Source:
Generated By: https://synbiohub.org/public/igem/igem2sbol/1
Created by: Chris French, Maria Kowal and Geraldine Avila
Date created: 2010-10-06 11:00:00
Date modified: 2015-05-08 01:12:00
cat + sacB for BRIDGE: A biobrick compatible markerless recombineering method
| Types | DnaRegion |
| Roles | engineered_region Composite |
| Sequences | BBa_K322922_sequence (Version 1) |
Description
BRIDGE stands for BioBrick Recombineering In Direct Genomic Editing. It is an alternative method for inserting BioBricks into the genome by using homologous recombination instead of restriction digestion, with the added bonus of not leaving a marker behind in the product.The first step of BRIDGE requires the deletion of existing DNA (probably a non-coding piece or a non-essential gene) to introduce a construct of two genes; one an antibiotic resistance gene cat BBa-K322210, the other sacB BBa-K322921, which prevents the host from growing on sucrose. After the first step we can select for cells which have taken up the construct by growing them on the relevant antibiotic.
Notes
BBa-K322210 Notes: The PCR product, P1601, was initially cloned in pSB1A2, which made it easy to confirm that the gene was active, since the transformants became chloramphenicol resistant. A considerable amount of upstream DNA was included in the hope that this would include the promoter region. In the course of the iGEM project, the same PCR product was cloned in pSB1C3 to comply with new submission rules. Since this BioBrick was originally prepared for internal use, it uses abbreviated forms of the prefix and suffix lacking the NotI sites, but is in all ways compatible with RFC10 assembly.BBa-K322921 Note: MABEL was used to mutate an internal EcoRI site to GAGTTC (underlined). Features: coding sequence runs from 13 to 1434, RBS 1 to 3, EcoRI site mutated 211 to 216.
Source
sacB BBa-K322921 Source: Bacillus subtilis 168 genomic DNA. Sequencecat BBa-K322210 Source: plasmid pTG262 (broad host range vector for Gram positive and Gram negative bacteria).
| Access | Instance | Definition |
| public public | BBa_K322210 BBa_K322921 | BBa_K322210 sacB |
| Sequence Annotation | Location | Component / Role(s) |
| BBa_K322210 BBa_K322921 | 1,1081 1090,2526 | BBa_K322210 sacB |