Source:
Generated By: https://synbiohub.org/public/igem/igem2sbol/1
Created by: Anthony Eckdahl
Date created: 2015-03-27 12:00:00
Date modified: 2015-08-31 04:08:29
Psimp2 Promoter with Four Consensus Variants
| Types | DnaRegion |
| Roles | Regulatory promoter |
| Sequences | BBa_J119377_sequence (Version 1) |
Description
Mutations were introduced into the Ptac promoter using NNNNNN in place of the TTGACA of the -35 region. Synthetic oligonucleotides carry the 4096 possible sequences were cloned via Golden Gate Assembly into pClone Red. A total of 81 different promoters were picked. The DNA sequence of the 81 mutant promoters was determined and their strength was measured by fluorometry (see BBa_J119375). A weighted method of consensus building was developed that assigned a score to each base in each -35 region based on the RFP expression of the mutant promoter that contained it. The resulting pClone -35 Consensus produced is shown below.
Four variants of the consensus were cloned into a simplified promoter sequence to test the effect of sequence context surrounding the -35 region. The variants are GATAAA, GACAAA, GATATA, and GACATA. The simplified promoter is called Psimp1 and is derived from De Mey et al. (2007) MC Biotechnol 7:34. The Experience tab shows images and fluorescent data that show the strength of the Psimp1 promoter with the TTGACA consensus and four variants of the pClone -35 Consensus sequence.
Notes
The consensus sequence from De Mey et al. shown below was used to generate a promoter that has a simple sequence by using W=A, N=G, R=G, and D=G, but avoiding runs of 4 or more Gs or Cs. The resulting promoter was named Psimp1.
Source
Synthetic oligonucleotides cloned into pClone Red (BBa_J119137).| Sequence Annotation | Location | Component / Role(s) |
| -10 region pClone -35 Consensus Variants -35 region | 43,48 20,25 20,25 | promoter feature/promoter sequence_alteration feature/mutation promoter feature/promoter |