Name: generating transposon insertion libraries in gram-negative bacteria for high-throughput sequencing
Uploaded: 2020-07-07T13:00:00
Description: Watch this Scientific Journal Video about Generating Transposon Insertion Libraries in Gram-Negative Bacteria for High-Throughput Sequencing at JoVE.com

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 fl...

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 border="0" cellpadding="0" cellspacing="0" style="width:793px;" width="792">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:393px;">10 mM ddCTP, 2&#8217;,3&#8217;-Dideoxycytidine-5&#8217;-Triphosphate</td>
			<td style="width:114px;">Affymetrix</td>
			<td style="width:119px;">77112</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">100 mM dCTP 2&#8217;-Deoxycytidine-5&#8217;-Triphosphate</td>
			<td style="width:114px;">Invitrogen</td>
			<td style="width:119px;">10217-016</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">100bp DNA Ladder Molecular Weight Marker</td>
			<td style="width:114px;">Promega</td>
			<td style="width:119px;">PR-G2101</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">100mm x 15mm Petri Dishes</td>
			<td style="width:114px;">Corning</td>
			<td style="width:119px;">351029</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">150mm x 15mm Petri Dishes</td>
			<td style="width:114px;">Corning</td>
			<td style="width:119px;">351058</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1X B&#38;W</td>
			<td style="width:114px;">N/A</td>
			<td style="width:119px;">N/A</td>
			<td>Dilute 2X B&#38;W by half to get 1X B&#38;W.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2,6-Diaminopimelic acid</td>
			<td style="width:114px;">Alfa Aesar</td>
			<td style="width:119px;">B2239103</td>
			<td>used at 600 &#181;M</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2X B&#38;W</td>
			<td style="width:114px;">N/A</td>
			<td style="width:119px;">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 height="21">
			<td height="21" style="height:21px;">50mL Conical Sterile Polypropylene Centrifuge Tubes</td>
			<td style="width:114px;">Fisher Scientific</td>
			<td style="width:119px;">12-565-271</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">9.5 mM dCTP/0.5 mM ddCTP</td>
			<td style="width:114px;">N/A</td>
			<td style="width:119px;">N/A</td>
			<td>9.5 ml 100 mM dCTP; 5 ml 10 mM ddCTP; 85.5 ml water. Store at -20&#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">AccuPrimeTM Pfx DNA Polymerase</td>
			<td style="width:114px;">Invitrogen</td>
			<td style="width:119px;">12344</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Acinetobacter baumannii ATCC 17978</td>
			<td style="width:114px;">ATCC</td>
			<td style="width:119px;">N/A</td>
			<td>AmpS, KanS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ampicillin (100 mg/L)</td>
			<td style="width:114px;">Fisher Scientific</td>
			<td style="width:119px;">BP1760</td>
			<td>used at 100 mg/L</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">AMPure XP PCR purification system</td>
			<td style="width:114px;">BECKMAN COULTER</td>
			<td style="width:119px;">A63881</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BioAnalyzer</td>
			<td style="width:114px;">Agilent</td>
			<td style="width:119px;">G2939B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bioanalyzer High Sensitivity DNA Analysis</td>
			<td style="width:114px;">Agilent</td>
			<td style="width:119px;">5067-4626</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Deoxynucleotide Solution Mix (dNTP)</td>
			<td style="width:114px;">New England Biolabs (NEB)</td>
			<td style="width:119px;">N0447L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DynaMag-2 Magnetic rack</td>
			<td style="width:114px;">Invitrogen</td>
			<td style="width:119px;">12321D</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">E.coli MFD Dap-</td>
			<td style="width:114px;">N/A</td>
			<td style="width:119px;">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 height="21">
			<td height="21" style="height:21px;">Ethanol</td>
			<td style="width:114px;">Fisher Scientific</td>
			<td style="width:119px;">A4094</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Externally Threaded Cryogenic Vials</td>
			<td style="width:114px;">Corning</td>
			<td style="width:119px;">09-761-71</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glass beads</td>
			<td style="width:114px;">Corning</td>
			<td>72684</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glycerol</td>
			<td style="width:114px;">Fisher Scientific</td>
			<td style="width:119px;">G33</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Inoculating loops</td>
			<td style="width:114px;">Fisher Scientific</td>
			<td style="width:119px;">22-363-602</td>
			<td>Scraping tool</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Kanamycin</td>
			<td style="width:114px;">Fisher Scientific</td>
			<td style="width:119px;">BP906</td>
			<td>used at 25 mg/L</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LB agar, Miller</td>
			<td style="width:114px;">Fisher Scientific</td>
			<td style="width:119px;">BP1425</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LB broth, Miller</td>
			<td style="width:114px;">Fisher Scientific</td>
			<td style="width:119px;">BP1426</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LoTE</td>
			<td style="width:114px;">N/A</td>
			<td style="width:119px;">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 height="21">
			<td height="21" style="height:21px;">Lysis buffer</td>
			<td style="width:114px;">N/A</td>
			<td style="width:119px;">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 height="21">
			<td height="21" style="height:21px;">Phenol/Chloroform/Isoamyl Alcohol (25:24:1 Mixture, pH 6.7/8.0, Liq.)</td>
			<td style="width:114px;">Fisher Scientific</td>
			<td style="width:119px;">BP1752I</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phosphate Buffered Saline, 10X Solution</td>
			<td style="width:114px;">Fisher Scientific</td>
			<td style="width:119px;">BP39920</td>
			<td>Diluted to 1X</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Qubit 4 Fluorometer</td>
			<td style="width:114px;">Thermo Fisher</td>
			<td>Q33238</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Qubit Assay Tubes</td>
			<td style="width:114px;">Thermo Fisher</td>
			<td>Q32856</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Qubit dsDNA HS Assay Kit</td>
			<td style="width:114px;">Thermo Fisher</td>
			<td>Q32851</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sonicator with refridgerated waterbath</td>
			<td>Qsonica Sonicators</td>
			<td>Q2000FCE</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Streptavidin Magnetic Beads</td>
			<td style="width:114px;">New England Biolabs (NEB)</td>
			<td style="width:119px;">S1420S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TE buffer</td>
			<td style="width:114px;">N/A</td>
			<td style="width:119px;">N/A</td>
			<td>10 mM Tris-HCl (pH 8.0); 1 mM EDTA (pH 8.0)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Terminal Deoxynucleotidyl Transferase (rTdt)</td>
			<td style="width:114px;">Promega</td>
			<td style="width:119px;">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 ...

