Source:
Generated By: https://synbiohub.org/public/igem/igem2sbol/1
Created by: Thomas Whittaker
Date created: 2012-09-20 11:00:00
Date modified: 2015-05-08 01:13:45
Synthesised Ratiometric Luciferase construct in non-standard plasmid
| Types | DnaRegion |
| Roles | engineered_region Reporter |
| Sequences | BBa_K911004_sequence (Version 1) |
Description
This part was designed as a ratiometric luciferase reporter. The first promoter, hyperSpank, is LacI - repressed and controls the transcription of a luxA gene that has been fused at the N-terminus to an mOrange gene via a flexible linker. This was described by Dachuan Ke and Shiao-Chun Tu (2011) as having an additional peak in its emission spectrum at 560 nm, whereas the normal peak is at 490 nm. This is terminated by b0015. Downstream, pVEG controls the translation of the entire normal lux operon, which is again terminated by b0015.This contruct has been designed to work in bacillus and E.coli, so all the RBSes are at the bacillus consensus.
During and after assembly of this sequence, unexpected toxicity issues were observed. This necessitated its assembly in a low copy number vector. This part is submitted to the registry as is, in the suitable backbone provided by the synthesis company. We strongly recommend that you do not attempt to assemble it in psB1C3, as it will kill your cells.
The toxicity of the construct causes strong selective pressure against it, and characterisation has been hampered by the tendency of cells to lose parts of the insert. We suspect that the repeated terminator may facilitate recombination, and another team might investigate whether replacing the second terminator aids stability.
The construct is not behaving entirely as expected, as the e.coli colonies are initially orange, despite mOrange being lacI-repressed. This is probably due to leakage, as the RBSes are very strong in both e.coli and bacillus.
Orange fluorescence co-segregates with luminescence: colonies that lose their orange colour also lose luminescence (colonies are constitutively luminescent)
We ran out of time before attempting to insert this into bacillus. Bacillus homology regions would need to be added, but on the upside, the much lower copy number would likely counteract the toxicity issues.
Further characterisation to follow
Notes
Sequence designed to be compatible with bacillus and e.coli. As such, all genes codon-optimised for bacillus, all RBSes are at bacillus consensus, all promoters are documented as having the same function in e.coli and bacillus.Source
Commercial synthesis by DNA 2.0| Sequence Annotation | Location | Component / Role(s) |
| HyperSpank Bacillus consensus RBS + Spacers mOrange no stop GS Linker LuxA (fused to mOrange) B0015 terminator pVEG Bacillus consensus RBS + Spacers LuxC Bacillus consensus RBS + Spacers LuxD Bacillus consensus RBS +Spacers LuxA (normal) Bacillus consensus RBS + Spacers LuxB Bacillus consensus RBS + Spacers LuxE Bacillus consensus RBS + Spacers LuxG B0015 terminator | 1,97 98,126 127,834 835,864 865,1932 1933,2062 2063,2159 2160,2188 2189,3622 3623,3651 3652,4569 4570,4598 4599,5666 5667,5695 5696,6670 6671,6699 6700,7836 7837,7865 7866,8585 8586,8715 | feature/promoter promoter ribosome_entry_site feature/rbs feature/cds CDS sequence_feature feature/misc feature/cds CDS stop_codon feature/stop promoter feature/promoter feature/rbs ribosome_entry_site CDS feature/cds feature/rbs ribosome_entry_site CDS feature/cds ribosome_entry_site feature/rbs CDS feature/cds ribosome_entry_site feature/rbs CDS feature/cds feature/rbs ribosome_entry_site CDS feature/cds feature/rbs ribosome_entry_site feature/cds CDS stop_codon feature/stop |