Difference between revisions of "Part:BBa J119376"
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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. | 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. | ||
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| + | [[File:Ptac_-35_Consensus.png|500px|]] | ||
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Four variants of the consensus were cloned into a simplified promoter sequence to test the effect of sequence context surrounding the -35 region. The simplified promoter is called Psimp1 and is derived from De Mey et al. (2007) MC Biotechnol 7:34. Below are 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. | Four variants of the consensus were cloned into a simplified promoter sequence to test the effect of sequence context surrounding the -35 region. The simplified promoter is called Psimp1 and is derived from De Mey et al. (2007) MC Biotechnol 7:34. Below are 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. | ||
Revision as of 12:54, 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 simplified promoter is called Psimp1 and is derived from De Mey et al. (2007) MC Biotechnol 7:34. Below are 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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]