Difference between revisions of "Part:BBa K343007"

Revision as of 01:19, 28 October 2010

Photosensor generator
This part consists of TetR repressed POPS/RIPS generator (including RBS), NpSopII-NpHtrII-StTar (M-fusion) and a dual terminator.

Data sheet of K343007

Download pdf

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.
For more background information and theory behind the part that is expressed via this generator, see the part K343003.

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 whether the part also functions with an internal retinal source, i.e. retinal synthesis in E. coli. Once the retinal has been added to the media, the cells will have to be incubated in the dark for at least two hours. This is necessary in order to obtain maximum output when the modified phototaxic organism is exposed to blue light.

Compatibility

This brick has been tested in the following plasmids and strains:

Chassis: E. coli MG1655.

Plasmids: PSB1C3 (high-copy), PSB3T3 (low-copy).

Risk-assesment

General use

This BioBrick poses no threat to the welfare of people working with it, as long as this is done in at least a level 1 safety lab by trained people. No special care is needed when working with this BioBrick.

Potential pathogenicity

This BioBrick consists of three different parts: The first 224 amino acid residues come from the NpSopII gene from Natronomonas pharaonis, encoding a blue-light photon receptor with 15 residues removed at the C-terminal. The following 9 amino acids are a linker. The last part is HtrII fused with Tar from E. coli. The complex' first 125 amino acid residues come from HtrII and the remaining 279 from Tar [1]. NpHtrII is thought to function in signal transduction and activation of microbial signalling cascades [2].

A single article has been written about haloarchaea in humans indicating that these played a role in patients with inflammatory bowel disease [3], but there is no evidence that the genes this BioBrick is made from or any near homologs are involved in any disease processes, toxic products or invasion properties. They do not regulate the immune system in any way.

Environmental impact

The BioBrick does not produce a product that is secreted into the environment, nor is it’s gene product itself toxic. It would not produce anything that distrupt natural occurring symbiosis.

The BioBrick might increase a bacteria’s ability to find nutrients and as such ease its ability to replicate and spread in certain dark environments. On the other hand, the BioBrick is very large and this will naturally slow down its replication rate. Generally, we do not believe this BioBrick will make its host able to outcompete naturally occurring bacteria, simply because its function is not something that will give its host a functional advantage.

Possible malign use

This BioBrick will not increase its hosts ability to survive in storage conditions, to be aerosoled, to be vaporized or create spores. None of its proteins regulate or affect the immune system or are pathogenic towards humans and animals.

Results

There is a wide range of motility assays for studying 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 suitable 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.

For a more detailed descriptions and protocols on how these experiments were carried out, visit our [http://2010.igem.org/Team:SDU-Denmark/project-p Team Wiki].

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 reducing the tumbling frequency.

Figure 1: E. coli MG1655 exposed to blue light on the left half and to darkness on the right half.

Figure 2: E. coli MG1655-pSB3T5-K343007 exposed to blue light on the left half and to darkness on the right half.

In this experiment, it was shown that blue light should decrease the tumbling frequency of the phototaxic bacteria. 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 for this is that at the microscopic scale, the agar creates a matrix-like structure forming 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, entailing them to be 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 [4]. This phenomen explains the results obeserved in Figure 1 and 2; the bacterial culture had spread out 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, the control cells did not show anything like the behaviour described above.

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

From left to right: Wildtype, Bacteria containing K343007 and DH5alpha (non-motile strain).

2. Videomicroscopy

The videomicroscopy indicates that blue light with a wavelength around 480nm 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 blue light than wildtype (E. coli MG1655) bacteria. The microscopy was done on a Nikon eclipse TE2000-S microscope with an optical magnification of 1000x. What then could be observed was bacteria expressing K343007 exposed to blue light with a wavelength around 480nm, were travelling farther than when not exposed to blue light. Furthermore, the bacteria expressing K343007 travelled farther than the wildtype bacteria both exposed and unexposed to blue light. These results stem from an analysis of the videos with the open-source software Celltrack [5], which gave informations on the sample's path, velocity and distance traveled. Because of some problems with the source material, only the path of the bacteria 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 PICTURE)

3. Stability assay:

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

As seen in the graph, almost all of the bacteria had shed 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. 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. However, a stability assay of a low copy plasmid expressing K343007 has not been carried out.

4. Growth assay:
OD at 550 nm was measured every hour for 12 hours and after 24 hours. In the experimental setup, no 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:

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

Sequence and Features

References

1. Jung KH, Spudich EN, Trivedi VD, Spudich JL. [http://www.ncbi.nlm.nih.gov/pubmed An archaeal photosignal-transducing module mediates phototaxis in Escherichia coli]. J. Bacteriol. 2001 Nov;183(21):6365-6371.
2. Mennes N, Klare JP, Chizhov I, Seidel R, Schlesinger R, Engelhard M. [http://www.ncbi.nlm.nih.gov/pubmed Expression of the halobacterial transducer protein HtrII from Natronomonas pharaonis in Escherichia coli.] FEBS Lett. 2007 Apr 3;581(7):1487-1494.
3. Oxley APA, Lanfranconi MP, Würdemann D, Ott S, Schreiber S, McGenity TJ, et al. [http://www.ncbi.nlm.nih.gov/pubmed Halophilic archaea in the human intestinal mucosa]. Environ Microbiol [Internet]. 2010 Apr 23 [cited 2010 Oct 26].
4. Derek L. Englert, Arul Jayaraman, Michael D. Manson,[http://www.springerlink.com/content/n386247071624387/fulltext.pdf Methods in Molecular Biology], 2009, Volume 571, 1-23.
   
5. Celltrack