Source:
Generated By: https://synbiohub.org/public/igem/igem2sbol/1
Created by: Vincent Leonardo
Date created: 2013-08-28 11:00:00
Date modified: 2015-06-08 09:31:00
HBsu-sfGFP
| Types | DnaRegion |
| Roles | Reporter engineered_region |
| Sequences | BBa_K1185001_sequence (Version 1) |
Description
This BioBrick codes for a HBsu protein attached to a superfolded green fluorescent protein (sfGFP). HBsu is a non-specific DNA binding protein that binds to DNA as a homodimer. The HBsu is joined to the sfGFP through ten amino acid flexible linker sequence. This allows the observation of Bacillus subtilis DNA using fluorescence microscopy. The integration strategy that we opted for was to clone in the BioBrick into the Multiple cloning site of pMutin4 backbone. First we attached HindIII restriction sites on 5'end and SacII restriction sites on 3'end of the BioBrick. We then cut the pMutin4 backbone and BioBrick using the previously mentioned restriction enzymes, this allowed us to ligate the plasmid and BioBrick together. We also attached a ~300bp amyE homology region onto the 3'end of the BioBrick after the SacII to allow the single cross over and integration of the whole plasmid into the genome. The pMutin4 plasmid contains a ery+ resistance marker for B.subtilis and amp+ for E.coli and also contains lacI, lacZ and Pspac promoter which is an IPTG induced promoter which regulated the transcription of this BioBrick. An alternative method to use this part would be to clone this BioBrick out and use any Assembly protocol to attach a desired promoter, RBS and antibiotic resistance genes.Notes
NoneSource
The source of the HBsu coding sequence was the B. subtilis strain 168 genome, SwissProtTM accession number: P08821. The sfGFP sequence was sourced from BBa_E0040 and the linker from: BBa_K105012.| Sequence Annotation | Location | Component / Role(s) |
| RBS-with post spacer hbs linker sequence BBa_K105012 sfGFP_BBa_I746916 3-frame stop codon | 1,20 21,296 297,326 327,1046 1047,1054 | ribosome_entry_site feature/rbs feature/protein CDS engineered_region feature/BioBrick engineered_region feature/BioBrick feature/stop stop_codon |