Difference between revisions of "Part:BBa K1638012"
 
(3 intermediate revisions by the same user not shown)
Line 2: Line 2:
 
<partinfo>BBa_K1638012 short</partinfo>
 
<partinfo>BBa_K1638012 short</partinfo>
  
−
The leucine zipper region of the yeast GCN4 protein.  
+
This part contains the leucine zipper region of the GCN4 protein from <i>Saccharomyces cerevisiae</i>. Leucine zippers is a common DNA-binding motif that is known to form a homodimer of two alfa-helix monomers.
  
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here
Line 11: Line 11:
 
<partinfo>BBa_K1638012 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K1638012 SequenceAndFeatures</partinfo>
  
 +
===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 <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.
 +
<br><br>
 +
Four different combinations were sequentially co-transformed into the BTH101-strain¤:
 +
<br>
 +
<ul>
 +
    <li>pSB1C3-T18+pSB1K3-T25</li>
 +
    <li>pSB1C3-T18+pSB1K3-T25-Zip</li>
 +
    <li>pSB1C3-T18-Zip+pSB1K3-T25</li>
 +
    <li>pSB1C3-T18-Zip+pSB1K3-T25-Zip</li>
 +
</ul>
 +
<br>
 +
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.
 +
 +
 +
[[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. ]]
 +
<br>
 +
<br>
 +
<br>
 +
<br>
 +
<br>
 +
<br>
 +
<br>
 +
<br>
 +
<br>
 +
<br>
 +
<br>
 +
<br>
 +
<br>
 +
<br>
 +
<br>
 +
<br>
 +
<br>
 +
<br>
 +
<br>
 +
<br>
 +
<br>
 +
<br>
 +
<br>
 +
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.
  
 
<!-- Uncomment this to enable Functional Parameter display  
 
<!-- Uncomment this to enable Functional Parameter display  

Latest revision as of 16:35, 18 September 2015

Leucine Zipper

This part contains the leucine zipper region of the GCN4 protein from Saccharomyces cerevisiae. Leucine zippers is a common DNA-binding motif that is known to form a homodimer of two alfa-helix monomers.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 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.