Source:
Generated By: https://synbiohub.org/public/igem/igem2sbol/1
Created by: Vincent Leonardo
Date created: 2013-10-25 11:00:00
Date modified: 2015-05-08 01:09:37
E. coli L-form Switch
| Types | DnaRegion |
| Roles | DNA sequence_feature |
| Sequences | BBa_K1185005_sequence (Version 1) |
Description
This is and engineered BioBrick derived from the BBa_K1185000 (L-form switch for Bacillus subtilis)which can be used as a switch to enable the model Gram-negative bacteria Escherichia coli to switch between cell-walled rod form and wall-less L-form. The BioBrick contains homologous regions to the ftsI (homolog to pbpb in Bacillus subtilis)and murE genes to allow integration into the chromosome of E. coli via homologous recombination. Within the BioBrick there is a IPTG inducible promoter Pspac. This promoter is upstream of the murE coding sequence when integrated into the chromosome of E. coli, placing murE operon expression under the control of IPTG. The murE operon is responsible for the synthesis of enzymes involved in production of peptidoglycan synthesis. Disruption of this pathway can be utilised to down-regulate production of the cell wall.After integration of the BioBrick, the expression of murE operon is controlled by the presence, or absence, of IPTG(via the Pspacpromoter). When IPTG is present, murE operon genes are expressed and functional cell wall is produced. When IPTG is not present the cell wall is no longer produced and cells can transition to the cell wall-less L-form phenotype.
The BioBrick also contains a cat gene, conferring Ampicilin resistance. This can be used to select for transformants in rod form.
This BioBrick only functions in enabling switching between walled cells and wall-deficient L-forms in E. coli. We are giving opportunities for future iGEM team to work with cell wall-less E. coli.
This BioBrick should work in a similar manner as the BBa_K1185000 (B. subtilis Switch BioBrick). The characterization of this BioBrick is something that future iGEM can do.
Notes
The BioBrick does not contain any illegal restriction sites. All of the sequences have been taken directly from E. coli genome so there is no need for codon optimization.Source
All the sequences come from the Escherichia coli K12 genomic sequence. The ftsI section in the BioBrick was taken from end of the ftsI gene 1268-1767bp (500bp). Sequence acquired through EcoCyc (EG10341). The murE section was taken from the start of the murE gene 1-500bp. Sequence from EcoCyc (EG10621). The Pspac promoter were taken from the registry designed by the 2007 Cambridge iGEM team (BBa_1746666). Ampicilin resistance marker gene was taken from the expression vector pSTAR (gb|AF047654.1|AF047654).| Sequence Annotation | Location | Component / Role(s) |
| ftsI Homologous region ampR BBa_1746665 murE endogenous murE Homologous region | 1,500 501,1818 1819,1876 1877,1893 1894,2393 | feature/dna sequence_feature feature/cds CDS engineered_region feature/BioBrick feature/rbs ribosome_entry_site feature/dna sequence_feature |