Source:
Generated By: https://synbiohub.org/public/igem/igem2sbol/1
Created by: Malcolm Campbell and Todd Eckdahl
Date created: 2012-08-19 11:00:00
Date modified: 2015-08-31 04:08:22
For Testing New Promoters via Golden Gate Assembly
| Types | DnaRegion |
| Roles | Measurement engineered_region |
| Sequences | BBa_J100091_sequence (Version 1) |
Description
The construct allows for the cloning and testing of new promoter sequences. It is a destination vector for Golden Gate Assembly using BsaI and Ligase, based on part [[Part:BBa_J100028]]. A new promoter can be derived from synthetic oligos, PCR, or a plasmid clone. The new promoter must be flanked by BsaI sites that produce the 4 nt overhangs required for assembly (see [[Part:BBa_J119022]]). Assembly replaces the double terminator in the destination vector with the new promoter. Upon assembly, a functional new promoter will be expected to cause RFP expression. The destination vector also incorporates the BD18 bicistronic translational junction (see [[Part:BBa_J119024]]) engineered by Vivek Mutalik and The BIOFAB Team at biofab.org.Notes
The major question is what plasmid to use. Since both [[Part:BBa_J119044]] and [[Part:BBa_J100091]] both contain a transcriptional terminator (TT), this may lead to recombination if more than two TTs are in the same plasmid. We have some evidence that [[Part:BBa_J119044]] can undergo some recombination when cloned into the plasmid pSB1A8 [[Part:BBa_J119043]]. Therefore, we recommend [[Part:BBa_J119044]] and [[Part:BBa_J100091]] be cloned into a modified version of pSB1A2 that has the Bsa I site removed from the ampicillin gene. We call this modified plasmid pSB1A2 BR for Bsa I Removed.Source
This part was engineered by Todd Eckdahl and Malcolm Campbell. They devised this as part of a national effort to have undergraduates contribute to a registry of functional promoters (RFP; Registry of Functional Promoters). There is a very similar part called J119044 [[Part:BBa_J119044]]. The only difference between these two parts is that J00091 uses E1010 [[Part:BBa_E1010]] version of RFP while [[Part:BBa_J119044]] uses an E. coli-optimized set of codons and was produced by GeneArt.| Sequence Annotation | Location | Component / Role(s) |
| BBa Prefix BsaI sticky end left BsaI cuts left TT B0014 BsaI cuts right BsaI sticky end right BD18 bicistron leader RBS RBS for RFP Stop for BD18 leader Start for RFP RFP E1010 Double Stop for E1010 BBa suffix PCR For Primer PCR Rev Primer | 1,22 23,26 28,33 34,128 129,134 136,139 140,227 157,165 210,218 223,225 225,227 225,905 900,905 906,926 1,20 232,251 | engineered_region feature/BioBrick feature/dna sequence_feature feature/misc sequence_feature feature/stem_loop stem_loop feature/misc sequence_feature feature/dna sequence_feature feature/misc sequence_feature feature/rbs ribosome_entry_site feature/rbs ribosome_entry_site feature/stop stop_codon start_codon feature/start CDS feature/protein feature/stop stop_codon feature/BioBrick engineered_region feature/primer_binding primer_binding_site feature/primer_binding primer_binding_site |