Difference between revisions of "Part:BBa K1890020"
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<b>Figure 1</b>: Construction of the biobrick K1890020 by means of two PCRs, using biobrick E0840 as a template.  
 
<b>Figure 1</b>: Construction of the biobrick K1890020 by means of two PCRs, using biobrick E0840 as a template.  
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<b>Figure 2</b>: Fluorescence spectrum of <i>E. coli</i> BL21 expressing this part at the excitation wavelength of 488 nm.  
 
<b>Figure 2</b>: Fluorescence spectrum of <i>E. coli</i> BL21 expressing this part at the excitation wavelength of 488 nm.  

Revision as of 19:45, 10 October 2016


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 is based on the part BBa_E0840, with an additional promoter. This part belongs to the following part collection, consisting of GFP under five consitutive promoters with different strengths:

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 7
    Illegal NheI site found at 30
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE 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).

Figure 1: Construction of the biobrick K1890020 by means of two PCRs, using biobrick E0840 as a template.

Characterization

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)

Figure 2: Fluorescence spectrum of E. coli BL21 expressing this part at the excitation wavelength of 488 nm.

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.