Difference between revisions of "Part:BBa K1890020"
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<h2>Construction</h2>
 
<h2>Construction</h2>
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This part is based on the part <partinfo>BBa_E0840</partinfo>, which already contains RBS, GFP gene and terminators. By means of PCR we added the strong constitutive promoter <partinfo>BBa_J23100</partinfo>. Two PCR reactions were performed with the following primers.
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This part is based on the part <partinfo>BBa_E0840</partinfo>, which already contains RBS, GFP gene and terminators. By means of PCR we added the strong constitutive promoter <partinfo>BBa_J23100</partinfo>. Two PCR reactions were performed with the following primers (Table 1).
  
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<b>Table 1:</b> Primers used to add promoter to GFP BioBrick.
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To prevent annealing of the primer containing the promoter, to the BioBrick prefix, a preliminary PCR was performed with a forward primer not containing the BioBrick prefix (Figure 1).
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Revision as of 18:39, 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.

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, to the BioBrick prefix, a preliminary PCR was performed with a forward primer not containing the BioBrick prefix (Figure 1).

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

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

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.