Difference between revisions of "Part:BBa J119376"

Latest revision as of 12:58, 27 March 2015

Psimp1 Promoter with Four Consensus Variants

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.

Sequence and Features