Source:
Generated By: https://synbiohub.org/public/igem/igem2sbol/1
Created by: Nicola Freyer, Lea Steinbeck, Svenja Meyer, Alexander Deitert
Date created: 2016-10-07 11:00:00
Date modified: 2016-10-08 12:13:18
mutated expression system for subtilisin E in E. coli (Y77W)
| Types | DnaRegion |
| Roles | Composite engineered_region |
| Sequences | BBa_K2020005_sequence (Version 1) |
Description
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, tyrosine77 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 tyrosine77 in the propeptide of subtilisin E is essential for the activity of the enzyme.
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 ([[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 tyrosine77 was substituted with TGG which codes for tryptophan.
Source
The sequences of the BioBrick parts were obtained through the Registry.{| class="wikitable" style="text-align:center"
|-
! part !! colspan="2"| BioBrick No.
|-
| LacI promoter || colspan="2"| [[Part:BBa_R0010|BBa_R0010]]
|-
| RBS || colspan="2"| [[Part:BBa_B0034|BBa_B0034]]
|-
| rowspan="2" | CDS || colspan="2"| [[Part:BBa_K2020001|BBa_K2020001]]
|-
| [[Part:BBa_J32015|BBa_J32015]] || [[Part:BBa_K2020000|BBa_K2020000]]
|-
| terminator || colspan="2"| [[Part:BBa_B0010|BBa_B0010]]
|-
|}
The construct was ordered at IDT.
| Sequence Annotation | Location | Component / Role(s) |
| LacI BBa_B0034 PelB propeptide TAT520TGG subtilisin E BBa_B0010 | 1,200 209,220 227,292 293,522 520,522 523,1351 1360,1439 | promoter feature/promoter feature/rbs ribosome_entry_site feature/tag tag CDS feature/cds feature/mutation sequence_alteration feature/cds CDS engineered_region feature/BioBrick |