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.

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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 production 1. Chemical methods to modify or degrade cellulose typically require strong acids and high temperatures. As such, enzymatic methods have become prominent in the bioconversion process. While the identification of active cellulases from bacterial and fungal isolates has been somewhat effective, the vast majority of microbes in nature resist laboratory cultivation. Environmental genomic, also known as metagenomic, screening approaches have great promise in bridging the cultivation gap in the search for novel bioconversion enzymes. Metagenomic screening approaches have successfully recovered novel cellulases from environments as varied as soils 2, buffalo rumen 3 and the termite hind-gut 4 using carboxymethylcellulose (CMC) agar plates stained with congo red dye (based on the method of Teather and Wood 5). However, the CMC method is limited in throughput, is not quantitative and manifests a low signal to noise ratio 6. Other methods have been reported 7,8 but each use an agar plate-based assay, which is undesirable for high-throughput screening of large insert genomic libraries. Here we present a solution-based screen for cellulase activity using a chromogenic dinitrophenol (DNP)-cellobioside substrate 9. Our library was cloned into the pCC1 copy control fosmid to increase assay sensitivity through copy number induction 10. The method uses one-pot chemistry in 384-well microplates with the final readout provided as an absorbance measurement. This readout is quantitative, sensitive and automated with a throughput of up to 100X 384-well plates per day using a liquid handler and plate reader with attached stacking system.

This protocol describes a high throughput screen for cellulolytic activity from a metagenomic library expressed in Escherichia coli. The screen is solution based and highly automated, and uses one-pot chemistry in 384 well microplates with the final readout as an absorbance measurement.

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 accelerating, promising greater depth and breadth. In light of these extraordinary advances, the need for fast, parallel methods to define gene function becomes ever more important. Collections of genome-wide deletion mutants in yeasts and E. coli have served as workhorses for functional characterization of gene function, but this approach is not scalable, current gene-deletion approaches require each of the thousands of genes that comprise a genome to be deleted and verified. Only after this work is complete can we pursue high-throughput phenotyping. Over the past decade, our laboratory has refined a portfolio of competitive, miniaturized, high-throughput genome-wide assays that can be performed in parallel. This parallelization is possible because of the inclusion of DNA 'tags', or 'barcodes,' into each mutant, with the barcode serving as a proxy for the mutation and one can measure the barcode abundance to assess mutant fitness. In this study, we seek to fill the gap between DNA sequence and barcoded mutant collections. To accomplish this we introduce a combined transposon disruption-barcoding approach that opens up parallel barcode assays to newly sequenced, but poorly characterized microbes. To illustrate this approach we present a new Candida albicans barcoded disruption collection and describe how both microarray-based and next generation sequencing-based platforms can be used to collect 10,000 - 1,000,000 gene-gene and drug-gene interactions in a single experiment.

We have developed comprehensive, unbiased genome-wide screens to understand gene-drug and gene-environment interactions. Methods for screening these mutant collections are presented.

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 a biorefinery. These raw materials would constitute a new industrial platform, which is both sustainable and carbon neutral, to replace 
the current dependency on fossil fuel. The recalcitrance to deconstruction observed in lignocellulosic materials is produced by several intrinsic properties 
of plant cell walls. Crystalline cellulose is embedded in matrix polysaccharides such as xylans and arabinoxylans, and the whole structure is encased by the 
phenolic polymer lignin, that is also difficult to digest 1. In order to improve the digestibility of plant materials we need to discover the main bottlenecks 
for the saccharification of cell walls and also screen mutant and breeding populations to evaluate the variability in saccharification 2. These tasks require 
a high throughput approach and here we present an analytical platform that can perform saccharification analysis in a 96-well plate format. This platform has 
been developed to allow the screening of lignocellulose digestibility of large populations from varied plant species. We have scaled down the reaction volumes 
for gentle pretreatment, partial enzymatic hydrolysis and sugar determination, to allow large numbers to be assessed rapidly in an automated system.
This automated platform works with milligram amounts of biomass, performing ball milling under controlled conditions to reduce the plant 
materials to a standardised particle size in a reproducible manner. Once the samples are ground, the automated formatting robot dispenses specified and recorded 
amounts of material into the corresponding wells of 96 deep well plate (Figure 1). Normally, we dispense the same material into 4 wells to have 4 replicates for 
analysis. Once the plates are filled with the plant material in the desired layout, they are manually moved to a liquid handling station (Figure 2). In this 
station the samples are subjected to a mild pretreatment with either dilute acid or alkaline and incubated at temperatures of up to 90&deg;C. The pretreatment solution 
is subsequently removed and the samples are rinsed with buffer to return them to a suitable pH for hydrolysis. The samples are then incubated with an enzyme
mixture for a variable length of time at 50&deg;C. An aliquot is taken from the hydrolyzate and the reducing sugars are automatically determined by the MBTH 
colorimetric method.

