Difference between revisions of "Part:BBa K1890020"
Line 38: Line 38:
 
      
 
      
 
<figure>
 
<figure>
−
<center><img src="https://static.igem.org/mediawiki/2016/4/45/T--TU_Delft--BBa_K1890020_construction.png" width="60%"></center>
+
<center><img src="https://static.igem.org/mediawiki/2016/4/45/T--TU_Delft--BBa_K1890020_construction.png" width="60%">
 
<figcaption>
 
<figcaption>
 
<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.  
−
</figcaption>
+
</figcaption></center>
 
</figure>
 
</figure>
  
Line 48: Line 48:
  
 
<figure>
 
<figure>
−
<center><img src="https://static.igem.org/mediawiki/2016/0/01/T--TU_Delft--Spectrum_BBa_K1890020.png" width="90%"></center>
+
<center><img src="https://static.igem.org/mediawiki/2016/0/01/T--TU_Delft--Spectrum_BBa_K1890020.png" width="90%">
 
<figcaption>
 
<figcaption>
 
<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.  
−
</figcaption>
+
</figcaption></center>
 
</figure>
 
</figure>
  
Line 59: Line 59:
  
 
<figure>
 
<figure>
−
<center><img src="https://static.igem.org/mediawiki/2016/e/e9/T--TU_Delft--Spectra_GFP.png" width="90%"></center>
+
<center><img src="https://static.igem.org/mediawiki/2016/e/e9/T--TU_Delft--Spectra_GFP.png" width="90%">
 
<figcaption>
 
<figcaption>
 
<b>Figure 3</b>: Fluorescence spectrum of <i>E. coli</i> BL21 expressing GFP under five different constitutive promoters, at the excitation wavelength of 488 nm.  
 
<b>Figure 3</b>: Fluorescence spectrum of <i>E. coli</i> BL21 expressing GFP under five different constitutive promoters, at the excitation wavelength of 488 nm.  
−
</figcaption>
+
</figcaption></center>
 
</figure>
 
</figure>
  

Revision as of 19:29, 14 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.

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).

Figure 3: Fluorescence spectrum of E. coli BL21 expressing GFP under five different constitutive promoters, at the excitation wavelength of 488 nm.

The fluorescence intensity of each of the individual strains is as expected, as compared to the promoter strengths.

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.