Difference between revisions of "Part:BBa K1638003"
 
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<partinfo>BBa_K1638003 short</partinfo>
 
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TThe T25 domain of CyaA to be used in the bacterial two-hybrid system. The expression of the gene is under control of a lac operator and is induced by Isopropyl β-D-1-thiogalactopyranoside (IPTG). When protein-coding genes is suffixed to the T25 and T18 domains of the bacterial two-hybrid system, the interaction of these two proteins can be examined. If the conjugated proteins associates, T25 and T18 associates too. This leads to the catalysation of the conversion of ATP to cAMP.
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This part contains the T25 domain of the adenylate cyclase <i>cyaA</i> from <i>Bordetella pertussis</i>. This part is intented to be used in the bacterial two-hybrid system. This system is based on the reconstitution of the adenylate cyclase. When protein-coding genes is suffixed to the T25 and T18 domains of <i>cyaA</i>, one can study the interaction of these two proteins. If the conjugated proteins interacts, T25 and T18 will be brought into close proximity. This will enable the two catalytic domains T18 and T25 to synthesize cyclic adenosinemonophosphate (cAMP).
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The rise in cyclic AMP can trigger the expression of genes by using a cAMP-induced promotor. This promotor could for example regulate the expression of a red flourescent protein (RFP). The presence of red-fluorescent cells can in turn be used to verify protein-protein interactions.
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The rise in cAMP can trigger the expression of genes by using a cAMP-induced promotor that induce the transcription of red fluorescent protein (RFP) (<partinfo>BBa_K861173</partinfo>). The presence of red-fluorescent cells can in turn be used to verify protein-protein interactions [1].
  
 
See <partinfo>BBa_K1638005</partinfo> for the T18 domain.  
 
See <partinfo>BBa_K1638005</partinfo> for the T18 domain.  
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
 
<partinfo>BBa_K1638003 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K1638003 SequenceAndFeatures</partinfo>
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===Characterization===
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To validate that our T18 and T25 domain constructs in fact can be used to study protein-protein interactions, we made a control experiment, where the leucine zipper region from the GCN4 yeast protein was fused to the T18 and T25 domains (T18-Zip+T25-Zip). Leucine zippers are known to interact by forming homodimers. If the system indeed works, their interaction will lead to functional complementation between the T18 and T25 domains. This leads to the synthesis of cAMP. By using the cAMP-induced <i>lacZ</i> reporter system, one can observe whether or not there is an interaction. This system is part of the <i>cyaA</i>-deficient <i>Escherichia coli</i> K12-strain BTH101 (MC1061-derived). The <i>lacZ</i> gene encodes a β-Galactosidase which is positively controlled by cAMP.
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Four different combinations were sequentially co-transformed into the BTH101-strain¤:
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    <li>pSB1C3-T18+pSB1K3-T25</li>
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    <li>pSB1C3-T18+pSB1K3-T25-Zip</li>
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    <li>pSB1C3-T18-Zip+pSB1K3-T25</li>
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    <li>pSB1C3-T18-Zip+pSB1K3-T25-Zip</li>
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These transformations were plated out on LB/X-gal plates with appropriate antibiotics (chloramphenicol 25 µg/ml and kanamycin 25 µg/ml) and 40 µg/ml X-gal (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside). X-gal produces a blue dye, when cleaved by β-Galactosidase. This will give a characteristic blue phenotype.
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[[File:BBa_K1638011_THvalidation.png‎|300px|thumb|left|Plate streaking of transformed BTH101 on LB/X-gal plates containing pSB1C3-T18+pSB1K3-T25, pSB1C3-T18-Zip+pSB1K3-T25, pSB1C3-T18-Zip+pSB1K3-T25 and pSB1C3-T18-Zip+pSB1K3-T25-Zip. Both the transformations and the streaks of the transformed BTH101 were incubated at 37<sup>o</sup>C overnight. ]]
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As expected, the results only showed complementation between T18 and T25 when the leucine zipper was fused to both of the domains.  These results prove that leucine zippers form homodimers, and that our T18/T25 constructs function as expected. This indicates that the system indeed can be used to study protein-protein interactions.
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¤Note: all of the constructs were under control by lac promoter, Plac.
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===References===
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[1] Karimova G, Pidoux J, Ullmann A, Ladant D. A bacterial two-hybrid system based on a reconstituted signal transduction pathway. Proceedings of the National Academy of Sciences of the United States of America. 1998;95(10):5752-6.
  
  

Latest revision as of 14:44, 18 September 2015

T25 domain of cyaA from Bordetella pertussis (IPTG inducible)

This part contains the T25 domain of the adenylate cyclase cyaA from Bordetella pertussis. This part is intented to be used in the bacterial two-hybrid system. This system is based on the reconstitution of the adenylate cyclase. When protein-coding genes is suffixed to the T25 and T18 domains of cyaA, one can study the interaction of these two proteins. If the conjugated proteins interacts, T25 and T18 will be brought into close proximity. This will enable the two catalytic domains T18 and T25 to synthesize cyclic adenosinemonophosphate (cAMP). The rise in cAMP can trigger the expression of genes by using a cAMP-induced promotor that induce the transcription of red fluorescent protein (RFP) (BBa_K861173). The presence of red-fluorescent cells can in turn be used to verify protein-protein interactions [1].

See BBa_K1638005 for the T18 domain.

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 843
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]

Characterization

To validate that our T18 and T25 domain constructs in fact can be used to study protein-protein interactions, we made a control experiment, where the leucine zipper region from the GCN4 yeast protein was fused to the T18 and T25 domains (T18-Zip+T25-Zip). Leucine zippers are known to interact by forming homodimers. If the system indeed works, their interaction will lead to functional complementation between the T18 and T25 domains. This leads to the synthesis of cAMP. By using the cAMP-induced lacZ reporter system, one can observe whether or not there is an interaction. This system is part of the cyaA-deficient Escherichia coli K12-strain BTH101 (MC1061-derived). The lacZ gene encodes a β-Galactosidase which is positively controlled by cAMP.

Four different combinations were sequentially co-transformed into the BTH101-strain¤:

  • pSB1C3-T18+pSB1K3-T25
  • pSB1C3-T18+pSB1K3-T25-Zip
  • pSB1C3-T18-Zip+pSB1K3-T25
  • pSB1C3-T18-Zip+pSB1K3-T25-Zip


These transformations were plated out on LB/X-gal plates with appropriate antibiotics (chloramphenicol 25 µg/ml and kanamycin 25 µg/ml) and 40 µg/ml X-gal (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside). X-gal produces a blue dye, when cleaved by β-Galactosidase. This will give a characteristic blue phenotype.


Plate streaking of transformed BTH101 on LB/X-gal plates containing pSB1C3-T18+pSB1K3-T25, pSB1C3-T18-Zip+pSB1K3-T25, pSB1C3-T18-Zip+pSB1K3-T25 and pSB1C3-T18-Zip+pSB1K3-T25-Zip. Both the transformations and the streaks of the transformed BTH101 were incubated at 37oC overnight.
























As expected, the results only showed complementation between T18 and T25 when the leucine zipper was fused to both of the domains. These results prove that leucine zippers form homodimers, and that our T18/T25 constructs function as expected. This indicates that the system indeed can be used to study protein-protein interactions.

¤Note: all of the constructs were under control by lac promoter, Plac.

References

[1] Karimova G, Pidoux J, Ullmann A, Ladant D. A bacterial two-hybrid system based on a reconstituted signal transduction pathway. Proceedings of the National Academy of Sciences of the United States of America. 1998;95(10):5752-6.