This method is significant because it can be used in diverse bacterial species to identify essential genes, antibiotic resistance genes and genes important for in vivo fitness during infection. The main advantage of this technique is the mechanical sharing of genomic DNA and Poly-CTL edition, which eliminate the need for expensive restriction enzymes, adaptor radiation and gel purification. The implications of this technique extend to the therapy of problematic bacterial infections, because genes identified as important for infection or antibiotic resistance, could serve as targets for novel anti-microbial therapeutics.This method can provide insight into complex genotype, phenotype relationships across gram-negative and gram-positive bacterial species, and improve our current understanding of bacterial genetics. To begin, transfer overnight cultures to 50 milliliter conical tubes, and pellet both recipient and donor cultures using centrifugation at 5, 000 times G for 7 minutes. Discard the supernatant.Use a 10 milliliter serological pipette to re-suspend the donor strain pellet in 4.5 milliliters of LB supplemented with diaminopimelic acid. Transfer the re-suspended donor strain into the recipient strain tube. And immediately distribute the mating suspension as individual 100 microliter droplets on LB agar plates, supplemented with diaminopimelic acid Incubate the plates at room temperature for 30 minutes.Then, carefully transfer the plates to at 37 degrees Celsius incubator and allow the cultures to mate for one hour. After the incubation add 1.5 milliliters of LB onto each plate and harvest the bacteria into a 50 milliliter conical tube. Pellet the mated cells using centrifugation at 5, 000 times G for seven minutes.Then, discard the supernatant and re-suspend the cells in 10 milliliters of LB, to remove residual diaminopimelic acid Pellet the cells again, and repeat the wash with LB.When finished use a 10 milliliter serological pipette to re-suspend the pellet in 10 milliliters of LB, supplemented with 25%glycerol. Spread the cells on plates, according to manuscript directions. Then, incubate the plates at 37 degrees Celsius, overnight.Create one milliliter aliquots of the remaining bacteria and store them at minus 80 degrees Celsius. Pour an aliquot of the frozen mating on ice. Then, use sterile glass beads to spread 150 microliters of the dilution, onto each 30 by 150 millimeter Luria-Bertani agar plate, supplemented with Kanamycin.Dispose of the used tube containing excess mating, and incubate plates at 37 degrees Celsius for 14 hours. After the incubation count the colony forming units on each plate to estimate the total mutants in the transposon library. Count 20%of at least three plates to determine the colony count estimate for the entire group of plates.After calculating the estimated colony yield, add three to five milliliters of LB to each plate, and scrape off the bacteria using a sterile scraping tool. Pull bacterial suspensions from all plates into 50 milliliter conical tubes, and pellet the suspensions by centrifuging at 5, 000 times G, for seven minutes. Discard the supernatant and re-suspend the pellet in five milliliters of LB, supplemented with 30%glycerol.Then, make one milliliter aliquots of the transposon library in cryovials and store them at minus 80 degrees Celsius. Dilute the gDNA with TE buffer to a concentration of 250 nanograms per microliter, in a total volume of 200 microliters, and place it in the water bath sonicator. Sonicate the DNA to yield fragments of approximately 300 nucleotides.Confirm that the DNA is sheared by running 10 microliters of unsheared DNA and 10 microliters of sheared DNA, on a 1%hog roast gel. Repeat the sonication, if needed. To add the Polly seed tail to the three prime end of the sheer DNA, set up the policy reaction as described in the text manuscript and incubate the reaction tubes for one hour at 37 degrees Celsius.To purify the policy reaction, add a 40 microliters of size selection paramagnetic beads to each sample and vortex the reaction tube. Incubate samples at room temperature for five minutes. Then, briefly centrifuge them to collect the liquid at the bottom of the tube.Transfer the tubes to a magnetic rack and incubate them at room temperature for two minutes or until the solution is clear. Carefully remove the supernatant and add 200 microliters of freshly prepared, 80%ethanol, without disturbing the beads. Incubate the samples until the solution is clear.Then remove the supernatant and repeat the ethanol wash. Briefly centrifuge the tubes and put them back in the magnetic rack, to remove any remaining liquid. Incubate the samples at room temperature, for two to five minutes, to allow them to dry.And add 25 microliters of water to each tube. Vortex them for approximately five seconds or pipette up and down. Then, centrifuge the tubes to collect liquid at the bottom.Transfer the tubes to the magnetic rack and let them sit for approximately two minutes until the solution is clear. Then, transfer the supernatant to a new tube without disturbing the beads. This protocol was used to generate a high density transposon library in A-baumannii strain ATCC 17978, through bacterial conjugation using E-coli MFD DAP auxotroph, which replicates the plasmid pJNW684.The pulled A-baumannii transposon mutant library was used to identify fitness factors, important for Colistin resistance under sub-inhibitory concentrations of the anti-microbial. After depleting the mutant library of genes that contribute to Colistin resistance, the total gDNA of the remaining library pull was isolated from control and experimental cultures. The gDNA was fragmented with mechanical shearing and a policy tail was added to the DNA fragments in preparation for sequencing.DNA concentrations were calculated and the samples were analyzed using chip based capillary electrophoresis, to confirm successful library builds. The most important thing to remember when attempting this procedure is to adjust the mating volume, based on CFU counts for specific target strains to generate a high resolution mutant library. Additionally, the recipient strain must be sensitive to the antibiotic resistance marker, using the transposon prior to mutagenesis.Following this procedure, gene deletion or sound saying method can be performed to knock out individual heads obtain from sequencing results and validate genes of interest under challenged conditions.

