IGEM:Stanford/2009/Project Homeostasis/CloningPlan
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| Project |
| Research Proposal |
| Systems Overview |
| Cloning Plan |
| Sequences & Primers |
| Anti-Inflammation |
| Device Overview |
| Parts Design |
| Challenges |
| Results |
| Anti-Immunosuppression |
| Device Overview |
| Parts Design |
| Challenges |
| Results |
| Protocols |
| Modeling |
| Overview |
| Notebook |
| Results |
| Future Work |
| Archived Work |
Working Construction Plan Documents
PDF of Construction Overview... Latest version
Device 1 Cloning Plan with Transposed Suffix
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| Cloning Step | Basic Parts | Detailed Parts | Antibiotics | Enzymes | Vector & Size (kb) | Insert & Size (kb) | Current Cloner | Picture |
|---|---|---|---|---|---|---|---|---|
| Step 1.1a | SoxR/S Promoter + GFP tag | RBS-SoxR-TT-SoxS Promoter + RBS-GFP-TT | Ant. | enzymes | Vector | ----Insert---- | Cloner | <a href="http://openwetware.org/images/1/19/1.1a.png"><img src="http://openwetware.org/images/1/19/1.1a.png" alt="1.1a" width="100" height="40"></img></a> |
| Progress Notes | ||||||||
| Step 1.1b | SoxR/S Promoter + Blh | RBS-SoxR-TT-SoxS Promoter + RBS-Blh | Ant. | enzymes | Vector | Insert | Cloner | <a href="http://openwetware.org/images/9/92/1.1b.png"> <img src="http://openwetware.org/images/9/92/1.1b.png" alt="1.1b" width="100" height="40"></img></a> |
| Progress Notes | ||||||||
| Step 1.1c | Blh + Terminator | RBS-Blh + TT | Ant. | enzymes | Vector | Insert | Cloner | <a href="http://openwetware.org/images/a/a5/1.1c.png"><img src="http://openwetware.org/images/a/a5/1.1c.png" alt="1.1c" width="100" height="40"></img></a> |
| Progress Notes | ||||||||
| Step 1.1d | Constitutive Promoter + Blh (1.1c) | Promoter +RBS-Blh-TT | Ant. | enzymes | Vector | Insert | Cloner | <a href="http://openwetware.org/images/9/92/1.1d.png"> <img src="http://openwetware.org/images/9/92/1.1d.png" alt="1.1d" width="100" height="40"></img></a> |
| Progress Notes | ||||||||
| Step 1.1e | Constitutive Promoter + Crt Cluster | Promoter + RBS-Crt | Ant. | enzymes | Vector | Insert | Cloner | <a href="http://openwetware.org/images/b/b9/1.1e.png"><img src="http://openwetware.org/images/b/b9/1.1e.png" alt="1.1e" width="100" height="40"></img></a> |
| Progress Notes | ||||||||
| Step 1.2a | Constitutive Promoter + SoxR/s w/ GFP (1.1a) | Promoter+ RBS-SoxR-TT-SoxSP-RBS-GFP-TT | Ant. | enzymes | Vector| | Insert | Cloner | <a href="http://openwetware.org/images/d/d1/1.2a.png"> <img src="http://openwetware.org/images/d/d1/1.2a.png" alt="1.2a" width="100" height="40"></img</a> |
| Progress Notes | ||||||||
| Step 1.2b | SoxR/S-Blh (1.1b) + RALDH II | RBS-SoxR-TT-SoxSP-RBS-Blh + RBS-RALDH II-TT | Ant. | enzymes | Vector | Insert | Cloner | <a href="http://openwetware.org/images/0/0f/1.22b.png"> <img src="http://openwetware.org/images/0/0f/1.22b.png" alt="1.2b" width="100" height="40"></img></a> |
| Progress Notes | ||||||||
