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Minnesota/30 June 2008
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|TetR/p22 mnt Promoter || 5' - GAATTCGCGGCCGCTTCTAGAGTCCC - 3' || 5' - CTGCAGCGGCCGCTACTAGTAGTGCTCAGTATCTAGGTCACCG -3' | |TetR/p22 mnt Promoter || 5' - GAATTCGCGGCCGCTTCTAGAGTCCC - 3' || 5' - CTGCAGCGGCCGCTACTAGTAGTGCTCAGTATCTAGGTCACCG -3' | ||
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| + | |6. '''Meeting with advisor''': | ||
| + | |- | ||
| + | |a. '''Discussed:''' design scheme for dual repressor parts, must buy p22 mnt, want least overlap in dually repressed parts so decided 2 combinations are LacI with LambdaCI (1) and TetR with P22 mnt (2), ordered new strain of E. Coli, performed double digest. | ||
| + | |- | ||
| + | |b. '''Suggestions for the week:''' need to test whether or not the base vectors transformed the plasmids/DNA and will know that plasmid attached correctly if E. Coli has growth, send design/construct to Yiannis and he will email rest of team, meet with Jon so can discuss how machinery works, Yiannis suggests that need to begin synbioSS and start simulating immediately - he wants to see excel spreads or plots from synbioSS (simulate by next Monday), when do the simulations will find delays and issues | ||
Revision as of 19:09, 30 June 2008
| 1. Set up Sequencher: Need to determine what will be synthesized and research p22 mnt sequence. |
| 2. Repick miniprep plate #3 |
| 3. Redo glycerol stock #7 |
| 4. Discuss vectors |
| 5. Use Vector NTI: Choose the primer base pairs (essentially 20 b.p.'s long to lessen chance of hairpins or errors). Using Vector NTI, we designed primers which will allow us to add the RBS sequence, to the TetR promoter/lamba cI gene construct, through PCR rather than cloning. Since the primer sequence is approximately 8 base pairs long, use of the cloning procedure would be highly ineffective. In addition, we designed primers that will allow us to isolate the sequence of each of our dually repressed promoters, with only the prefix and suffix tails. |
- Designed Primers
| Sequence Name | Forward Primer | Reverse Primer |
|---|---|---|
| lambda cI | 5’ – TCACACAGGAAAGTACTAGATGAGCACAAAAAAGAAACC – 3’ (RBS tail)
5' - GAATTCGCGGCCGCTTCTAGAGTCACACAGGAAAGTACTAGATGAGC - 3' (Prefix tail) | 5’ – CTGCAGCGGCCGCTACTAGTATTATTAAGC – 3’ |
| LacI/λcI Promoter | 5' - GAATTCGCGGCCGCTTCTAGAGGC - 3' | 5' - CTGCAGCGGCCGCTACTAGTATGTGTGAAATTGTTATCCGC - 3' |
| TetR/p22 mnt Promoter | 5' - GAATTCGCGGCCGCTTCTAGAGTCCC - 3' | 5' - CTGCAGCGGCCGCTACTAGTAGTGCTCAGTATCTAGGTCACCG -3' |
| 6. Meeting with advisor: |
| a. Discussed: design scheme for dual repressor parts, must buy p22 mnt, want least overlap in dually repressed parts so decided 2 combinations are LacI with LambdaCI (1) and TetR with P22 mnt (2), ordered new strain of E. Coli, performed double digest. |
| b. Suggestions for the week: need to test whether or not the base vectors transformed the plasmids/DNA and will know that plasmid attached correctly if E. Coli has growth, send design/construct to Yiannis and he will email rest of team, meet with Jon so can discuss how machinery works, Yiannis suggests that need to begin synbioSS and start simulating immediately - he wants to see excel spreads or plots from synbioSS (simulate by next Monday), when do the simulations will find delays and issues |
