The overall aim of the following experiment is to evaluate the ability of two bacterial species to compete with each other when thriving in the same niche. In particular, we will evaluate the role of the type six secretion system or type six in this competition process. This is achieved by preparing assay input plates with either donor cells with an active type six or prey cells, which will become blue OnX scale plates.
Then assay output plates are prepared on which the donor and praise strains are combined. Next, the plates are incubated to allow type six activity to occur. Then serial dilution are prepared from the input and output plates, and the diluted cultures are spotted on readout input and output plates.
Results are obtained that show differences in the dominance of the blue color in each spot based on a simple visual observation indicating the survival of the prey cells in each case. The more blue means the more prey survival and thus poor killing activity of the donor cells. The main advantage of this technique over existing methods like counting colony forming units, is that this method is easy non time consuming and allows a direct evaluation of the type six activity against a given prey.
So this technique evaluates bacteria seal activity in mixed cultures and therefore it may help to understand why some chronic polymicrobial infections ultimately become dominated by one single bacterial species, such as is the case with pseudomona shin in the lungs of cystic fibrosis patients. To begin gross pseudomonas aerogen donor cells that are type six active D positive or type six inactive D negative on LB agar or LBA plates overnight at 37 degrees Celsius. In the example presented here, we used a pseudomonas aerogen RET S strain, possessing a constitutively active H one type six, and an isogenic mutant with a deletion of the H one type six gene cluster engineer, an E coli recipient cell or prey by transforming DH five alpha with a plasmid, allowing the alpha complementation of the lacked gene.
Isolate the bacteria from a glycerol stock on LB with XGL and an appropriate antibiotic and incubate overnight at 37 degrees Celsius the following day. Select and pick blue transformants and prepare an overnight culture in five milliliters of tryptychs soy broth or TSB with antibiotic grow under agitation at 37 degrees Celsius for the next day's experiment prepare LBA plates labeled as assay input for strains, D positive, D, negative and P and assay output for D negative plus P and D positive plus P.For each input cell culture, measure the optical density and calculate the volume required to obtain a cell density equivalent to one unit OD 600. Collect each culture in a sterile 1.5 milliliter micro tube.
After piloting the bacterial samples at 13, 000 RPM for one minute and Resus suspending the pellets in 100 microliters of fresh TSB inoculate 10 microliters of each strain as a single spot on the A input plate. Next, gently mix 30 microliters of D positive with 30 microliters of P and in a second tube, 30 microliters of D negative with 30 microliters of P.Then inoculate the A output of plate with a 20 microliter spot of each mixture. After allowing the spots to dry near abus and burner incubate the plate at 37 degrees Celsius to allow bacterial killing to take place.
To analyze bacterial killing performance, prepare LBA plates containing 40 micrograms per milliliter of xal. For the assay readout, label the plates readout input to spot isolated bacteria or out output to spot mixed bacterial cultures on the back of each plate divided into four equal parts, and label the section zero 10 to the minus one, 10 to the minus two, and 10 to the minus three. Each dilution will allow a semi-quantitative evaluation of the blue white bacteria ratio within the same spot.
Using a sterile loop, collect the individual bacterial spots from the A input plate and the A output plate and resuspend each spot in separate 1.5 milliliter micro tubes containing one milliliter of TSB. Shake the tubes for 30 minutes to resuspend the bacteria while the cultures are shaking. Prepare five sets of three micro tubes each containing 900 microliters of TSB.
Then starting with each resuspended spot, prepare tenfold serial dilutions using fresh tips and briefly vortexing in between dilution spot each dilution on LVA xal plates, beginning with the most diluted sample and ending with the undiluted sample. Plating the A input tubes on the R input plates and the A output dilution on the R output plates per reproducibility spot 20 microliters in triplicate within each quadrant. To quantify the competition essay at this stage plate 100 microliters of the 10 to the minus three dilutions from the A output samples in triplicate on LBA xga plates incubate overnight at 37 degrees Celsius and count blue colonies in sterile area leave the plates open to allow excess liquid to absorb and incubator gain overnight at 37 degrees Celsius, at which time killing is still occurring.
As both strains are still in contact for the output analysis image or scan the plates. Spots that remain largely blue indicate that e coli has not been killed. As is typical when cultured with the type six defective posis strain shown, here are serial dilution for the readout input plates for the strains used in this assay.
As expected, the e coli pre spots P over expressing the lacked gene appear blue on media supplemented with XGL, while the donor p aerogen noses strains D positive type six active and D negative type six inactive remain white. The readout output plates on which the mix between the prey and a type six active strain D positive P has been spotted show the disappearance of the blue prey, thus indicating it has been killed. This demonstrates the ability of the donor to outcompete the prey.
The persistence of the blue color on the D negative P plate demonstrates the inability of an inactive type six donor to kill the blue prey. So while attempting this procedure, it is important to remember that the requirements for growth or for type six secretion activity may vary from one bacterial species to another, and therefore it may need a little bit of adjustment of the experimental setup. In addition, you have also to remember that you, if you want to assess the type six secretion activity in one single species, it is very important as well to strictly compare the activity of a type six secretion mutant with the parental strain whenever it's possible.
After watching this video, you should have a better understanding of how to set up a visual essay to assess the survival of pre cells after they're being in contact with the type six proficient or a type six defective strain.