| Step 1.3a | Constitutive Promoter + SoxR/S w/ Blh and RaldhII | Promoter+ RBS-SoxR-TT-SoxSP-RBS-Blh-RBS-RALDH II-TT | Ant. | enzymes | Vector | Insert | Cloner | <a href="http://openwetware.org/images/6/6a/1.32b.png"> <img src="http://openwetware.org/images/6/6a/1.32b.png" alt="1.3a" width="100" height="40"></img></a> |
| Progress Notes | ||||||||
| Controls W/ PQ (+) | Constitutive Promoter + GFP tag | Promoter+ RBS-GFP-TT | Ant. | enzymes | Vector | Insert | Cloner | <a href="http://openwetware.org/images/f/fb/1%2BC.png"><img src="http://openwetware.org/images/f/fb/1%2BC.png" alt="1.3a" width="100" height="40"></img></a> |
| Progress Notes | ||||||||
| Controls W/ PQ (-) | GFP tag w/ No Promoter | RBS-GFP-TT | Ant. | enzymes | Vector | Insert | Cloner | <a href="http://openwetware.org/images/e/e0/1-C.png"><img src="http://openwetware.org/images/e/e0/1-C.png" alt="1.3a" width="100" height="40"></img></a> |
| Progress Notes | ||||||||
| Controls Blh and Raldh II (+) | Constitutive Promoter + Blh and RaldhII | Promoter+ RBS-Blh-TT-RBS-RALDH II-TT | Ant. | enzymes | Vector | Insert | Cloner | <a href="http://openwetware.org/images/4/4f/1%2BC2.png"><img src="http://openwetware.org/images/4/4f/1%2BC2.png" alt="1.3a" width="100" height="40"></img></a> |
| Progress Notes | ||||||||
| Controls Blh and Raldh II (-) | Blh and RaldhII w/ no Promoter | RBS-Blh-TT-RBS-RALDH II-TT | Ant. | enzymes | Vector | Insert | Cloner | <a href="http://openwetware.org/images/3/37/1-C2.png"><img src="http://openwetware.org/images/3/37/1-C2.png" alt="1.3a" width="100" height="40"></img></a> |
| Progress Notes | ||||||||
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Device 1 Cloning Plan with Proper Sequences
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| Cloning Step | Basic Parts | Detailed Parts | Antibiotics | Enzymes | Vector & Size (kb) | Insert & Size (kb) | Current Cloner | Picture |
|---|---|---|---|---|---|---|---|---|
| Step 1.1a | SoxR/S Promoter + GFP tag | RBS-SoxR-TT-SoxS Promoter + RBS-GFP-TT | Ant. | enzymes | Vector | ----Insert---- | Cloner | <a href="http://openwetware.org/images/1/19/1.1a.png"><img src="http://openwetware.org/images/1/19/1.1a.png" alt="1.1a" width="100" height="40"></img></a> |
| Progress Notes | ||||||||
| Step 1.1b | SoxR/S Promoter + Blh | RBS-SoxR-TT-SoxS Promoter + RBS-Blh | Ant. | enzymes | Vector | Insert | Cloner | <a href="http://openwetware.org/images/9/92/1.1b.png"> <img src="http://openwetware.org/images/9/92/1.1b.png" alt="1.1b" width="100" height="40"></img></a> |
| Progress Notes | ||||||||
| Step 1.1c | Blh + Terminator | RBS-Blh + TT | Ant. | enzymes | Vector | Insert | Cloner | <a href="http://openwetware.org/images/a/a5/1.1c.png"><img src="http://openwetware.org/images/a/a5/1.1c.png" alt="1.1c" width="100" height="40"></img></a> |
| Progress Notes | ||||||||
| Step 1.1d | Constitutive Promoter + Blh (1.1c) | Promoter +RBS-Blh-TT | Ant. | enzymes | Vector | Insert | Cloner | <a href="http://openwetware.org/images/9/92/1.1d.png"> <img src="http://openwetware.org/images/9/92/1.1d.png" alt="1.1d" width="100" height="40"></img></a> |
| Progress Notes | ||||||||
