This protocol is useful for performing genetic manipulation on symbiotic bacteria, in this case, specifically on Burkholderia, and identifying the genes essential for colonizing an insect host. Transposon insertion sequencing is a powerful tool for identifying a large number of conditionally essential genes simultaneously in a single experiment. Begin by inoculating a fresh donor culture of E.coli donor in 10 milliliters of LB medium supplemented with Kanamycin and DAP under a sterile hood.
Inoculate Burkholderia gladioli strain A recipient cells in five milliliters of KB medium. Incubate the cultures at 30 degrees Celsius overnight on a shaker at 250 RPM. After overnight growth, centrifuge four milliliters of each of the cultures at 9, 600 times G for six minutes to pellet the cells and discard the supernatant.
Under a sterile hood, wash the pelleted cell cultures in KB medium containing DAP. And finally, resuspend the cultures separately in four milliliters of KB plus DAP medium. In a fresh 15 milliliter tube, mix 250 microliters of the washed E.coli donor cells with one milliliter of the washed Burkholderia gladioli strain A recipient cells.
Spot 10 microliters of this conjugation cell mixture on KB agar plates containing DAP. Allow the plate to rest undisturbed in the sterile hood at room temperature for one hour. Incubate the plates with the conjugation spots at 30 degrees Celsius for 12 to 18 hours.
After incubation, add two to four milliliters of 1X PBS onto the plates under a sterile hood and use a cell scraper to release the grown bacterial conjugation spots from the agar. Then pipette the conjugated cell mix into two milliliter microfuge tubes. Pellet the cells by centrifuging at 9, 600 times G for two minutes.
Discard the supernatant and wash the pellet twice in one milliliter of 1X PBS by pipetting up and down. Resuspend the final pellet in 1, 200 microliters of 1X PBS. Mix well and spread 100 microliters of the cell mixture on large KB agar plates supplemented with Kanamycin and incubate at 30 degrees Celsius overnight.
Count the total number of transconjugant colonies on the three plates and extrapolate to calculate the approximate number of mutants obtained in all the plates. To increase the chances of obtaining a representative library, ensure that the total number of colonies is several fold higher than the total number of genes in the genome. To confirm the success of the conjugation, perform a PCR targeting the insertion cassette using 10 to 20 sample colonies as described in the manuscript.
Under a sterile hood, scrape the colonies from the plates by adding one to two milliliters of 1X PBS on the agar. Pool the cell mixture scraped off from the plates into 50 milliliter tubes. Vortex the library to mix thoroughly, and then split one milliliter of the pooled mutant library into several cryo tubes.
Add one milliliter of 70%glycerol to the tubes and store at minus 80 degrees Celsius. Select an L.villosa egg clutch and count the number of eggs continuing if the clutch contains more than 100 eggs. To sterilize the entire egg clutch, add 200 microliters of 70%ethanol and gently wash the eggs for five minutes, then remove the ethanol and wash the eggs twice with autoclaved water.
Add 200 microliters of 12%bleach and gently wash the eggs for 30 seconds. Remove the bleach immediately and wash the eggs again three times with 200 microliters of autoclaved water. Infect the eggs in the sterilized egg clutch using two times 10 to the six cells per microliter of the washed mutant library in PBS.
Two days after the infected beetle larvae hatch, collect 100 second instar larvae per 1.5 milliliter microfuge tube and store at minus 80 degrees Celsius. To generate the in vitro control library, inoculate 250 microliters of two times 10 to the six cells per microliter of the mutant library in 10 milliliters of KB medium containing Kanamycin. After extracting DNA from the in vivo and in vitro grown mutant libraries, share the DNA with an ultrasonicator.
To check if the DNA was sheared to the desired size range, load five microliters of the unsheared and sheared DNA after mixing with gel loading dye in a one-to-one ratio on a 1.6%agarose gel run at 250 volts for 40 minutes. To prepare the fragment ends required for adapter ligation, start by adding three microliters of the enzyme mix and seven microliters of reaction buffer to 50 microliters of the sheared DNA and mix well by pipetting. Set a thermocycler with a heated lid at greater than or equal to 75 degrees Celsius and incubate the samples for 30 minutes at 20 degrees Celsius and 30 minutes at 65 degrees Celsius, then hold the temperature at four degrees Celsius.
For adapter ligation, add 30 microliters of ligation master mix, one microliter of ligation enhancer, and 2.5 microliters of diluted adapter to the products of the end preparation step. Mix thoroughly by pipetting and incubate the sample for 15 minutes at 20 degrees Celsius in the thermocycler with the heated lid off. After 15 minutes, add three microliters of the enzyme.
Mix well by pipetting and incubate the sample for 15 minutes at 37 degrees Celsius in a thermocycler with the lid heated at greater than or equal to 47 degrees Celsius. To select the size of adapter ligated DNA targeting fragments of 250 base pairs, start by vortexing the magnetic bead solution and place it at room temperature for 30 minutes before use. Add 0.3 times of beads to 96.5 microliters of the ligated DNA mixture and mix by pipetting thoroughly.
Incubate the bead mixture for five minutes. Place the tubes on a magnetic stand to pull down the beads and remove DNA fragments of unwanted size. Let the beads settle for five minutes and then transfer the clear supernatant to a new microfuge tube.
Add 0.15 times of fresh beads to the supernatant and mix by pipetting well. Incubate the bead mixture for five minutes and then place the tubes on a magnetic stand to pull down the beads bound to the target DNA. Wait for five minutes and then discard the supernatant, keeping the beads.
With the beads on the magnetic stand, add 200 microliters of freshly prepared 80%ethanol and wait for 30 seconds before discarding the ethanol without disturbing the beads. Remove the tubes from the stand and add 17 microliters of 0.1X TE or Low TE, then mix by pipetting 10 times and incubate the mixture at room temperature for two minutes. Place the tube on the magnetic stand and take 15 microliters of the size-selected DNA for PCR-1.
Vortex streptavidin beads and place them at room temperature for at least 30 minutes. Take 32 microliters of the beads and wash them with 500 microliters of 1X bind and wash buffer. Add 32 microliters of 2X bind and wash buffer and resuspend the beads.
To this, add 32 microliters of the cleaned up PCR-1 products. Mix thoroughly and incubate at room temperature for 30 minutes. Place the bead DNA mixture on a magnetic stand for two minutes.
Pipette out the supernatant as biotin-tagged DNA containing the insertion edge binds to streptavidin on the beads. Wash the beads with 500 microliters of 1X bind and wash buffer, and then wash the beads with 200 microliters of Low TE.Resuspend the DNA bound beads in 17 microliters of Low TE.The DNA of the in vivo and in vitro grown mutant libraries extracted and fragmented in an ultrasonicator showed majority of the fragments span between 100 and 400 base pairs. The location of transposon-mediated insertions across the four replicons of Burkholderia gladioli strain A genome can be observed in the pod.
A summary of the sequencing output and the number of unique insertions and genes hit in the three replicate in vivo and in vitro libraries can be observed in the table. The important thing to remember is to calculate the population bottleneck size during host colonization to get an appropriate representation of the library during infection. Also, adjust for a compatible number of bacterial generations in vitro and in the host.
After generating the transposon mutant library, it can be screened on selective media to recover mutants based on phenotypic differences. Specific genes important for a condition can thus be identified.
