Team:SDU-Denmark/project-p

Our Parts

<groupparts>iGEM010 SDU-Denmark</groupparts>

Characterization of parts

BBa_K343003

The part BBa_K343003 (from now on shortly called PS) is a generator for the SopII-HtrII photosensor from Natronomonas Pharaonis coupled to E.Colis chemotaxis pathway via the Salmonella protein Tar. This part's effect on the system is to make E.Coli phototaxic, so that it becomes aware of different light conditions, so for characterizationof this part we chose to look at the motility and motility patterns as the first priority and afterwards the plasmid stability and growth of the cells.
There is a wide range of motility assays for chemotaxis in bacteria, which meant that we had a broad spectrum of experiments to choose from, which just had to be tweaked for making them suited for the analysis of phototaxis. The two experiments we chose for analysing the effect of this part (PS), were growth of the bacterial cultures in semi-solid agar and computer analysis of swimming motility through video microscopy:

1. Growth of bacterial culture on semi-solid agar plates (Experiment 1):
The bacteria and plates were prepared after our own protocol, which you can find here: [1]
Since exposure to blue light should decrease the phototaxic bacterias tumbling frequency, the expected result was that the colony which was placed between the light and dark half of the plate would spread out in the darkness and would not move further when it reached the light. This is counter-intuitive, since decreased tumbling should lead to a longer distance traveled. What happens at the microscopic scale in semisolid agar is that the agar creates a matrix like structure where there a channels through the agar, which the bacteria can swim through. The decrease in tumbling frequency of the bacteria will make it harder for them to find the channels in the agar to swim through them, which leads to them being trapped where they were placed. The result is that a colony which shows an increase run time, will look as if it it was non-motile on these plates. Our results showed exactly this, the bacterial culture had spread out to on the dark half of the plate and did not get nearly as far on the half exposed to light. This experiment was done at the same time with a normal wildtype Mg1655 and a non-motile strain of E.Coli, DH5alpha, as controls. Thse cells did not show anything like the behavior described above, which indicates that the effect stems from the modification to our photosensor bacteria. These results were useable, but not totally conclusive, since there were some non-optimal conditions present in this experiment. We used ambient light instead of pure blue light, and the exposure to light for the multiple samples was not exactly even. Therefore we had to improve our experiment setup and see if we could reproduce these results with a more reliable setup.

2. Growth of bacterial culture on semi-solid agar plates (Experiment 1):