Source:
Generated By: https://synbiohub.org/public/igem/igem2sbol/1
Created by: Frederic Grenier
Date created: 2015-09-13 11:00:00
Date modified: 2015-09-18 12:43:05
araC-PBAD-vcrx028-ampR
| Types | DnaRegion |
| Roles | Composite engineered_region |
| Sequences | BBa_K1744000_sequence (Version 1) |
Description
This part is designed for clean deletion recombineering experiments. It has an arabinose induced toxin for negative selection and an ampicillin resistance gene for positive selection. This part is designed to be amplified using primer with homology to the targeted region and then integrated in that region using lambda red recombineering systems available commercially such as the heat-inducible pSIM plasmid family. The toxin vcrx028 is toxic for the cell. To repress its expression you must put glucose in the medium (we use 5%w/v). To activate the arabinose killswitch, we use 1 %w/v arabinose in the medium. To do the clean deletion (without DNA scar in the sequence) through recombineering, you must first insert the cassette in the genome and select the recombinants with ampicillin without forgetting to use medium containing glucose to avoid unwanted toxin expression. Then you make it pop out using a fusion PCR of both adjacent regions to the one you want to delete and counterselect the cells that did not lose the part with arabinose (triggering the killswitch).Notes
The protein induced is a toxin, so you MUST repress this arabinose killswitch by adding glucose. Otherwise, you will get either no positive clones or highly mutated toxin that won't work well. In fact, the toxin sequence used is mutated from the native one, but proven good killing efficiency when induced. The part could not be BioBrick standardized for XbaI and PstI. It can still be digested using NotI or EcoRI + SpeI.Source
The part was constructed using pVCR94's toxin coding sequence and cloning it in the plasmid pBAD30. The RBS was selected through rational design and added with the primer.| Sequence Annotation | Location | Component / Role(s) |
| forward priming site araC operator O2 araC promoter operator O1 CAP site operator I2 and I1 PBAD promoter TSS T > G RBS vcrx028 A > T, was the native stop codon of vcrx028 rrnB terminator T1 T2 bla bla promoter and RBS reverse priming site | 1,22 23,901 930,947 1052,1080 1088,1109 1131,1144 1140,1178 1177,1204 1213,1213 1205,1205 1234,1245 1246,1623 1596,1596 1649,2074 2365,3027 2167,2364 3028,3047 | feature/primer_binding primer_binding_site feature/cds CDS feature/operator operator feature/promoter promoter operator feature/operator non_covalent_binding_site feature/binding feature/operator operator promoter feature/promoter feature/start start_codon feature/mutation sequence_alteration feature/rbs ribosome_entry_site CDS feature/cds sequence_alteration feature/mutation stem_loop feature/stem_loop feature/cds CDS promoter feature/promoter feature/primer_binding primer_binding_site |