Difference between revisions of "Part:BBa K1497024"
 
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The GBD domain and its ligand (<a href="/Part:BBa_K771106">BBa_K771106</a>) are suitable tools for protein colocalizaion. Initially, the domain was a part of the N-WASP protein (GTPase binding domain) in <i> Rattus rattus</i>. The GBD domain is used as a binding unit of the so-called protein scaffold published by Dueber et al. in 2012. The scaffold (<a href="/Part:BBa_K1497033">BBa_K1497033</a>) is composed of different binding units, which enable the assembly of multiple target proteins. This BioBrick adds a BglII and BamHI restriction site in front of and behind the previously constructed domain sequence (<a href="/Part:BBa_K771105">BBa_K771105</a>). <br> <br> <br>
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The GBD domain and its ligand (<a href="/Part:BBa_K771106">BBa_K771106</a>) are suitable tools for protein colocalizaion. Initially, the domain was a part of the N-WASP protein (GTPase binding domain) in <i> Rattus rattus</i>. The GBD domain is used as a binding unit of the so-called protein scaffold published by Dueber et al. in 2009. The scaffold (<a href="/Part:BBa_K1497033">BBa_K1497033</a>) is composed of different binding units, which enable the assembly of multiple target proteins. The Domain was initially edited by Team BioX-Shanghai 2012 (<a href="/Part:BBa_K771105">BBa_K771105</a>). The iGEM Team TU Darmstadt 2014 modified this BioBrick by adding a BglII and BamHI restriction site in front of and behind the previously constructed domain sequence and codon optimized it for expression in <i> E. coli</i>. <br> <br> <br>
 
  Now, different binding units of the scaffold protein can be fused together without the introduction of restriction sites.  This allows the easy construction of BioBricks of different permutations of the scaffold protein domains.
 
  Now, different binding units of the scaffold protein can be fused together without the introduction of restriction sites.  This allows the easy construction of BioBricks of different permutations of the scaffold protein domains.
 
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<b>Attention!</b> This part is not a twin. We had problems with the implementation, so the wrong DNA sequence was added. You can find the right DNA sequence on the design page.
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The here presented BioBricks are improved for the construction of scaffold proteins by introducing BglII and BamHI restriction sites flanking the domain sequence. Additionally, C-terminal linker domains consisting of glycine and serine residues were added to the scaffold domains. New scaffold sequences can be constructed by standard cloning approaches (see Figure 2). As usual, a backbone is ligated with an insert to create the desired sequence. For example a new scaffold protein consisting of two domains can be constructed by ligating a backbone vector including the desired N-terminal scaffold domain with an insert containing the desired C-terminal domain.  For this, the backbone has to be digested with the restriction enzymes BamHI and PstI cleaving the plasmid downstream of the first domain. In contrast, the insert has to be extracted from its vector by digestion with BglII and PstI. The overlap sequcences of the BglII and BamHI restriction sites are complementary. Thus, the insert can be ligated behind the first domain into the backbone. The scar sequence resulting from a combination of a BglII with BamHI restriction site cannot be recognized by nether of the enzymes. Therefore, a single ligation creates a new scaffold BioBrick immediately, which is again flanked by a BglII and BamHI sequence. Of course, more sophisticated scaffold BioBricks can therefore be constructed from composite BioBricks containing more than one domain or again by iterative cloning of single domains behind an initial domain.
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The here presented BioBrick is improved for the construction of scaffold proteins by introducing BglII and BamHI restriction sites flanking the domain sequence. Additionally, C-terminal linker domains consisting of glycine and serine residues were added to the scaffold domain. New scaffold sequences can be constructed by standard cloning approaches (see Figure 2). As usual, a backbone is ligated with an insert to create the desired sequence. For example a new scaffold protein consisting of two domains can be constructed by ligating a backbone vector including the desired N-terminal scaffold domain with an insert containing the desired C-terminal domain.  For this, the backbone has to be digested with the restriction enzymes BamHI and PstI cleaving the plasmid downstream of the first domain. In contrast, the insert has to be extracted from its vector by digestion with BglII and PstI. The overlap sequcences of the BglII and BamHI restriction sites are complementary. Thus, the insert can be ligated behind the first domain into the backbone. The scar sequence resulting from a combination of a BglII with BamHI restriction site cannot be recognized by nether of the enzymes. Therefore, a single ligation creates a new scaffold BioBrick immediately, which is again flanked by a BglII and BamHI sequence. Of course, more sophisticated scaffold BioBricks can therefore be constructed from composite BioBricks containing more than one domain or again by iterative cloning of single domains behind an initial domain.
 
