Part:BBa_K1890020
GFP with strong constitutive promoter, RBS and terminator
Indroduction
Green fluorescent protein (GFP) from the jellyfish Aequorea victoria. This mutant represents a series of mutations resulting in enhanced maturation and emission [1]. It is expressed under control of the strong constitutive promoter BBa_J23100, strong RBS BBa_B0030 and terminators BBa_B0010 and BBa_B0012. This part belongs to the following part collection, consisting of GFP under five consitutive promoters with different strengths:
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 30 - 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 700
Construction
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_J23100. Two PCR reactions were performed with the following primers (Table 1).
Table 1: Primers used to add promoter to GFP BioBrick.
| Primer name | Sequence |
|---|---|
| E0840_FW | ATTAAAGAGGAGAAATACTAGATGCGTAAAGG |
| J23100-E0840_FW | CGGCGAATTCGCGGCCGCTTCTAGAGTTGACGGCTAGCTCAGTCCTAGGTACAGTGCTAGCATTAAAGAGGAGAAATACTAGATGCGTAAAGG |
| VR | ATTACCGCCTTTGAGTGAGC |
To prevent annealing of the primer containing the promoter (J23100-E0840_FW), to the BioBrick prefix, a preliminary PCR was performed with a forward primer not containing the BioBrick prefix (E0840_FW) (Figure 1).
Characterization
In order to validate the fluorescence of the gene product, a fluorescence spectrum was measured at the excitation wavelength of 488 nm. The spectrum was recorded in a plate reader and for comparison, the results were normalized by dividing by the OD600.
References
[1] Cormack, B. P., Valdivia, R. H., & Falkow, S. (1996). FACS-optimized mutants of the green fluorescent protein (GFP). Gene, 173(1), 33-38.
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