Talk:20.109(S11):Complete DNA design (Day2): Difference between revisions
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|We are substituting all -10 and -35 regions in the Or1 and Or2 regions to C's. This way, frameshift mutations can be prevented. They also avoid the restrict enzyme interference. | |We are substituting all -10 and -35 regions in the Or1 and Or2 regions to C's. This way, frameshift mutations can be prevented. They also avoid the restrict enzyme interference. | ||
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| | |Yellow | ||
| | |5' CCCGGGTAAGCACCTGTAGGATCGTACAGGTTTACGCAAGAAAATGGTTTGTTATAGTCGAATAACACCGTGCGTGTTTGCTATTTTTACCTCTGGCGGTGATATTGGATCC 3' | ||
| | 5’GGATCCAATATCACCGCCAGAGGTAAAAATAGCAAACACGCACGGTGTTATTCGACTATAACAAACCATTTTCTTGCGTAAACCTGTACGATCCTACAGGTGCTTACCCGGG 3’ | ||
|We decided to change 2 nucleotides in the -35 region of P(R), and 1 nucleotide in the -10 region. We also added an extra basepair to between these two regions, increasing its length. These changes make the promoter less ideal of a binding site, therefore making it a weaker promoter. We chose the changes in nucleotides based on the lac promoter consensus sequence. | |||
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Revision as of 12:46, 10 March 2011
High-level designs and implementation with primers
T/R
| Group | Forward primer; <br>Reverse primer | Summary of approach | |
|---|---|---|---|
| Blue | 5’CCGGGTAAGCACCTGTAGGATCGTACAGGTTTACGCAAGAAAATGGTTTGTTATAGTCGAATAACACCGTGCGTGTTGTTTTACCTCTGGCGGTGATAGGG3’ 5’GATCCCCTATCACCGCCAGAGGTAAAACAACACGCACGGTGTTATTCGACTATAACAAACCATTTTCTTGCGTAAACCTGTACGATCCTACAGGTGCTTAC3’ |
The Plux-lamda promoter may be leaky due to the high basal expression of the Pr promoter. We are deleting part of the -35 region (after Or2) in the Pr promoter and adding 2 G's in the middle of -10 region (right after Or1) in order to reduce the strength of the promoter and decrease the AT richness in the sequence. | |
| Pink | 5'CCCGGGTAAGCACCTGTAGGATCGTACAGGTTTACGCAAGAAAATGGTTTGTTATAGTCGAATACCTCTGGCGGTGATATAACACCGTGCGTGTTGACTATTTTACCTCTGGCGGTGATAGGATCC3' 5'AAATCCTATCACCGCCAGAGGTAAAATAGTCAACACGCACGGTGTTATATCACCGCCAGAGGTATTCGACTATAACAAACCATTTTCTTGCGTAAACCTGTACGATCCTACAGGTGCTTACCCGGG 3' |
We are inserting a second copy of the Or1 sequence directly infront of Or2 to hopefully improve repressing of the operons | |
| Green | 5’CCCGGGTAAGCACCTGTAGGATCGTACAGGTTTACGCAAGAAAATGGTTTGTTATAGTCCCCTAACACCGTGCGTGTTGCCCCTTTTACCTCTGGCGGTGATACCGGATCC3’ 3'GGGCCCATTCGTGGACATCCTAGCATGTCCAAATGCGTTCTTTTACCAAACAATATCAGGGGATTGTGGCACGCACAACGGGGAAAATGGAGACCGCCACTATGGCCTAGG5' |
We are substituting all -10 and -35 regions in the Or1 and Or2 regions to C's. This way, frameshift mutations can be prevented. They also avoid the restrict enzyme interference. | |
| Yellow | 5' CCCGGGTAAGCACCTGTAGGATCGTACAGGTTTACGCAAGAAAATGGTTTGTTATAGTCGAATAACACCGTGCGTGTTTGCTATTTTTACCTCTGGCGGTGATATTGGATCC 3'
5’GGATCCAATATCACCGCCAGAGGTAAAAATAGCAAACACGCACGGTGTTATTCGACTATAACAAACCATTTTCTTGCGTAAACCTGTACGATCCTACAGGTGCTTACCCGGG 3’ |
We decided to change 2 nucleotides in the -35 region of P(R), and 1 nucleotide in the -10 region. We also added an extra basepair to between these two regions, increasing its length. These changes make the promoter less ideal of a binding site, therefore making it a weaker promoter. We chose the changes in nucleotides based on the lac promoter consensus sequence. | |
W/F
| Group | Forward primer; <br>Reverse primer | Summary of approach |
|---|---|---|