Research

JoVE Journal

Biology

A High Throughput Screen for Biomining Cellulase Activity from Metagenomic Libraries

Cellulose, the most abundant source of organic carbon on the planet, has wide-ranging industrial applications with increasing emphasis on biofuel ...

Competitive Genomic Screens of Barcoded Yeast Libraries

By virtue of advances in next generation sequencing technologies, we have access to new genome sequences almost daily.  The tempo of these advances is ...

High-throughput Saccharification Assay for Lignocellulosic Materials

Polysaccharides that make up plant lignocellulosic biomass can be broken down to produce a range of sugars that subsequently can be 
used in establishing ...

RNA Secondary Structure Prediction Using High-throughput SHAPE

Understanding the function of RNA involved in biological processes requires a thorough knowledge of RNA structure. Toward this end, the methodology dubbed ...

Generation of High Quality Chromatin Immunoprecipitation DNA Template for High-throughput Sequencing (ChIP-seq)

Generation of High Quality Chromatin Immunoprecipitation DNA Template for High-throughput Sequencing ChIP-seq

ChIP-sequencing (ChIP-seq) methods directly  offer whole-genome coverage, where combining chromatin immunoprecipitation  (ChIP) and massively parallel ...

Highly Efficient Ligation of Small RNA Molecules for MicroRNA Quantitation by High-Throughput Sequencing

MiRNA cloning and high-throughput sequencing, termed miR-Seq, stands alone as a transcriptome-wide approach to quantify miRNAs with single nucleotide ...

Recovery of Adult Zebrafish Hearts for High-throughput Applications

Use of the zebrafish model system for studying development, regeneration, and disease is expanding toward use of adult hearts for cell dissociation and ...

High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays ...

A Protocol for Functional Assessment of Whole-Protein Saturation Mutagenesis Libraries Utilizing High-Throughput Sequencing

Site-directed mutagenesis has long been used as a method to interrogate protein structure, function and evolution. Recent advances in massively-parallel ...