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 12:30:28
mutated expression system for subtilisin E in E. coli (Y77X)
| Types | DnaRegion |
| Roles | Composite engineered_region |
| Sequences | BBa_K2020006_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 the amber stop codon UAG. Therefore, the expression of the enzyme is interrupted.This composite part was created to integrate a non-canonical amino acid into the sequence of the propeptide of subtilisin E by adding an orthogonal tRNA/aminoacyl-synthetase pair that is capable of incorporating the non-canonical amino acid of choice at the UAG codon.
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 the stop codon TAG.
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 TGG520TAG subtilisin E BBa_B0010 | 1,200 209,220 227,292 293,522 520,522 523,1351 1360,1439 | promoter feature/promoter ribosome_entry_site feature/rbs tag feature/tag CDS feature/cds feature/mutation sequence_alteration CDS feature/cds feature/BioBrick engineered_region |