Difference between revisions of "Part:BBa K566004:Experience"

Revision as of 04:14, 29 September 2011

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Experience

Testing and characterization for composite K566004 and K566006 in E. coli JT2 strain under different  light intensities and  irradiation times of 30 minutes at 532 nm.

Experimental

G (+) = E.coli JT2 carrying pJT122, pPLPCB(S), K566004 (CFP), K566006 (Green light genetic inversor)

G (-) = E.coli JT2 carrying pJT122, K566004(CFP), K566006 (Green light genetic inversor)

A light irradiation experiment was carried out in JT2 cultures under two controlled environments, one exposed to light and the other in complete darkness. The samples G (+) and G (-) were incubated overnight at 37°C and then 200uL of the samples were inoculated by triplicate for each intensity.

Both samples were measured in a Fluorometer BioTek Synergy, using special filters for CFP detection and measurement (Exitation filter 420nm ±50 and Emsion filter 460 ±40) before being irradiated to observe an initial fluorescence. After been introduced to the Light Machine, where the exposure time started with 30 minutes of Far-red light(710 nm) to reset the photoreceptors that could be switched during the night at the end of this period the fluorescence was measured. Then began to expose the samples to green light (532 nm) for 2 hours and every 30 minutes a portion of the sample is taken to measure the fluorescence gain or lost.

Figure 2. CFP tends to increase over time.

Results

At the end of two hours the following data was obtained:

Plate Control (-)

• • Always in the dark

For measurements of the samples in the Fluorometer, LB medium was removed (it tends to absorb wavelengths near the ultraviolet range, which can interfere with CFP measurements) and then resuspended in sterile MiliQ water, the sample then was treated with liquid nitrogen to lyse the cells without using chemicals or buffers that could interfiere in the fluorescence measurements.

Conclusions

During this experiment it was demonstrated that the new part K566004 which contains a new Cyan Fluorescent Protein controlled by Mnt regulable promoter emits cyan fluorescence. This new CFP was optimized for better expression in E. coli based on use of preferential codons, therefore, comparation tests against other CFP would be ideal. Also, during this experiment it was observed that synthetic construction for CFP protein, may be regulated using light specific wavelengths. Demonstrating capacity of the system to be light-regulated.

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