Difference between revisions of "Part:BBa K1497024:Design"
 
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===Design Notes===
 
===Design Notes===
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The follow DNA sequence is the right DNA sequence of this part. After the giant jamborre we are able to replace the wrong sequence against this sequence.
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<pre>
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ATGAGATCTACCAAAGCAGATATTGGCACCCCGAGCAATTTTCAGCATATTGGTCATGTTGGTTGGGACCCGAATACCGG
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TTTTGATCTGAATAATCTGGACCCGGAACTGAAAAACCTGTTTGATATGTGTGGTATTAGCGAAGCACAGCTGAAAGATC
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GTGAAACCAGCAAAGTGATCTATGACTTTATCGAAAAAACCGGTGGTGTTGAAGCCGTGAAAAATGAACTGCGTCGTCAG
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GCACCGGGTAGCGGTAGCGGTTCAGGTTCTGGTTCAGGTAGCGGCAGTGGTAGCGGATCC</pre>
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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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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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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.
 
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.
 
<br><br>
 
<br><br>
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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. <br><br>
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Wu, X.; Knudsen, B.; Feller, S. M.; Zheng, J.; Sali, A.; Cowburn, D. et al. (1995): Structural basis for the specific interaction of lysine-containing proline-rich peptides with the N-terminal SH3 domain of c-Crk. In Structure 3 (2), pp. 215–226.
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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.
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Latest revision as of 19:16, 17 October 2014

GBD-Domain


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]


Design Notes

The follow DNA sequence is the right DNA sequence of this part. After the giant jamborre we are able to replace the wrong sequence against this sequence. 

ATGAGATCTACCAAAGCAGATATTGGCACCCCGAGCAATTTTCAGCATATTGGTCATGTTGGTTGGGACCCGAATACCGG
TTTTGATCTGAATAATCTGGACCCGGAACTGAAAAACCTGTTTGATATGTGTGGTATTAGCGAAGCACAGCTGAAAGATC
GTGAAACCAGCAAAGTGATCTATGACTTTATCGAAAAAACCGGTGGTGTTGAAGCCGTGAAAAATGAACTGCGTCGTCAG
GCACCGGGTAGCGGTAGCGGTTCAGGTTCTGGTTCAGGTAGCGGCAGTGGTAGCGGATCC

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


Source

Rattus rattus.

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.

Wu, X.; Knudsen, B.; Feller, S. M.; Zheng, J.; Sali, A.; Cowburn, D. et al. (1995): Structural basis for the specific interaction of lysine-containing proline-rich peptides with the N-terminal SH3 domain of c-Crk. In Structure 3 (2), pp. 215–226.