Difference between revisions of "Part:BBa K1497025"
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<p class="MsoCaption" align="text-align:justify"><span lang="EN-US"><b>Figure 1:</b></span></a><span lang="EN-US"> | <p class="MsoCaption" align="text-align:justify"><span lang="EN-US"><b>Figure 1:</b></span></a><span lang="EN-US"> | ||
| − | Crystal structure of the SH3 domain (blue) of the c-Crk protein from Mus musculus binding the SOS peptide PPPVPPRRRR (white) a sequence from Homo sapiens (Wu et al. 1995). PDB entry 1CKB.. PDB entry 1T84.</span></p> | + | Crystal structure of the SH3 domain (blue) of the c-Crk protein from <i>Mus musculus</i> binding the SOS peptide PPPVPPRRRR (white) a sequence from Homo sapiens (Wu et al. 1995). PDB entry 1CKB.. PDB entry 1T84.</span></p> |
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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 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. | 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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<p class="MsoCaption" align="text-align:justify"><span lang="EN-US"><b>Figure 2:</b></span></a><span lang="EN-US"> | <p class="MsoCaption" align="text-align:justify"><span lang="EN-US"><b>Figure 2:</b></span></a><span lang="EN-US"> | ||
| − | 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. | + | 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. |
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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. | ||
Latest revision as of 18:45, 17 October 2014
SH3-Domain
The SH3 domain and its ligand (BBa_K771108) are suitable tools for protein colocalizaion. Initially, the domain was a part of the Crk protein in Mus musculus. The SH3 domain is used as a binding unit of the so-called protein scaffold published by Dueber et al. in 2012. 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_K771107). 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 SH3 domain (blue) of the c-Crk protein from Mus musculus binding the SOS peptide PPPVPPRRRR (white) a sequence from Homo sapiens (Wu et al. 1995). PDB entry 1CKB.. 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:
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 1
Illegal BamHI site found at 226 - 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
References
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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. 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. |