A simple, rapid method for determining the saccharification potential of large numbers of plant biomass samples is described. The automated platform for this analysis involves the preparation of the plant biomass for analysis in 96 well plates and the subsequent performance of pretreatment, hydrolysis and quantification of the sugars released.

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

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 sequencing can be utilized to identify the repertoire of mammalian DNA sequences bound by transcription factors in vivo. "Next-generation" genome sequencing technologies provide 1-2 orders of magnitude increase in the amount of sequence that can be cost-effectively generated over older technologies thus allowing for ChIP-seq methods to directly provide whole-genome coverage for effective profiling of mammalian protein-DNA interactions.
For successful ChIP-seq approaches, one must generate high quality ChIP DNA template to obtain the best sequencing outcomes. The description is based around experience with the protein product of the gene most strongly implicated in the pathogenesis of type 2 diabetes, namely the transcription factor transcription factor 7-like 2 (TCF7L2). This factor has also been implicated in various cancers.
Outlined is how to generate high quality ChIP DNA template derived from the colorectal carcinoma cell line, HCT116, in order to build a high-resolution map through sequencing to determine the genes bound by TCF7L2, giving further insight in to its key role in the pathogenesis of complex traits.

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

The combination of chromatin immunoprecipitation and ultra-high-throughput sequencing (ChIP-seq) can identify and map protein-DNA interactions in a given tissue or cell line. Outlined is how to generate a high quality ChIP template for subsequent sequencing, using experience with the transcription factor TCF7L2 as an example.

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 resolution. This technique captures miRNAs by attaching 3&#8217; and 5&#8217; oligonucleotide adapters to miRNA molecules and allows de novo miRNA discovery. Coupling with powerful next-generation sequencing platforms, miR-Seq has been instrumental in the study of miRNA biology. However, significant biases introduced by oligonucleotide ligation steps have prevented miR-Seq from being employed as an accurate quantitation tool. Previous studies demonstrate that biases in current miR-Seq methods often lead to inaccurate miRNA quantification with errors up to 1,000-fold for some miRNAs1,2. To resolve these biases imparted by RNA ligation, we have developed a small RNA ligation method that results in ligation efficiencies of over 95% for both 3&#8217; and 5&#8242; ligation steps. Benchmarking this improved library construction method using equimolar or differentially mixed synthetic miRNAs, consistently yields reads numbers with less than two-fold deviation from the expected value. Furthermore, this high-efficiency miR-Seq method permits accurate genome-wide miRNA profiling from in vivo total RNA samples2.

MicroRNAs (miRNAs) are a widely conserved class of regulatory molecules. Here we describe a miRNA cloning method that relies upon two potent ligation steps followed by high-throughput sequencing. Our method permits accurate genome-wide quantitation of miRNAs.

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 purification of RNA, DNA, and proteins. All of these applications demand the rapid recovery of significant numbers of zebrafish hearts to avoid gene regulatory, metabolic, and other changes that begin after death. Adult zebrafish hearts are also required for studying heart structure for a variety of mutants and for studying heart regeneration. However, the traditional zebrafish heart dissection is slow and difficult and requires specialized tools, making large-scale dissection of adult zebrafish hearts tedious. Traditional methods also harbor the risk of damaging the heart during the dissection. Here, we describe a method for dissection of adult zebrafish hearts that is fast, reproducible, and preserves heart architecture. Furthermore, this method does not require specialized tools, is painless for the zebrafish, can be performed on fresh or fixed specimens, and can be performed on zebrafish as young as one month old. The approach described expands the use of adult zebrafish for cardiovascular research.

Use of zebrafish for cardiovascular research is expanding towards research on adult hearts. For these applications, quick and simple isolation of cardiac tissues is key to avoid post-mortem changes and to obtain an adequate number of samples. Here, we describe a fast and reproducible method for dissecting adult zebrafish hearts.

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 focus on transcriptional regulation, being essentially aimed at the identification of promoter-targeting molecules. As a result, post-transcriptional mechanisms are largely uncovered by gene expression targeted drug development. Here we describe a cell-based assay aimed at investigating the role of the 3&#39; untranslated region (3&#8217; UTR) in the modulation of the fate of its mRNA, and at identifying compounds able to modify it. The assay is based on the use of a luciferase reporter construct containing the 3&#8217; UTR of a gene of interest stably integrated into a disease-relevant cell line. The protocol is divided into two parts, with the initial focus on the primary screening aimed at the identification of molecules affecting luciferase activity after 24 hr of treatment. The second part of the protocol describes the counter-screening necessary to discriminate compounds modulating luciferase activity specifically through the 3&#8217; UTR. In addition to the detailed protocol and representative results, we provide important considerations about the assay development and the validation of the hit(s) on the endogenous target. The described cell-based reporter gene assay will allow scientists to identify molecules modulating protein levels via post-transcriptional mechanisms dependent on a 3&#8217; UTR.

