Source:
Generated By: https://synbiohub.org/public/igem/igem2sbol/1
Created by: Daniel Goodman
Date created: 2008-10-29 12:00:00
Date modified: 2015-05-08 01:08:37
Gram-positive Shuttle Vector for Chromosomal Integration
| Types | DnaRegion |
| Roles | plasmid Plasmid |
| Sequences | BBa_K090403_sequence (Version 1) |
Description
This is pBSINT1, a BioBrick-compatible Shuttle Vector that allows for single-copy chromosomal insertion into Bacillus subtilis. It contains the replication origin and Biobrick cut site from pSB1A3 for normal replication in E. coli. It also holds the 5' and 3' flanking regions of the AmyE gene in Bacillus subtilis, and uses these regions of homology to integrate and replace the contents of that locus on the chromosome via a double crossover event. It has two selectable resistance markers, one for Chloramphenicol (5??g/ml) in Bacillus subtiis, and one for Ampicillin (100 ??g/ml) in E. coli.This vector can be used to construct large BioBrick parts using traditional gram-negative-based transformation and minipreps, and then also be used to integrate the completed part into the genome of a gram-positive chassis.
As the figure explains, this vector was made via InFusion assembly from several pieces at once.
[[Image:PBSINT1_final.png]]
For a detailed protocol of transformation in Bacillus and how to detect integration events at the AmyE locus, see the Cambridge iGEM 2008 wiki protocols page:
http://2008.igem.org/Team:Cambridge/Bacillus_subtilis_transformation
Notes
We attempted to design the PCR ends to not interfere with the AmyE flanking regions, and also to have the same annealing temperatures. To see the primers we used, see our Primer page under the Bacillus subproject:http://2008.igem.org/Team:Cambridge/Bacillus/Primers
Source
As explained in the image, this vector was assembled at once from 4 separate pieces. The green and red regions (biobrick cut site and E. coli replication/Amp resistance) come from PBS1A3. The blue pieces are the flanking AmyE regions from ECE112, which was obtained via the Bacillus Genetic Stock Center.| Sequence Annotation | Location | Component / Role(s) |
| BioBrick prefix chloramphenicol acetyltransferase amyE back; partial alpha-amylase; similar to Genbank U46196 pBR322 origin beta-lactamase (ampicillin resistance) beta-lactamase (ampR) promoter BioBrick suffix | 1,21 419,1069 1449,2468 3070,3689 3844,4704 4746,4774 5611,5632 | sequence_feature feature/dna CDS feature/cds CDS feature/cds sequence_feature feature/dna CDS feature/cds feature/promoter promoter feature/dna sequence_feature |