Source:
Generated By: https://synbiohub.org/public/igem/igem2sbol/1
Created by: Chris Hirst
Date created: 2008-09-11 11:00:00
Date modified: 2015-05-08 01:10:23
5??? Integration Sequence for the epsE locus of B. subtilis
| Types | DnaRegion |
| Roles | DNA sequence_feature |
| Sequences | BBa_K143005_sequence (Version 1) |
Description
Integration sequences allow DNA to be incorporated into the chromosome of a host cell at a specific locus using leading (5') and trailing (3') DNA sequences that are the same as those at a specific locus of the chromosome.The 5' integration sequence can be added to the front of a Biobrick construct and the 3' integration sequence specific for this locus (Part BBa_K143006) to the rear of the Biobrick construct to allow integration of the Biobrick construct into the chromosome of the gram positive bacterium B.subtilis.The EpsE (aka YveO) locus has to our knowledge never been used for integration into B.subtilis before, but is useful in that it knocks out the potential molecular clutch EpsE gene [1]. In particular, both the 5' and 3' integration sequences for the EpsE locus conatin in-frame stop codons to prevent translation of the gene (if nothing is integrated into the locus, integration also prevents correct EpsE expression). The 5' and 3' integration sequences for the EpsE locus were used to integrate over the EpsE gene and prevent its expression in the Imperial 2008 iGEM project B.sutbilis host.
References
1. Blair KM, Turner L, Winkelman JT, Berg HC, and Kearns DB. A molecular clutch disables flagella in the Bacillus subtilis biofilm. Science 2008 Jun 20; 320(5883) 1636-8. doi:10.1126/science.1157877 pmid:18566286
Notes
The EpsE integration sequences were designed from the EpsE (aka YveO) gene's Genbank entry[2] and identification of the sequence directly upstream of the gene on the chromosome (found using NCBI's sequence viewer). The upstream and EpsE gene sequence was analysed for restriction sites and primers (with biobrick prefix and suffix sequences) for two approximately equally sized integration sequences were desgined. The integration sequences were then produced by PCR cloning with Pfu DNA polymeraseSource
The 5??? integration sequence was taken from the B.subtilis chromosome and is homologous to the chromosme from a few hundred bp upstream of the gene's start codon until 300 bp into the gene. It was produced by PCR cloning with Pfu DNA polymerase| Sequence Annotation | Location | Component / Role(s) |
| 5' EpsE Integration Sequence In frame stop codon | 1,498 495,497 | sequence_feature feature/misc feature/stop stop_codon |