Difference between revisions of "Part:BBa K2020005"

Revision as of 20:41, 8 October 2016

mutated expression system for subtilisin E in E. coli (Y77W)

Once introduced into Escherichia coli, this BioBrick is able to produce an inactive version of subtilisin E and simultaneously secret the enzyme into the periplasm of the cell. By performing a site-directed mutagenesis, tyrosine⁷⁷ in the propeptide of the enzyme was exchanged against tryptophan. Therefore, the enzyme loses its proteolytic activity.

Usage and Biology

Subtilisin E is an alkaline serine protease which non-specifically digests proteins. It is naturally produced by Bacillus subtilis.

This composite part consists of the promoter BBa_R0010, the ribosome binding site BBa_B0034, the newly created BioBrick part BBa_K2020001 and the terminator BBa_B0010. BioBrick BBa_K2020001 is a composite part itself and includes the secretion tag pelB (BBa_J32015) and a subtilisin E gene optimized for E. coli codon usage (BBa_K2020000).

Subtilisin E has to autoprocess itself to become functional. At first, the enzyme exists as a precursor, namely the pre-pro-subtilisin. The pre-sequence serves as a recognition sequence for secretion across the cytoplasmic membrane and is cleaved off in the course of the process. The pro-peptide acts as an intramolecular chaperone and facilitates the folding of the protease. Folding is essential for the activity of an enzyme. Still, the maturation process of Subtilisin E is not completed, as the pro-peptide covers the substrate binding site and inhibits activity. However, enough proteolytic activity is achieved to autoprocess the IMC-domain and therefore cleave off the pro-peptide. Yet, the C-terminal end of the pro-peptide continues to block the substrate binding site. After the degradation of the pro-peptide, the substrate-binding site is cleared and the protease is completely active.

This mutated version of the BioBrick BBa_K2020002 was created to prove that tyrosine⁷⁷ in the propeptide cleavage-site of subtilisin E is essential for the activity of the enzyme.

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
INCOMPATIBLE WITH RFC[25]
Illegal NgoMIV site found at 280
1000
COMPATIBLE WITH RFC[1000]

@@ Line 3: / Line 3: @@
 <partinfo>BBa_K2020005 short</partinfo>
-This composite part consists of the promoter [[Part:BBa_R0010|BBa_R0010]], the ribosome binding site [[Part:BBa_B0034|BBa_B0034]], the newly created BioBrick part [[Part:BBa_K2020001|BBa_K2020001]] and the terminator [[Part:BBa_B0010|BBa_B0010]]. BioBrick BBa_K2020001 is a composite part itself and includes the secretion tag pelB ([[Part:BBa_J32015|BBa_J32015]]) and a subtilisin E gene optimized for ''Escherichia coli'' codon usage ([[Part:BBa_K2020000|BBa_K2020000]]). Once introduced into ''E. coli'', this BioBrick is able to produce an inactive version of subtilisin E and simultaneously secret the enzyme into the periplasm of the cell. Subtilisin E is an alkaline serine protease which non-specifically digests proteins. By performing a site-directed mutagenesis, tyrosine<sup>77</sup> in the propeptide of the enzyme was exchanged against tryptophan. Therefore, the enzyme loses its proteolytic activity.
+Once introduced into ''Escherichia coli'', this BioBrick is able to produce an inactive version of subtilisin E and simultaneously secret the enzyme into the periplasm of the cell. By performing a site-directed mutagenesis, tyrosine<sup>77</sup> in the propeptide of the enzyme was exchanged against tryptophan. Therefore, the enzyme loses its proteolytic activity.
-With this composite part, iGEM Aachen 2016 was able to prove that tyrosine<sup>77</sup> in the propeptide of subtilisin E is essential for the activity of the enzyme.
-<!-- Add more about the biology of this part here
 ===Usage and Biology===
-<!-- -->
+Subtilisin E is an alkaline serine protease which non-specifically digests proteins. It is naturally produced by ''Bacillus subtilis''.
+This composite part consists of the promoter [[Part:BBa_R0010|BBa_R0010]], the ribosome binding site [[Part:BBa_B0034|BBa_B0034]], the newly created BioBrick part [[Part:BBa_K2020001|BBa_K2020001]] and the terminator [[Part:BBa_B0010|BBa_B0010]]. BioBrick BBa_K2020001 is a composite part itself and includes the secretion tag pelB ([[Part:BBa_J32015|BBa_J32015]]) and a subtilisin E gene optimized for ''E. coli'' codon usage ([[Part:BBa_K2020000|BBa_K2020000]]).
+Subtilisin E has to autoprocess itself to become functional. At first, the enzyme exists as a precursor, namely the pre-pro-subtilisin. The pre-sequence serves as a recognition sequence for secretion across the cytoplasmic membrane and is cleaved off in the course of the process. The pro-peptide acts as an intramolecular chaperone and facilitates the folding of the protease. Folding is essential for the activity of an enzyme. Still, the maturation process of Subtilisin E is not completed, as the pro-peptide covers the substrate binding site and inhibits activity. However, enough proteolytic activity is achieved to autoprocess the IMC-domain and therefore cleave off the pro-peptide. Yet, the C-terminal end of the pro-peptide continues to block the substrate binding site. After the degradation of the pro-peptide, the substrate-binding site is cleared and the protease is completely active.
+This mutated version of the BioBrick [[Part:BBa_K2020002|BBa_K2020002]] was created to prove that tyrosine<sup>77</sup> in the propeptide cleavage-site of subtilisin E is essential for the activity of the enzyme.
 <span class='h3bb'>Sequence and Features</span>
 <partinfo>BBa_K2020005 SequenceAndFeatures</partinfo>