Difference between revisions of "Part:BBa K1890020"
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<h2>Indroduction</h2>
 
<h2>Indroduction</h2>
 
Green fluorescent protein (GFP) from the jellyfish <i>Aequorea victoria</i>. This mutant represents a series of mutations resulting in enhanced maturation and emission [1]. It is expressed under control of the strong constitutive promoter <partinfo>BBa_J23100</partinfo>, strong RBS <partinfo>BBa_B0030</partinfo> and terminators <partinfo>BBa_B0010</partinfo> and <partinfo>BBa_B0012</partinfo>.
 
Green fluorescent protein (GFP) from the jellyfish <i>Aequorea victoria</i>. This mutant represents a series of mutations resulting in enhanced maturation and emission [1]. It is expressed under control of the strong constitutive promoter <partinfo>BBa_J23100</partinfo>, strong RBS <partinfo>BBa_B0030</partinfo> and terminators <partinfo>BBa_B0010</partinfo> and <partinfo>BBa_B0012</partinfo>.
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This part belongs to the following part collection, consisting of GFP under five consitutive promoters with different strengths:
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* <partinfo>BBa_K1890020</partinfo>
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* <partinfo>BBa_K1890021</partinfo>
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* <partinfo>BBa_K1890022</partinfo>
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* <partinfo>BBa_K1890023</partinfo>
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* <partinfo>BBa_K1890024</partinfo>
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<h2><span class='h3bb'>Sequence and Features</span></h2>
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<partinfo>BBa_K1890020 SequenceAndFeatures</partinfo>
  
 
<h2>Construction</h2>
 
<h2>Construction</h2>
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<figure>
 
<figure>
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<img src="https://static.igem.org/mediawiki/2016/4/45/T--TU_Delft--BBa_K1890020_construction.png" width="80%">
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<img src="https://static.igem.org/mediawiki/2016/4/45/T--TU_Delft--BBa_K1890020_construction.png" width="70%">
 
<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>
 
</figure>
 
</figure>
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</html>
 
  
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<h2><span class='h3bb'>Sequence and Features</span></h2>
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<h2>Characterization</h2>
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<partinfo>BBa_K1890020 SequenceAndFeatures</partinfo>
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<p>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.</p>
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</html>
  
 
<h2>References</h2>
 
<h2>References</h2>

Revision as of 19:00, 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 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, 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.