The overall goal of this procedure is to identify novel genes associated with the production of the exo polysaccharide alginate in pseudomonas aerogen using mini hamar one transposon mediated mutagenesis. This is accomplished by first growing two separate overnight cultures of the donor strain, e coli Lambda P carrying the PAC plasmid and the recipient cells pseudomonas aerogen, PAO one. Next, the PAC plasmid is conjugated by co-culture e coli Lambda peer with pseudomonas aerogen.
PAO one and trans conjugates are selected on pseudomonas isolation agar then moid colonies are identified and isolated on PIA plates. Finally, the location and orientation of the mini he mar one transpose on insertion site are identified within the pseudomonas aerogen PAO one genome. Ultimately, the use of mini he R one transpose on mediated mutagenesis identifies and characterizes those genes associated with the production of alginate in pseudomonas aerogen.
This method can help answer key questions in the fields of microbiology and bacteria genetics, such as identifying genetic loci involved in the regulation of different cellular functions demonstrating the procedure will be run withers. A graduate student from my laboratory, The mini he R one Mariner transpose on vector P fac contains 2 27 base pair inverted repeats with TA insertion sites flanking the A CC one Gentamycin resistance cassette with its Sigma 70 dependent promoter upstream of a multiple cloning site. The PFAC vector also contains genes encoding the highly active he R one transposes beta-lactamase and the TRA transfer operation.
The TA insertion sites allow for efficient integration into the genome of pseudomonas aerogen strains. Accuracy in identifying and selecting OID mutants is critical for the overall success in determining gene loci involved with alginate regulation. To prepare bacterial strains begin by inoculating e coli SM 10 Lambda peer P fac in five milliliters of lb, supplemented with 15 micrograms per milliliter of Gentamycin and place in a shaking incubator overnight at 37 degrees Celsius on the same day.
Inoculate posa strain PO one in five milliliters of LB and place in a shaking incubator overnight at 42 degrees Celsius the following day. Measure the OD 600 of the overnight cultures and mix equal amounts of PAO one and E coli P fac, such that the final volume is between one milliliter and 1.4 milliliters. Centrifuge the mixture at 6, 000 times gravity for five minutes.
Meanwhile, dry LB plates for conjugation by leaving them open in an incubator at 37 degrees Celsius for 10 to 15 minutes. Remove all but 50 microliters of the supernatant from the cell mixture. Then Resus.
Suspend the cell pellet in the remaining 50 microliters of supernatant and pipette onto a dry LB plate. In one compact droplet carefully place the plate in a fume hood to dry. Do not let cells spread over the plate.
This will significantly decrease the efficiency of the conjugation. Once the droplet is dry, invert the LB plate and incubate at 37 degrees Celsius for four to six hours while the cell mixture is incubating. Prepare large pseudomonas isolation agar or PIA plates with 300 micrograms per milliliter of gentamycin.
After the incubation, use a sterile inoculation loop to collect this single droplet of cells into a micro centrifuge tube containing one milliliter of lb vortex or pipette to mix thoroughly before spreading the cells evenly onto the large PIA plates. Incubate the plates overnight at 37 degrees Celsius. Remove the plates from the incubator and examine for OID colonies.
Isolate a single moid colony and streak for isolation on small plates with PIA and 300 micrograms per milliliter of Gentamycin. The next day, isolate a single moid colony and repeat the streaking. Repeat the single colony isolation two more times to determine the stability of the moid isolate after the initial isolation.
Step inoculate 4.5 milliliters of LB with a single OID colony and incubate in a shaker overnight at 37 degrees Celsius. The next day, label three micro centrifuge tubes for each OID mutant. Prepare two 1.25 milliliter aliquots of overnight culture for genomic DNA extraction and identification of the transposon insertion site.
Additionally, archive each OID mutant by pipetting one milliliter of overnight culture into an appropriately labeled cryo vial containing an equal volume of 10%skim milk. Store at negative 86 degrees Celsius. After extracting the GDNA from the MOID mutant.
Using any preferred method and determining its concentration, digest a total of two micrograms with one microliter of the restriction Endonuclease S one in a total volume of 50 microliters overnight at 37 degrees Celsius. Once the digested DNA is purified using any preferred method into a total volume of 25 microliters, prepare a ligation reaction using about 11 microliters of DNA and incubate the mixture for at least 15 minutes at room temperature. Then inactivate it at 70 degrees Celsius for 15 minutes, perform IPCR on the ligation product using the following forward and reverse primers and thermocycling conditions.
Then verify amplification on an AROS gel before finally using the GM three out and GM five out primers to sequence the reaction. Examples of MOID and non MOID isolates are shown in this figure. Based on the protocol demonstrated in this video, each conjugation should produce a bank of about 20, 000 mutants over 15 to 20 large plates due to the overproduction of alginate.
The moid colony should be clear or white in color and have a creamy or slimy appearance. If mutagenesis and DNA amplification is successful, there should be a single amplicon at the appropriate size as illustrated in this figure. There is an absence of IPCR amplicon in PAO one.
However, we detect a single amplicon of about 1, 400 base pairs and about 1000 base pairs in PA, O one VE two, and PA O one VE 13 respectively. The size of these amplicons corresponds to the insertion of the HE R one mariner transposon into the promoter region of PA 4 0 3 3 in PA oh one VE two, and internally in PA 5 4 84 in PA oh one VE 13 PA oh one genomic DNA. That has undergone restriction, digestion, and ligation can be used as a negative control.
So once mastered, this technique can be performed in three or four days if it is performed properly. While attempting this procedure, it is important to remember to incubate the bacterial strains at appropriate temperature prior to bio parental conjugation.