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Latest revision as of 18:45, 17 October 2014

GBD-Domain

The GBD domain and its ligand (BBa_K771106) are suitable tools for protein colocalizaion. Initially, the domain was a part of the N-WASP protein (GTPase binding domain) in Rattus rattus. The GBD domain is used as a binding unit of the so-called protein scaffold published by Dueber et al. in 2009. The scaffold (BBa_K1497033) is composed of different binding units, which enable the assembly of multiple target proteins. The Domain was initially edited by Team BioX-Shanghai 2012 (BBa_K771105). The iGEM Team TU Darmstadt 2014 modified this BioBrick by adding a BglII and BamHI restriction site in front of and behind the previously constructed domain sequence and codon optimized it for expression in E. coli.


Now, different binding units of the scaffold protein can be fused together without the introduction of restriction sites. This allows the easy construction of BioBricks of different permutations of the scaffold protein domains.

Figure 1 Crystal structure of the GBD domain from the N-WASP protein (red) of Rattus rattus binding the small molecule Wiskostatin (1-(3,6-Dibromo-carbazol-9-yl)-3-dimethylamino-propan-2-ol). The GBD domain is locked by Wiskostatin in its autoinhibited binding conformation (Peterson et al. 2004). PDB entry 1T84.

Usage and Biology

Attention! This part is not a twin. We had problems with the implementation, so the wrong DNA sequence was added. You can find the right DNA sequence on the design page.


The here presented BioBrick is improved for the construction of scaffold proteins by introducing BglII and BamHI restriction sites flanking the domain sequence. Additionally, C-terminal linker domains consisting of glycine and serine residues were added to the scaffold domain. New scaffold sequences can be constructed by standard cloning approaches (see Figure 2). As usual, a backbone is ligated with an insert to create the desired sequence. For example a new scaffold protein consisting of two domains can be constructed by ligating a backbone vector including the desired N-terminal scaffold domain with an insert containing the desired C-terminal domain. For this, the backbone has to be digested with the restriction enzymes BamHI and PstI cleaving the plasmid downstream of the first domain. In contrast, the insert has to be extracted from its vector by digestion with BglII and PstI. The overlap sequcences of the BglII and BamHI restriction sites are complementary. Thus, the insert can be ligated behind the first domain into the backbone. The scar sequence resulting from a combination of a BglII with BamHI restriction site cannot be recognized by nether of the enzymes. Therefore, a single ligation creates a new scaffold BioBrick immediately, which is again flanked by a BglII and BamHI sequence. Of course, more sophisticated scaffold BioBricks can therefore be constructed from composite BioBricks containing more than one domain or again by iterative cloning of single domains behind an initial domain.

Figure 2: Cloning scheme for the construction of scaffold proteins. To assemble domains for the construction of a new scaffold protein, the backbone containing the N-terminal domain(s) can be digested with BamHI and PstI and the C-terminal domain(s) can be cut from the plasmid with BglII and PstI. The ligation of the two DNA fragments creates a new BioBrick, which can also be used for the construction of new scaffold proteins. Further scaffold proteins can be elongated by adding domains through the C-terminal BamHI site. The variability of the scaffold proteins can be increased by assembly of different domains.

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]


References


Dueber, John E.; Wu, Gabriel C.; Malmirchegini, G. Reza; Moon, Tae Seok; Petzold, Christopher J.; Ullal, Adeeti V. et al. (2009): Synthetic protein scaffolds provide modular control over metabolic flux. In Nat. Biotechnol. 27 (8), pp. 753–759. DOI: 10.1038/nbt.1557.

Kim, A. S.; Kakalis, L. T.; Abdul-Manan, N.; Liu, G. A.; Rosen, M. K. (2000): Autoinhibition and activation mechanisms of the Wiskott-Aldrich syndrome protein. In Nature 404 (6774), pp. 151–158. DOI: 10.1038/35004513.

Peterson, Jeffrey R.; Bickford, Lincoln C.; Morgan, David; Kim, Annette S.; Ouerfelli, Ouathek; Kirschner, Marc W.; Rosen, Michael K. (2004): Chemical inhibition of N-WASP by stabilization of a native autoinhibited conformation. In Nat. Struct. Mol. Biol. 11 (8), pp. 747–755. DOI: 10.1038/nsmb796.