So neurminic acid derivative are only present in pathogenic bacteria, aminiglycosylated flagella contain this sugar. In frame deletion of region contained in the genes can help to find flagella glycosylation clusters. This technique, the latest specific region or genes, such as glycosyltransferases which have high homology between them and are involved in different processes, and analyze its involvement in flagella glycosylation process.
This technique can also help to find the role of glycosyltransferases involved in the biosynthesis of relevant polysaccharides in pathogenic bacteria and also the role of gene encoding proteins involved in flagella formation. Demonstrating the procedure will Maite Polo, a technician of my laboratory. Design two pairs of primers that amplify DNA regions upstream and downstream of the selected gene or region to be deleted.
Primers A and D need to include at the five prime end, a restriction site for an endonuclease that allows cloning in pDM4. Ensure that primers B and C are within the gene to be deleted or inside the first and last gene of the region to be deleted. Ensure that both primers are in frame and located between five to six codons inside the gene.
Ensure that both primers include a 21 base pairs complimentary sequence at the five prime end to join DNA amplicons AB and CD.Use 100 nanograms of purified chromosomal DNA as the template in two sets of asymmetric PCRs with AB and CD primers followed by the analysis of resultant products in 1%agarose gel. Purify and quantify the excised amplicons from electrophoretic gel. Mix 100 nanograms of each amplicon with PCR reagents without primer.
After extending it, add A and D primers and amplify them as a single fragment. Next analyze, purify and quantify the resultant products as previously described. Digest one microgram of pDM4 and 400 nanograms of AD amplicon with an endonuclease.
Combine the digested pDM4 with AD amplicon at a molar vector to insert ratio of one to four and prepare the ligation reaction. Incubate overnight at 20 degrees Celsius. Later, inactivate T4 ligase at 70 degrees Celsius for 20 minutes.
Inoculate Escherichia coli strain into Luria Bertani Miller broth and incubate at 30 degrees Celsius with shaking at 200 RPM until an optical density between 0.4 and 0.6 is observed. Pellet the bacteria culture by centrifugation at 5, 000 times G and four degrees Celsius for 15 minutes. Suspend the pellet in chilled distilled water and repeat the cleaning process two more times.
Transfer the bacteria suspension to microfuge tubes and repeat the centrifugation at 14, 000 times G and four degrees Celsius for five minutes. After resuspending the pellet in chilled water, add approximately 3.5 microliters of the ligation mixture and incubate on ice for five minutes. Transfer the contents to a chilled two millimeter gap electroporation cuvette.
After electroporation add the SOC medium and transfer the contents to a culture tube. Incubate for one hour at 30 degrees Celsius with shaking at 200 RPM. Plate the transformed cells on LB agar plates supplemented with chloramphenicol.
Check insertion of the deletion construct into pDM4 by PCR using primers that flank the pDM4 cloning side. Prepare overnight cultures so the recipient Aeromonas strain rifampicin resistant and two different strains of Escherichia coli. The donor strain with the pDM4 recombinant plasmid, and the helper strain with the PRK 2073 plasmid at 30 degrees Celsius.
Suspend the same number of colonies of the donor, recipient, and helper strains in three LB tubes with 150 microliters of LB.Then on an LB agar plate without antibiotics mix the three bacterial suspensions in two drops at a recipient to helper to donor ratio of five to one to one, and incubate the plate face up overnight at 30 degrees Celsius. Harvest bacteria by adding one milliliter of LB broth to the LB agar plate. Transfer the bacterial suspension to a conical 1.5 milliliter microfuge tube and vigorously vortex.
Spread 100 microliters of the bacterial suspension on LB agar plates with rifampicin and chloramphenicol. Spin down 200 microliters and 600 microliters of the samples, suspend the pellet in 100 microliters of LB broth and plate on LB agar with rifampicin and chloramphenicol followed by incubation. Perform an oxidase test to confirm that the colonies are Aeromonas.
Further confirm the insertion of pDM4 recombinant plasmid into the specific chromosomal region by PCR using the E and F primers. Grow the recombinant colonies in two milliliters of LB with 10%sucrose and without antibiotics overnight at 30 degrees Celsius. After preparing different culture dilutions, plate 100 microliters of the bacterial cultures and dilutions on LB agar plate with 10%sucrose and incubate overnight at 30 degrees Celsius.
The next day, pick up the colonies and transfer them to LB plates with and without chloramphenicol. After incubating them overnight at 30 degrees Celsius, select the chloramphenicol sensitive colonies. Analyze the sensitive colonies by PCR using the EF primers pair and select the colonies whose PCR product correlated with the size of the deleted construct.
Put a small drop of silicone in the four corners of the cover slide and mount overnight grown culture of Aeromonas on a cover slide. Place an excavated slide on the cover slide and turn the slide upside down. Analyze swimming motility by light microscopy using an optic microscope, then transfer three microliters of the culture onto the center of a soft agar plate.
Incubate the plate face up overnight between 25 and 30 degrees Celsius. Using a ruler at the end of the incubation, measure the bacterial migration from the plate center toward the periphery through the agar. Culture Aeromonas strains overnight in 900 milliliters TSB between 25 to 30 degrees Celsius.
After centrifugation, suspend the pellet in 20 milliliters of 100 millimolar Tris hydrogen chloride buffer of pH 7.8. To remove the anchored flagella, sheer the suspension in a vortex with a glass bar for three to four minutes, and then pass repetitively through a 21 gauge syringe. Pellet the bacteria by two consecutive centrifugations at four degrees Celsius and collect the supernatant into a separate tube.
After ultracentrifuging the supernatant, suspend the pellet in 150 microliters of 100 millimolar Tris hydrogen chloride containing two millimolar EDTA buffer. Transfer 150 microliters of the enriched fraction of flagella to a thin walled ultra clear tube, and fill it with 12 milliliters of cesium chloride solution. Ultracentrifuge the tube at 60, 000 times G for 48 hours at four degrees Celsius in a swinging bucket rotor.
Collect the flagella band into the cesium chloride gradient with a syringe and dialyze against 100 millimolar Tris hydrogen chloride plus two millimolar EDTA followed by analysis by electrophoresis and Coomassie blue staining or mass spectrometry. In the following figure, lane WT is Aeromonas piscicola AH3. Lanes one to eight shows chloramphenicol sensitive colonies of the second recombination.
ST is hyper bladder 1KB marker. Lanes one to three and five to eight show the colonies with wild type FGI4 gene as lane WT.Lane four shows a colony with the deleted FGI4 gene. Light microscopy assays showed that polar or lateral flagella modifications only lead to reductions in the migration diameters, thus, AH-3 FGI mutant and AH-3 FGI-4 mutants show only reduced motility in relation to the wild type strain.
The figure shows polar flagellum from AH-3 of lane one, and the no mutants in the FGI region of lane two and FGI-4 gene of lane three, wherein both mutants have polar flagellins with lower molecular weight than the wild type strain, which suggests alterations in the flagella glycan. It is important to ensure that mutations do not affect downstream genes and leads the inability to the flagella assembly or the inability of filaments of flagella. When change in flagellin proteins are not detectable, mass spectrometry analysis are required using the total protein of flagellin peptides in order to know the glycan mass.
