GFP with medium constitutive promoter, RBS and terminator
Green fluorescent protein (GFP) originates from the jellyfish Aequorea victoria. This part contains a series of mutations compared to the wildtype GFP resulting in enhanced maturation and emission . It is expressed under control of the medium constitutive promoter BBa_J23105, strong RBS BBa_B0030 and terminators BBa_B0010 and BBa_B0012.
This part is based on the part BBa_E0840, with an additional promoter and belongs to the following part collection, consisting of GFP under five consitutive promoters with different strengths:
Sequence and Features
- 10COMPATIBLE WITH RFC
- 12INCOMPATIBLE WITH RFCIllegal NheI site found at 7
Illegal NheI site found at 30
- 21COMPATIBLE WITH RFC
- 23COMPATIBLE WITH RFC
- 25COMPATIBLE WITH RFC
- 1000INCOMPATIBLE WITH RFCIllegal BsaI.rc site found at 700
This part is based on the part BBa_E0840, which already contains RBS, GFP gene and terminators. By means of PCR we added the strong constitutive promoter BBa_J23105. Two PCR reactions were performed with the following primers (Table 1).
Table 1: Primers used to add promoter to GFP BioBrick.
To prevent annealing of the primer containing the promoter (J23105-E0840_FW) to the BioBrick prefix, a preliminary PCR was performed with a forward primer not containing the BioBrick prefix (E0840_FW) (Figure 1).
In order to validate the fluorescence of the gene product, the plasmid was expressed in E. coli BL21, which was grown in eM9 medium. A fluorescence spectrum was measured in a plate reader at the excitation wavelength of 488 nm (Figure 2).
As documented by Cormack et al. the emission peak is indeed at 511 nm.
In order to compare this part to the other members of its collection, the spectra were measured in the same plate reader and the results were normalized by dividing by the OD600 (Figure 3).
The fluorescence intensity of each of the individual strains is as expected, as compared to the promoter strengths.
 Cormack, B. P., Valdivia, R. H., & Falkow, S. (1996). FACS-optimized mutants of the green fluorescent protein (GFP). Gene, 173(1), 33-38.