Team:Edinburgh/Bacterial/Blue light sensor

From 2010.igem.org







Overview: The blue light sensor


The blue-light sensor, which is composed of the LovTAP hybrid protein designed by Strickland et al. (2008) and made available by Professor Sosnick, was BioBricked by EPF-Lausanne in iGEM 2009. It is based on a α-helical domain linker between the Lov2 domain (the photoactive protein) and the E. coli trp repressor, which acts as a conduit for allosteric signals. The effective response of the sensor is at a wavelength of 470nm (as documented by the aforementioned Lausanne iGEM team).

The blue-light sensor (LovTAP) consists of five parts:

  1. Photoreceptor1 (the shared helix between the Lov domain and the TrpR domain)
    • Dark blue when contacting the Lov domain (dark state).
    • Red when contacting the TrpR domain (light state).
  2. Photoreceptor2 (falvin monoucleotide-FMN cofactor)
  3. Lov domain-orange (photoactive protein)
  4. TrpR domain-grey (DNA regulator)
  5. Operator DNA



Figure 1: The mechanism of action of the LovTAP allosteric light sensor.

Image: Strickland et al. (2008)


Figure 1 above shows the whole process regarding how the light sensor works, from the dark state (A) to the light-activated state (B → C) and then returning to the stable state (D → A). In the dark state, the shared helix contacts the Lov2 domain, and the inactivated TrpR dissociates from the DNA; in the light state, the Lov2 domains absorb the blue light proton and form a covalent adduct between the FMN cofactor and a conserved cysteine residue, destroying the shared helix in the Lov domain and binding / populating an active formation of the TrpR domain. This in turn leads to LovTAP binding the DNA and repressing lambda-cI. However, this binding is not stable, and thus it will eventually return to the initial state.



Strategy


Our original plan was to obtain and revive EPF-Lausanne 2009's BBa_K191009. Once we had done so, we aimed to use it to create a blue light sensing system, and to transform cells for characterisation of the system and for analysis of their compatibility with the mutated blue luciferases. Further characterization of LovTAP would include investigation of the influence of tryptophan on the system, and therefore a TrpR mutant strain of E. coli was also transformed with the LovTAP-reporter construct.





Problems


We were unable to successfully characterise the LovTAP that we initially received from Lausanne due to a frameshift mutation. Eventually, we received a new version of LovTAP from our collaborators at Mexico UNAM-Genomics, so that we could perform characterisation tests on it for them. Unfortunately, we were again unable to elicit a clear response from our sensor.

Our major problem was that transformants often grew the wrong colour. LovTAP controls the production of RFP in the cells; when LovTAP is inactive (i.e. in the dark), the cells should be producing RFP and hence should be growing red. When LovTAP is active (i.e. in white or blue light), the cells stop producing RFP and hence should produce white colonies. At the moment some cells in the light are still growing red, although some are definitely growing white, and vice versa in the dark. One suggestion was that the plasmids are unstable and dividing randomly, altering the intensity of the colour in some cells. Attempts to stabilise the colours of the colonies are ongoing (see the video in the gallery which demonstrates one of the innovative methods of providing proper culture conditions).



BioBricks


As stated above, our blue light sensor is based on a modified version of Lausanne 2009's LovTAP part (BBa_K191009) developed by our collaborators at Mexico UNAM-Genomics: BBa_K360121. We have coupled this with a simple reporter system (RFP) in order to perform characterisation tests.

BBa_K322999: LovTAP with RFP reporter system, based on Mexico UNAM-Genomics BBa_K360121



Characterisation


The protocol that we developed for LovTAP characterization is as follows:

  1. Liquid cultures (same as for minipreps) were grown overnight in 37C with antibiotics. The next day, bottles with 3.5 ml of broth were inoculated with the 0.5 ml of appropriate cultures (0.5. ml of broth added as a control).
  2. samples designated DARK were wrapped in the aluminium foil straight after the inoculation; the cap was not wrapped (not see through anyway)so that samples could be easily taken without unwrapping the bottles from the foil.
  3. All the samples were then taken to the shaker in the hot room and placed in prepared spots: for the dark- anywhere on an easily accessible holder, for light- in 2 rows of holders..
  4. Light samples were covered with the cardboard box with blue LEDs attached on the sides(470 nm wavelength; 2200 mcd/B; V=4.5 V; RS466-3548), in a way to optimize light accession to the samples. Box was securely taped to the shaker.
  5. Measurements were taken every half an hour: 200ul of the samples were placed in the numbered cuvette and then 800ul of water was added. Readings of fluorescence (green mode) were taken in standard fluorescence units (SFU, two readings each). Then optical density was measured (in the incubator room) at 360 nm wavelength. Control was used to calibrate the machine (set ref button). Also 2 readings were taken. Samples were shaken by inversion prior to taking measurement.
  6. Experiment was stopped after 3 hours (6 sets of reading taken).

Results analysis (to generate the data for the graph):

  1. means were calculated from 2 readings of optical density and fluorescence.
  2. (mean SFU minus background SFU) was divided by (mean OD minus background OD, if different from 0).
  3. table was made for the samples at different points of time- e.g. sample 1 after 30 min, 60 min, 90 min, 120 min etc.
  4. graph was made with series named the same as the samples.



Figure 2: Results obtained for our LovTAP activation experiment.








Figure 3: Results obtained for our LovTAP TrpR mutant activation experiment.



Figure 2 shows the response of two clones of LovTAP under blue light (controls were not included in this experiment). There is no clear difference in response (measured by fluorescence/optical density) over time. Expected results would show higher fluorescence in the dark than in the light.

Figure 3 shows the same experiment performed on the TrpR mutant - to see if there is any difference in LovTAP activation under blue light illumination. Again, fluorescence should be higher in the dark state - due to RFP reporter being suppressed by blue light - of LovTAP activation. Again, however, results are inconclusive.

We believe that there is a need to test the system under different conditions: temperature, different strength of promoters, etc. Further experiments are being carried out at the present time. For further details, please see the lab book.



References


Strickland, D., Moffat, K. & Sosnick, T. R. (2008). Light-activated DNA binding in a designed allosteric protein. Proceedings of the National Academy of Sciences 105, 10709-10714.

Schüttrigkeit, T. A., Kompa, C. K., Salomon, M., Rüdiger, W. & Michel-Beyerle, M. E. (2003). Primary photophysics of the FMN binding LOV2 domain of the plant blue light receptor phototropin of Avena sativa. Chemical Physics 294, 501-508.

Wu, Y. I., D. Frey, et al. (2009). A genetically encoded photoactivatable Rac controls the motility of living cells. Nature Vol 461

EPF Lausanne 2009 team wiki, http://2009.igem.org/Team:EPF-Lausanne.




Throughout this wiki there are words in bold that indicate a relevance to human aspects. It will become obvious that human aspects are a part of almost everything in iGEM.