Source:
Generated By: https://synbiohub.org/public/igem/igem2sbol/1
Created by: Rachel Neal
Date created: 2016-06-27 11:00:00
Date modified: 2016-06-29 03:21:08
rClone Red Version 2 with RBS: Device for GGA Cloning and Testing RBS elements and Riboswitches
| Types | DnaRegion |
| Roles | engineered_region Reporter |
| Sequences | BBa_J100282_sequence (Version 1) |
Description
rClone Red Version 2 (V2) allows users to clone and test new RBS elements and riboswitches without gel purification or other preparation of DNA. It is a destination vector for Golden Gate Assembly (GGA) using BsaI and ligase. A new RBS or riboswitch can be derived from synthetic oligos, PCR, or a plasmid clone. For proper assembly, the new insert must have the appropriate 4 nt sticky ends or be flanked by BsaI sites that produce the sticky ends. With reference to the top strand, the left site must be 5' CGAC 3' and the right site must be 5' GCGG 3'. BsaI always produces a 5' overhang sticky end. Successful GGA assembly replaces the reverse promoter driving GFP expression with the new RBS or riboswitch. Transcription will be initiated in the direction of the RFP coding sequence. The level of expression of RFP will depend on the efficiency of the newly cloned RBS or riboswitch. Hopefully by removing the hairpin the in the original rClone Red it will increase translation of RFP. By adding the RBS this should glow red with the rClone red RFP.
Notes
When designing oligonucleotides for use with rClone Red V2, make sure they result in 5' overhang sticky ends that are CGAC (left) and GCGG (right). Also make sure the oligonucleotides do not contain binding sites for BsaI. Finally, make sure the RBS element ends immediately before the GCGG right sticky end. This will ensure a spacing of 6 bases between the RBS and the ATG start codon of RFP. Below is an example.
| Sequence Annotation | Location | Component / Role(s) |
| RBS RFP sticky end P5 promoter sticky end | 41,51 58,738 37,41 1,36 52,55 | ribosome_entry_site feature/rbs CDS feature/cds feature/dna sequence_feature feature/promoter promoter feature/dna sequence_feature |