Part:BBa_K343007

Photosensor generator

Background

This part constitutively expresses the protein from the coding sequence of BBa_K343003, which in turn makes E.Coli phototaxic when exposed to the right conditions. The promoter is inhibited by TetR, which in turn will not be active in the presence of tetracyclin.

Usage and parameters

The part requires retinal to work in E.Coli. This can be achieved through adding retinal to the liquid growth medium and/or the plates. Currently we are doing experiments on wether the part also functions with an internal retinal source, ie retinal synthesis in E. Coli. On top of adding retinal the cells will have to be grown in the dark for at least two hours after the addition of retinal to the growth medium if you want to see an effect. This is so that the photosensor is not exposed to light before the experiments and will result in maximum output if exposed to blue light.

Results

There is a wide range of motility assays for chemotaxis in bacteria, this 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. Semi-solid agar plates motility assay:

Our results from the experiments with semi-solid agar confirms that the biobrick does indeed couple itself to the bacterial chemotaxis pathway and modify the bacterial motility pattern by modifying the tumbling frequency.

In this experiment it was shown that blue light should decreased 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. The reason at the microscopic scaleis that the agar creates a matrix like structure where there are channels through the agar, which the bacteria can swim through. The decrease in tumbling frequency, which happens when the bacteria are exposed to light, will make it harder for them to find the channels in the agar to swim through, which leads to them being trapped where they were placed. The result is that a colony which shows an increased run time, will look as if it it was non-motile on these plates [1]. This is phenomenom is what Figure 1 and 2 show; 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 with a normal wildtype MG1655 and a non-motile strain of E.coli, DH5alpha, as controls. As expected these cells did not show anything like the behavior described above.

(PICETURES HERE)

A second modified experiment showed exactly the same behavior, were again one half of the plates was exposed to light and the other half was in the dark. Afterwards 5 ul of bacteria culture was placed on each half. We then observed that the bacteria that were exposed to light again did not spread out and the cultures growing in the dark spread normally.

(2. PICETURES HERE)

2. Videomicroscopy

The videomicroscopy indicates that blue light with a wavelength around 500nm leads to CheA's autophosphorylation being downregulated. This means that the bacterial tumbling frequency gets reduced and the bacteria will spend an increased time in the "run" mode of propulsion, so that bacteria containing K343007 will travel further when exposed to bluelight, than wildtype (E. Coli MG1655) bacteria. Thee microscopy was done on a Nikon eclipse TE2000-S microscope with an optical magnification of 1000x. What then could be observed was bacteria containing K343007 that were exposed to blue light with a wavelength around 480nm, were traveling further than bacteriacontaining the part that were not exposed to blue and wildtype bacteria both exposed and unexposed to blue light. These results stem from an analysis of the videos with the open-source software Celltrack (2), which gave informations on the sample's path, velocity and distance traveled. Because of some problems with the source material, only the bacteria's path gave 100% reliable information in this experiment:

From left to right, trajectory of: E.Coli with photosensor exposed to blue light, E.Coli with photosensor exposed to red light and E.Coli Mg1655 Wildtype exposed to blue light: (Blue dots show the location of the cell in the given frame, so the number of dots equals the number of frames from the sample.)
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.

Another more accurate experiment was done with Unisensor A/S's prototype for tracking particle movement in liquids. The analysis of these data is still ongoing, but when the bacteria are exposed to a light gradient they seem to travel along the gradient towards the source of light.

(GRADIENT PICETURE)

3. Stability assay:

The stability of pSB1C3-K343007 is most likely <20 generations, which was determined throuh a stability assay.

(GRAF)

As seen in the graph, almost all of the bacteria had shedded the plasmid after 20 generations, suggesting that the plasmid is only stable within the cell for a few generations (<20). This is presumably due to the strain brought upon the bacteria by the plasmid. Thereby when the bacteria are carrying a high-copy plasmid like pSB1C3-K343007 it is plausible that the bacteria will quickly shed the plasmid when no longer exposed to a selection pressure.

4. Growth assay:
OD at 550 nm was measured every hour for 12 hours and after 24 hours. In the experimental setup , lag phase was observed in any of the measurements. The graph below shows the growth of our wild type E. coli strain MG1655, the MG1655/pSB3T5-K343007 and MG1655/pSB1C3-K343007 respectively:

(GROWTH GRAF)

From our data we see no significant difference between the plasmid carrying bacteria and the wild type. This can be said to be quite conteradictory to our results obtained from the stability assay.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NotI site found at 1863
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 153
  Illegal NgoMIV site found at 411
  Illegal AgeI site found at 1665
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 1107
  Illegal BsaI.rc site found at 1380
  Illegal SapI site found at 881
  Illegal SapI.rc site found at 1881