The overall goal of this experimental procedure is to create a high density transposon insertion library in Escherichia coli or Shigella flexneri using bacterial conjugation. This technique allows the creation of hundreds of thousands of unique bacterial mutants by the random genomic insertion of a Tn10 transposon. The main advantage of this technique is that the transfer of the plasma at harboring the transposon is done via bacterial conjugation instead of other less efficient methods, such as electroporation.
Additionally, the Tn10 transposon itself inserts into the genome with less bias than other transposons. Using sterile technique, add one milliliter of overnight culture of the donor strain into a sterile eppendorf tube. The tube should be labeled for example with a D, for donor.
Do the same for the recipient strain removing one milliliter of overnight culture of the recipient strain into a sterile eppendorf tube. Again for example, labeled with R, for recipient. Centrifuge both tubes for one minute at 14, 000 times G at room temperature.
This can be done at a benchtop centrifuge. While the centrifuge is running, prepare the filters for conjugation. Use sterile forceps to place a nitrocellulose filter onto a labeled LB agar plate containing no antibiotics.
Alternatively, in place of nitrocellulose filter, sterile blotting paper may be used. Place each filter onto a separate LB agar plate in order to minimize contamination. Ensure that each filter is placed onto an LB agar plate that is appropriately labeled.
Make three plates, each containing a filter. One for the donor strain, one for the recipient strain, and one for the library. Once the centrifuge has finished, remove the overnight cultures.
Remove all of the growth media containing antibiotics from the eppendorf tube and discard. Take care not to disrupt or disturb the bacterial cell pallette, yet try to remove all of the growth media. Make sure to work aseptically and change tips between each sample.
To each eppendorf tube, add 110 microliters of fresh LB media without antibiotics and pipette up and down to resuspend the culture. Make sure to work aseptically. By pipetting up and down several times to ensure that no cell clumps remain.
This step is done to concentrate the bacterial culture and remove any antibiotics left from the overnight culture. Using a pipette, place 50 microliters of the concentrated donor culture onto the filter labeled Donor. Slowly add the 50 microliters to the filter in a drop wise fashion.
Do the same for the recipient strain adding 50 microliters of the resuspended culture, the concentrated resuspended culture onto the recipient filter in a drop wise fashion. For the conjugation to occur, both the recipient strain and the donor strain are added to the same filter paper so they are in close physical contact. In order to do this, add 50 microliters of both the donor strain and the recipient strain to the same filter on the plate labeled library.
Place these agar plates containing the filters at 37 degrees for six hours. The process of conjugation occurs over six hours at 37 degrees Celsius. During conjugation, the pJA1 plasmin moves into the recipient strain.
After conjugation, the bacteria are disassociated from the filter by vortexing and induced with one millimolar IPTG. IPTG induces the transposase and the transposon containing the gene for Kanamycin resistance is randomly inserted into the genome of the recipient strain. Bacteria are plated on LB agar plates containing kanamycin and another antibiotic to select for the recipient strain containing the transposon.
The recipient strain is Lambda pir negative, so the pJA1 plasmin is lost. After six hours of growth at 37 degrees Celsius, conjugation is complete. Remove the plates containing the filters from the incubator.
Use sterile forceps to remove the filter containing the negative donor control from the LB agar plate. Tap the tube to get the filter paper to the bottom of the conical vial and ensure that the filter is fully submerged in the LB media. This tube contains two milliliters of LB media with IPTG and a final concentration of one millimolar.
Vortex the tube for one minute to disassociate the bacteria from the nitrocellulose filter. The LB media should become cloudy with bacterial cells. Repeat this for the recipient control and the library as well.
After the filters containing the bacterial cells have been thoroughly vortexed to disassociate the bacteria from the filter, the bacterial cells are plated on selected media. At this step, bacteria are plated into LB agar plates containing kanamycin and another antibiotic such as nalidixic acid. Kanamycin is used to select for recipient cells that have the transposon containing the kanamycin resistance marker successfully integrated into the genome.
Recipient cells that have not had the marker integrated into the genome are selected against. Another antibiotic such as nalidixic acid is used to select against the donor strain. The donor strain used here is sensitive to nalidixic acid, while the recipient strain is resistant to nalidixic acid.
Using this second antibiotic, it is possible to remove the donor strain. At this step, several dilutions should be plated to determine a dilution of the library that yields many colonies that are adequately spaced. After counting and recording the number of colonies on all plates containing the transposon library, some users may want to pool or combine the library into one tube.
This can be done using a sterile spreader. Add one milliliter of LB media to the LB agar plate containing the transposon library. Once the media has been added to the plate, use the sterile spreader to scrape off and disassociate the bacteria from the plate.
It is often not possible to get all bacteria off of the plate. Remove the bacterial suspension and place in a 50 mil or 15 mil conical vile. Repeat this for all plates.
Once all plates have been scraped, vortex the pooled bacterial suspension for a full minute to break up any cell clumps. This now constitutes the pooled library. After growth at 37 degrees for 18 hours, there should be no growth on the donor or recipient control plates.
The plates containing the mutant library ideally contain many colonies with a variety of sizes. Different colony sizes indicated clones of varying fitness and are a good sign the protocol has worked. The creation of a dense transposon mutagenesis library in bacteria is potentially advantageous for many downstream applications, ranging from discoveries of virulence genes and bacteria pathogens to study of essential genes to the identification of genetic interaction networks.
All of these downstream applications rely on the construction of a dense transposon insertion library. The method presented here provides a simple, fast, and inexpensive procedure for making a very dense transposon library in enterobacterial strains such as E coli or Shigella flexneri.
