Composite
CFP

Part:BBa_K566004:Experience

Designed by: Daniel Rodriguez   Group: iGEM11_UANL_Mty-Mexico   (2011-09-26)

This experience page is provided so that any user may enter their experience using this part.
Please enter how you used this part and how it worked out.

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 (Excitation filter 420nm ±50 and Emission 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.

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

Plate Control (-)

    • Always in the dark
CFPgraficauno.png
Figure 2. CFP tends to increase over time.




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 the use of preferential codons; therefore comparison with other CFPs would be ideal. It was observed that the system K566004/K566006 may be perturbed by green light. However, further experiments must be done to determine the behavior of the composite system correctly. UNIQac740257edcc8a91-partinfo-00000000-QINU UNIQac740257edcc8a91-partinfo-00000001-QINU