Here we describe a cell-based reporter gene assay as a valuable tool to screen chemical libraries for compounds modulating post-transcriptional control mechanisms exerted through 3&#8217; UTR.

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 sequencing technology have opened up the possibility of assessing the functional or fitness effects of large numbers of mutations simultaneously. Here, we present a protocol for experimentally determining the effects of all possible single amino acid mutations in a protein of interest utilizing high-throughput sequencing technology, using the 263 amino acid antibiotic resistance enzyme TEM-1 &#946;-lactamase as an example. In this approach, a whole-protein saturation mutagenesis library is constructed by site-directed mutagenic PCR, randomizing each position individually to all possible amino acids. The library is then transformed into bacteria, and selected for the ability to confer resistance to &#946;-lactam antibiotics. The fitness effect of each mutation is then determined by deep sequencing of the library before and after selection. Importantly, this protocol introduces methods which maximize sequencing read depth and permit the simultaneous selection of the entire mutation library, by mixing adjacent positions into groups of length accommodated by high-throughput sequencing read length and utilizing orthogonal primers to barcode each group. Representative results using this protocol are provided by assessing the fitness effects of all single amino acid mutations in TEM-1 at a clinically relevant dosage of ampicillin. The method should be easily extendable to other proteins for which a high-throughput selection assay is in place.

We present a protocol for the functional assessment of comprehensive single-site saturation mutagenesis libraries of proteins utilizing high-throughput sequencing. Importantly, this approach uses orthogonal primer pairs to multiplex library construction and sequencing. Representative results using TEM-1 &#946;-lactamase selected at a clinically relevant dosage of ampicillin are provided.

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

High-throughput Screening for Protein-based Inheritance in S. cerevisiae

The encoding of biological information that is accessible to future generations is generally achieved via changes to the DNA sequence. Long-lived inheritance encoded in protein conformation (rather than sequence) has long been viewed as paradigm-shifting but rare. The best characterized examples of such epigenetic elements are prions, which possess a self-assembling behavior that can drive the heritable manifestation of new phenotypes. Many archetypal prions display a striking N/Q-rich sequence bias and assemble into an amyloid fold. These unusual features have informed most screening efforts to identify new prion proteins. However, at least three known prions (including the founding prion, PrPSc) do not harbor these biochemical characteristics. We therefore developed an alternative method to probe the scope of protein-based inheritance based on a property of mass action: the transient overexpression of prion proteins increases the frequency at which they acquire a self-templating conformation. This paper describes a method for analyzing the capacity of the yeast ORFeome to elicit protein-based inheritance. Using this strategy, we previously found that &gt;1% of yeast proteins could fuel the emergence of biological traits that were long-lived, stable, and arose more frequently than genetic mutation. This approach can be employed in high throughput across entire ORFeomes or as a targeted screening paradigm for specific genetic networks or environmental stimuli. Just as forward genetic screens define numerous developmental and signaling pathways, these techniques provide a methodology to investigate the influence of protein-based inheritance in biological processes.

High-throughput Screening for Protein-based Inheritance in S. cerevisiae

This protocol describes a high-throughput methodology to functionally screen for protein-based inheritance in S. cerevisiae.

The encoding of biological information that is accessible to future generations is generally achieved via changes to the DNA sequence. Long-lived ...

Modified Methods for Loading of High-Throughput DNA Extraction Plates Reduce Potential for Contamination

High-throughput DNA sequencing techniques have contributed substantially to advances in our understanding of relationships among microbial communities, host characteristics, and broader ecosystem functions. With this rapid increase in breadth and depth of sequencing capabilities have come methods to extract, amplify, analyze, and interpret environmental DNA successfully with maximum efficiency. Unfortunately, performing DNA extractions quickly can come at the cost of increasing the risk of contamination among samples. In particular, high-throughput extractions that are based on samples contained in a 96-well plate offer a relatively quick method, compared to single-tube extractions, but also increase opportunities for well-to-well cross-contamination. To minimize the risk of cross-contamination among samples, while retaining the benefits of high-throughput extraction techniques, we developed a new method for loading environmental samples into 96-well plates. We used pierceable PCR sealing films to cover each plate while loading samples and added samples first to PCR tubes before moving them into wells; together, these practices reduce the risk of sample drift and unintended double loading of wells. The method outlined in this paper provides researchers with an approach to maximize available high-throughput extraction techniques while reducing the risk of cross-contamination inherent to 96-well plates. We provide a detailed step by step outline of how to move from sample collection to DNA extraction while minimizing the risk of unwanted cross-contamination.

Current methods for loading 96-well plates for DNA extractions can be prone to contamination. We detail a new method for loading 96-well plates that reduces risk of cross-contamination among wells. Our method will help other researchers capitalize on the efficiency of high-throughput DNA extraction techniques and minimize risk of contamination.

High-throughput DNA sequencing techniques have contributed substantially to advances in our understanding of relationships among microbial communities, ...