA-3 gene is initially excluded to minimize the potential polar effect of kanamycin cassette. To create the gene deletion constructs, high-throughput PCR amplification and purification are performed in a 96-well plate format. A linear recombinant PCR amplicon for each gene deletion will be made up through four PCR reactions using high-fidelity DNA polymerase. The initial exponential growth phase of S. sanguinis cultured in Todd Hewitt broth supplemented with 2.5% inactivated horse serum is used to increase competence for the transformation of PCR-recombinant constructs. Under this condition, up to 20% of S. sanguinis cells can be transformed using ~50 ng of DNA. Based on this approach, 2,048 mutants with single-gene deletion were ultimately obtained from the 2,270 genes in S. sanguinis excluding four gene ORFs contained entirely within other ORFs in S. sanguinis SK36 and 218 potential essential genes. The technique on creating gene deletion constructs is high throughput and could be easy to use in genome-wide single gene deletions for any transformable bacteria.

Genome-wide Gene Deletions in Streptococcus sanguinis by High Throughput PCR

An efficient genome-wide single gene mutation method has been established using Streptococcus sanguinis as a model organism. This method has achieved via high throughput recombinant PCRs and transformations.

Transposon  mutagenesis and single-gene deletion are two methods applied in genome-wide  gene knockout in bacteria 1,2. Although transposon mutagenesis is ...

Gene Digital Circuits Based on CRISPR-Cas Systems and Anti-CRISPR Proteins

Synthetic gene Boolean gates and digital circuits have a broad range of applications, from medical diagnostics to environmental care. The discovery of the CRISPR-Cas systems and their natural inhibitors-the anti-CRISPR proteins (Acrs)-provides a new tool to design and implement in vivo gene digital circuits. Here, we describe a protocol that follows the idea of the "Design-Build-Test-Learn" biological engineering cycle and makes use of dCas9/dCas12a together with their corresponding Acrs to establish small transcriptional networks, some of which behave like Boolean gates, in Saccharomyces cerevisiae. These results point out the properties of dCas9/dCas12a as transcription factors. In particular, to achieve maximal activation of gene expression, dSpCas9 needs to interact with an engineered scaffold RNA that collects multiple copies of the VP64 activation domain (AD). In contrast, dCas12a shall be fused, at the C terminus, with the strong VP64-p65-Rta (VPR) AD. Furthermore, the activity of both Cas proteins is not enhanced by increasing the amount of sgRNA/crRNA in the cell. This article also explains how to build Boolean gates based on the CRISPR-dCas-Acr interaction. The AcrIIA4 fused hormone-binding domain of the human estrogen receptor is the core of a NOT gate responsive to &#946;-estradiol, whereas AcrVAs synthesized by the inducible GAL1 promoter permits to mimic both YES and NOT gates with galactose as an input. In the latter circuits, AcrVA5, together with dLbCas12a, showed the best logic behavior.

CRISPR-Cas systems and anti-CRISPR proteins were integrated into the scheme of Boolean gates in Saccharomyces cerevisiae. The new small logic circuits showed good performance and deepened the understanding of both dCas9/dCas12a-based transcription factors and the properties of anti-CRISPR proteins.

Synthetic gene Boolean gates and digital circuits have a broad range of applications, from medical diagnostics to environmental care. The discovery of the ...

Bioengineering

Generation of Enterobacter sp. YSU Auxotrophs Using Transposon Mutagenesis

Prototrophic bacteria grow on M-9 minimal salts medium supplemented with glucose (M-9 medium), which is used as a carbon and energy source. Auxotrophs can be generated using a transposome. The commercially available, Tn5-derived transposome used in this protocol consists of a linear segment of DNA containing an R6K&#947; replication origin, a gene for kanamycin resistance and two mosaic sequence ends, which serve as transposase binding sites. The transposome, provided as a DNA/transposase protein complex, is introduced by electroporation into the prototrophic strain, Enterobacter sp. YSU, and randomly incorporates itself into this host&#8217;s genome. Transformants are replica plated onto Luria-Bertani agar plates containing kanamycin, (LB-kan) and onto M-9 medium agar plates containing kanamycin (M-9-kan). The transformants that grow on LB-kan plates but not on M-9-kan plates are considered to be auxotrophs. Purified genomic DNA from an auxotroph is partially digested, ligated and transformed into a pir+ Escherichia coli (E. coli) strain. The R6K&#947; replication origin allows the plasmid to replicate in pir+ E. coli strains, and the kanamycin resistance marker allows for plasmid selection. Each transformant possesses a new plasmid containing the transposon flanked by the interrupted chromosomal region. Sanger sequencing and the Basic Local Alignment Search Tool (BLAST) suggest a putative identity of the interrupted gene. There are three advantages to using this transposome mutagenesis strategy. First, it does not rely on the expression of a transposase gene by the host. Second, the transposome is introduced into the target host by electroporation, rather than by conjugation or by transduction and therefore is more efficient. Third, the R6K&#947; replication origin makes it easy to identify the mutated gene which is partially recovered in a recombinant plasmid. This technique can be used to investigate the genes involved in other characteristics of Enterobacter sp. YSU or of a wider variety of bacterial strains.

Generation of Enterobacter sp. YSU Auxotrophs Using Transposon Mutagenesis

Enterobacter sp. YSU grows in glucose minimal salts medium. Auxotrophs are generated by transforming it with a transposome which randomly inserts itself into the host genome. Mutants are found by replica plating from complex medium to minimal medium. Interrupted genes are identified by gene rescue and sequencing.

Prototrophic bacteria grow on M-9 minimal salts medium supplemented with glucose (M-9 medium), which is used as a carbon and energy source. Auxotrophs can ...

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

Summary

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Chapters in this video

Automated Gel Size Selection to Improve the Quality of Next-generation Sequencing Libraries Prepared from Environmental Water Samples

Determination of the Optimal Chromosomal Location(s) for a DNA Element in Escherichia coli Using a Novel Transposon-mediated Approach

Generating Transgenic Plants with Single-copy Insertions Using BIBAC-GW Binary Vector

Genetic Mapping of Thermotolerance Differences Between Species of Saccharomyces Yeast via Genome-Wide Reciprocal Hemizygosity Analysis

The Production of C. elegans Transgenes via Recombineering with the galK Selectable Marker

Creation of a Dense Transposon Insertion Library Using Bacterial Conjugation in Enterobacterial Strains Such As Escherichia Coli or Shigella flexneri

High-throughput Parallel Sequencing to Measure Fitness of Leptospira interrogans Transposon Insertion Mutants During Golden Syrian Hamster Infection

Genome-wide Gene Deletions in Streptococcus sanguinis by High Throughput PCR

Gene Digital Circuits Based on CRISPR-Cas Systems and Anti-CRISPR Proteins

Generation of Enterobacter sp. YSU Auxotrophs Using Transposon Mutagenesis