Difference between revisions of "Part:BBa K2020005:Design"
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The sequence of the subtilisin E gene from ''Bacillus subtilis'' was codon optimized for ''E. coli'' with the DNA and protein sequence analysis software "Geneious" and additionally with the "Codon Optimization Tool" from IDT. | The sequence of the subtilisin E gene from ''Bacillus subtilis'' was codon optimized for ''E. coli'' with the DNA and protein sequence analysis software "Geneious" and additionally with the "Codon Optimization Tool" from IDT. | ||
The sequence was partly ordered from IDT ([[Part:BBa_K2020001|BBa_K2020001]] + [[Part:BBa_B0010|BBa_B0010]]) and then cloned into [[Part:BBa_J04500|BBa_J04500]], a protein expression backbone which already carries the LacI promoter [[Part:BBa_R0010|BBa_R0010]] and the ribosome binding site [[Part:BBa_B0034|BBa_B0034]]. Afterwards, a mutation in the active site of the enzyme was introduced by performing a site-directed mutagenesis. The codon TAT of tyrosine<sup>77</sup> was substituted with TGG which codes for tryptophan. | The sequence was partly ordered from IDT ([[Part:BBa_K2020001|BBa_K2020001]] + [[Part:BBa_B0010|BBa_B0010]]) and then cloned into [[Part:BBa_J04500|BBa_J04500]], a protein expression backbone which already carries the LacI promoter [[Part:BBa_R0010|BBa_R0010]] and the ribosome binding site [[Part:BBa_B0034|BBa_B0034]]. Afterwards, a mutation in the active site of the enzyme was introduced by performing a site-directed mutagenesis. The codon TAT of tyrosine<sup>77</sup> was substituted with TGG which codes for tryptophan. | ||
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The construct was ordered at IDT. | The construct was ordered at IDT. | ||
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===References=== | ===References=== | ||
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Latest revision as of 20:45, 8 October 2016
mutated expression system for subtilisin E in E. coli (Y77W)
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 280
- 1000COMPATIBLE WITH RFC[1000]
Design Notes
The sequence of the subtilisin E gene from Bacillus subtilis was codon optimized for E. coli with the DNA and protein sequence analysis software "Geneious" and additionally with the "Codon Optimization Tool" from IDT. The sequence was partly ordered from IDT (BBa_K2020001 + BBa_B0010) and then cloned into BBa_J04500, a protein expression backbone which already carries the LacI promoter BBa_R0010 and the ribosome binding site BBa_B0034. Afterwards, a mutation in the active site of the enzyme was introduced by performing a site-directed mutagenesis. The codon TAT of tyrosine77 was substituted with TGG which codes for tryptophan.
Source
The sequences of the BioBrick parts were obtained through the Registry.
| part | BioBrick No. | |
|---|---|---|
| LacI promoter | BBa_R0010 | |
| RBS | BBa_B0034 | |
| CDS | BBa_K2020001 | |
| BBa_J32015 | BBa_K2020000 | |
| terminator | BBa_B0010 | |
The construct was ordered at IDT.