Team:SDU-Denmark/project-r

Results

Photosensor

Motility assay:
In this experiment we shone blue light on one half of the plate, while the other half was in the dark. We then placed one colony in each half and observed their motility pattern after 24 hours. We did this for three different strains of E.Coli - DH5alpha, Wildtype Mg1655 and Mg1655 containing a plasmid with our photosensor constitutively on. See the results for yourself:
Light shone on the right half of the plates, the left half was in the dark.

MG1655

Photosensor

DH5alpha

Interpreting these results is not totally obvious since the effect of different motility patterns can be counter intuitive in semisolid agar. An explanation as to how different motility patterns show up on semisolid agar plates is explained by Englert et al. in the book "Microfluidic techniques for the analysis of bacterial chemotaxis":

Polymerized agar consists of chains of extended polymers permeated by water-filled channels. At low agar concentrations (0.25– 0.4% semisolid agar) the channels are sufficiently large that the bacteria can swim through them. In the absence of chemoeffectors, cells conduct a 3D random walk through the agar matrix much as they would in liquid and spread out randomly from the point of inoculation. Since the cells are typically growing, the result is an expanding colony that forms within the agar. This “pseudotaxis” occurs in the absence of any tactic behavior. Mutant cells that only swim smoothly get trapped cul de sacs in the agar matrix (16), and their colonies do not expand significantly faster than those of nonmotile cells.

This means that if the photosensor leads to a reduced tumbling rate when exposed to blue light, it should look a lot like the colony of DH5alpha whichw as exposed to blue light. This is exactly the case which proves that the photosensor has an effect on the tumbling rate of the bacteria. The photosensor colony in the dark behaved like the Mg1655 colony in the dark and spread out. This is normal as cells that do not tend to either tumble or run a lot, will spread the most.

Video microscopy results:
Bacteria containing the photosensor will exhibit a lowered tumbling rate when exposed to blue light (wavelengths around 350nm - 450nm). This was analysed with the help of video microscopy and the open source software "CellTrack". The individual cells trajectory was tracked and their speed measured. The tracking results are as follows:

Photosensor bacteria exposed to blue light

Photosensor bacteria exposed to red light (~580nm wavelength)

Wildtype bacteria exposed to blue light

The phototaxic bacteria move more in a straight line when exposed to bluelight, as can be seen when comparing the trajectories of the thee bacteria given earlier. These were taken from a batch of 10 cells tracked per sample.

Retinal

UV-Vis spectrophotometer determination of beta-carotene production:
In this experiment cells was prepare and harvested according to protocol [1]. This experiment was preformed with four different strain of E. Coli – wildtype Top10, wildtype MG1655, Top10 containing PSB1A2 with K274210 constitutively active and MG1655 containing PSB1A2 with K274210 constitutively active. The resulting graphs is presented beneath the text

Wildtype bacteria exposed to blue light

In order assess the results a standard dilution of beta-carotene: 1mM, 100 µM, 50 µM, 25 µM, 10 µM, 5 µM, 1 µM, 100 nM and 10 nM. The standart dilutions was also measured on the spectrophotometer. The resulting graphs is presented beneath the text

When combining the graphs of the samples and the standard dilutions it is possible to tell that the spectres are uniform. This indicates that it is Beta-carotene which is produced because of the similarity in the spectres. The graphs also indicate the amount of Beta-carotene produced by the MG1655 and the Top10 E. Coli cells. The resulting graphs is presented beneath the text