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

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.