Difference between revisions of "Part:BBa K1638009"
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<partinfo>BBa_K1638009 short</partinfo>
 
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T18 domain of the catalytic active domain of CyaA from Bordetella pertussis linked together with GFP through a flexible 10 aa linker. This indirectly allows detection of correctly transcription and translation of both T18 domain and GFP (by the presence of green fluorescence).
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T18 domain of the catalytic active domain of CyaA from <i>Bordetella pertussis</i> linked together with green fluorescent protein (GFP) through a flexible 10 aa linker. This part allows detection of expression of the gene encoding the T18-GFP fusion protein. Additionally, the presence of green fluorescence will verify correct folding of the protein fused to the T18-domain through the flexible aa linker. Correct folding of our T18-domain is obviously improtant for the bacterial two-hybrid system to work.
  
 
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<partinfo>BBa_K1638009 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K1638009 SequenceAndFeatures</partinfo>
  
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===Characterization===
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GFP was fused to the two components of the two-hybrid system, T18 and T25. This allows us to detect expression of these two components that are both under control of a <i>lac</i> promoter. Additionally, the presence of green fluorescence will verify that proteins can be fused to T18 and T25, and still fold into the correct structure.
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<br>
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The two constructs pSB1C3-T18-<i>gfp</i> and pSB1C3-T25-<i>gfp</i> were transformed into the competent E. coli K12-strain TOP10. The following fluorescence microscopy images confirm the presence of green fluorescence, which means that both T18 and T25 are expressed under the lac promoter, and that proteins fused to these two constructs will fold into the correct structure.
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[[File:BBa_K1638009_Fluorescenceimages.png‎|400px|thumb|left|Fluorescence microscopy images: (A) pSB1C3-T25-GFP, (B) pSB1C3-T18-GFP and (C) pSB1C3-T18 transformed into TOP10 (E. coli K12-strain). Both (A) and (B) show as expected green fluorescence, while (C) does not. In preparation for imaging, the cells were washed two times with PBS.]]
  
 
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Latest revision as of 15:13, 18 September 2015

T18 domain of cyaA from Bordetella pertussis with GFP reporter

T18 domain of the catalytic active domain of CyaA from Bordetella pertussis linked together with green fluorescent protein (GFP) through a flexible 10 aa linker. This part allows detection of expression of the gene encoding the T18-GFP fusion protein. Additionally, the presence of green fluorescence will verify correct folding of the protein fused to the T18-domain through the flexible aa linker. Correct folding of our T18-domain is obviously improtant for the bacterial two-hybrid system to work.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 711
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 252
    Illegal NgoMIV site found at 662
    Illegal AgeI site found at 468
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 1384

Characterization

GFP was fused to the two components of the two-hybrid system, T18 and T25. This allows us to detect expression of these two components that are both under control of a lac promoter. Additionally, the presence of green fluorescence will verify that proteins can be fused to T18 and T25, and still fold into the correct structure.
The two constructs pSB1C3-T18-gfp and pSB1C3-T25-gfp were transformed into the competent E. coli K12-strain TOP10. The following fluorescence microscopy images confirm the presence of green fluorescence, which means that both T18 and T25 are expressed under the lac promoter, and that proteins fused to these two constructs will fold into the correct structure.

Fluorescence microscopy images: (A) pSB1C3-T25-GFP, (B) pSB1C3-T18-GFP and (C) pSB1C3-T18 transformed into TOP10 (E. coli K12-strain). Both (A) and (B) show as expected green fluorescence, while (C) does not. In preparation for imaging, the cells were washed two times with PBS.