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== Results ==
== Results ==
As mentioned in the section above, in the attempts to inhibit the gene expression of LacZ, we got the opposite of what was expected. Therefore we investigated and characterised the EnvZ-OmpR two component system.

Revision as of 14:19, 12 October 2009

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Overall project

In this project, we originally intended to create an E.coli system that is sensitive to multiple wavelengths of light and produce a colour indication of the specific wavelength it is exposed to. However due to limitations in the time and number of people available, and due to unexpected results the project changed significantly.

In the end, our project became: characterizing existing parts, such as BBa_R0082 for beta-galactocidase activity depending on time from the reaction start as well as wavelength and intensity of exposed light.

Project Details

The first experiment we carried to familiarise ourselves with the strain, was to repeat the light sensing ability of RU1012 as described in the Levskaya et al. paper[1], in its most simple form. Red light switch off the photoreceptor through inhibiting autophosphorylation, and therefore switching off the expression for Lac Z production. We shown borad spectrum light on one sample and kept the other in the dark so that gene expression can not be interefered. The samples were put on seperate agar plates with X-gal on it which acts as a substrate for Lac Z and producing a blue colouration. To our surprise, the result were opposite to what we expected: the illuminated plate produced more pigment than the one in the dark, whereas it should have been the opposite. No control was used in this experiment.

Sheff Initial Experiment.jpg

The same experiment was repeated with a control strain to confirm that the result was systematic. Later on we realise that the wavelength that inhibits the autophosphorylation has to be a specific wavelength, a broadspectrun light that contains that wavelength does not have the same effect. Too solve that problem we have tried various measures:

Hydrogen lamp

The Balmer lamp provided a narrower band of wavelength, we hoped that it can characterise the E.coli system better than broad spectrum light. Experiments were repeated with Hydrogen lamp, conducted at room temperature - due to set up of the lamp, the experiment can not be carried out in an incubator at 37C. Unfortunately, this has not changed the outcome of the result, the gene expression was still not switched off.

To compensate the fact that the experiment was conducted at room temperature, L-arabinose was added to the agar plates as an inducer for the conversion of heam to phycocyanobillin - forms part of the photoreceptor. However, results still remained unchaged. Further investigation was carried out, we contacted authors of the article and asked for their help and extracted the fact that to deactivate the phytochrom of the transformed E.coli RU1012, the wavelength used can only be very narrow band of red light. We also extracted that the intensity of the light is also significant to the deactiation.

The response from the authors inspired us to characterise the two gene system further in terms of the optimum wavelength and intensity for gene expression deactivation. The gene expression is also apparently leaky


Filter paper

A range of filter paper which only let through certain band of wavelength was used; blue(450-495nm),red(620-750) and green(495-570). All samples was were wrapped in different filter paper and incubated for 12hrs at 37C, photos of samples were taken as a time series to show the progress of the colouration. Result at 12hrs was left out of the time series because it was identical to 9hrs results.

Sheff results colourfilter.jpg
Graduation of colouration can be seen clearly in this results table, the sample under red filter has the least colouration, while the sample in the dark has the most colouration. Blue and green has similar level of colouration judging by eye. This result matches the theory in the paper; that red light inhibits the gene expression the most. However, to complete the charaterisation of the system, quantitative measurements is required. Miller Assay quantifies the amount of beta-galactocidase, this can act as an indicator of how active the gene expression is.

Varying light intensity

The light intensity can also effect the amount of gene expressed in this system, according to the paper. To investigate the optimum light intensity for the maximum inhibition of gene expression, Miller Assay was used to measure the amount of beta-galactocidase produced at each intensity.Cultures were placed at the following light intensities uEins/m2/s: 2.5, 6, 7, 10 At each intensity, 4 sets of culture were placed: 3 experimental and 1 control.


As mentioned in the section above, in the attempts to inhibit the gene expression of LacZ, we got the opposite of what was expected. Therefore we investigated and characterised the EnvZ-OmpR two component system.


Further Work

Due to time restrictions, and the few people we had available for this project. We would to continue characterising the parts of this system, as well as repeat our the Miller assay for more values of light intensities and wavelengths.

We also have the thought of new directions to develop the project after contacting Christopher A. Voigt.



  1. Engineering Escherichia coli to see light
    Nature 24 November 2005 DOI:10.1038/nature04405
    A. Levskaya et al.
    URL Pubmed Hubmed|}