Part:BBa_K331033:Experience

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Method

In order to characterize the tetracycline repressible CFP (BioBrick BBa_K331033), cyan fluorescent protein (CFP-BBa_E0020) with an oligoarginine tag fused to the C-terminus (BBa_K331025) (and preceded by a ribosomal binding site – BBa_B0034) was synthesized. We used our Red/White 3-Antibiotic assembly method to add a tetracycline repressible promoter (BBa_R0040) for constitutive expression of the fusion protein. This construction yielded BioBrick BBa_K331033.

The BioBrick containing plasmid was transformed into Escherichia coli DH5α cells. These cells were grown to an OD₆₀₀ of approximately 0.7 in LB media, and diluted 1:10 with MilliQ H₂O immediately prior to analysis by fluorescent spectroscopy.

This dilution of cells was excited at 439 nm, and the emission spectra was read from 444 nm to 650 nm.

Results

Fluorescence at 476 nm was observed. This peak, along with a shoulder occurring at approximately 510 nm is consistent with results obtained by McRae¹ et al. in their rapid purification of ECFP.

Conclusion

The BioBrick BBa_K331033 that we constructed generates CFP in the absence of tetracycline, as expected.

Reference

¹Förster T. (1948), Zwischenmolekulare Energiewanderung und Fluoreszenz. Annalen der Physik, 437: 55-75.

²McRae S.R., Brown C.L., Bushell G.R. (2005), Rapid Purification of EGFP, EYFP, and ECFP with high yield and purity. Protein Expression and Purification 41. 1 121-127.