| Step 1.1e | Constitutive Promoter + Crt Cluster | Promoter + RBS-Crt | Ant. | enzymes | Vector | Insert | Cloner | <a href="http://openwetware.org/images/b/b9/1.1e.png"><img src="http://openwetware.org/images/b/b9/1.1e.png" alt="1.1e" width="100" height="40"></img></a> |
| Progress Notes | ||||||||
| Step 1.2a | Constitutive Promoter + SoxR/s w/ GFP (1.1a) | Promoter+ RBS-SoxR-TT-SoxSP-RBS-GFP-TT | Ant. | enzymes | Vector| | Insert | Cloner | <a href="http://openwetware.org/images/d/d1/1.2a.png"> <img src="http://openwetware.org/images/d/d1/1.2a.png" alt="1.2a" width="100" height="40"></img</a> |
| Progress Notes | ||||||||
| Step 1.2b | SoxR/S-Blh (1.1b) + RALDH II | RBS-SoxR-TT-SoxSP-RBS-Blh + RBS-RALDH II-TT | Ant. | enzymes | Vector | Insert | Cloner | <a href="http://openwetware.org/images/0/0f/1.22b.png"> <img src="http://openwetware.org/images/0/0f/1.22b.png" alt="1.2b" width="100" height="40"></img></a> |
| Progress Notes | ||||||||
| Step 1.3a | Constitutive Promoter + SoxR/S w/ Blh and RaldhII | Promoter+ RBS-SoxR-TT-SoxSP-RBS-Blh-RBS-RALDH II-TT | Ant. | enzymes | Vector | Insert | Cloner | <a href="http://openwetware.org/images/6/6a/1.32b.png"> <img src="http://openwetware.org/images/6/6a/1.32b.png" alt="1.3a" width="100" height="40"></img></a> |
| Progress Notes | ||||||||
| Controls W/ PQ (+) | Constitutive Promoter + GFP tag | Promoter+ RBS-GFP-TT | Ant. | enzymes | Vector | Insert | Cloner | <a href="http://openwetware.org/images/f/fb/1%2BC.png"><img src="http://openwetware.org/images/f/fb/1%2BC.png" alt="1.3a" width="100" height="40"></img></a> |
| Progress Notes | ||||||||
| Controls W/ PQ (-) | GFP tag w/ No Promoter | RBS-GFP-TT | Ant. | enzymes | Vector | Insert | Cloner | <a href="http://openwetware.org/images/e/e0/1-C.png"><img src="http://openwetware.org/images/e/e0/1-C.png" alt="1.3a" width="100" height="40"></img></a> |
| Progress Notes | ||||||||
| Controls Blh and Raldh II (+) | Constitutive Promoter + Blh and RaldhII | Promoter+ RBS-Blh-TT-RBS-RALDH II-TT | Ant. | enzymes | Vector | Insert | Cloner | <a href="http://openwetware.org/images/4/4f/1%2BC2.png"><img src="http://openwetware.org/images/4/4f/1%2BC2.png" alt="1.3a" width="100" height="40"></img></a> |
| Progress Notes | ||||||||
| Controls Blh and Raldh II (-) | Blh and RaldhII w/ no Promoter | RBS-Blh-TT-RBS-RALDH II-TT | Ant. | enzymes | Vector | Insert | Cloner | <a href="http://openwetware.org/images/3/37/1-C2.png"><img src="http://openwetware.org/images/3/37/1-C2.png" alt="1.3a" width="100" height="40"></img></a> |
| Progress Notes | ||||||||
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Device 2 Cloning Plan with Transposed Suffix
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| Cloning Step | Basic Parts | Detailed Parts | Antibiotics | Enzymes | Vector & Size (kb) | Insert & Size (kb) | Current Cloner | Picture |
|---|---|---|---|---|---|---|---|---|
| Step 2.1a | hIl-6-HlyAs + Terminator | hIl-6-HlyAs + TT | Ant. | enzymes | Vector | ----Insert---- | Cloner | <a href="http://openwetware.org/images/9/99/2.1aw.png"><img src="http://openwetware.org/images/9/99/2.1aw.png" alt="1.1a" width="100" height="40"></img></a> |
| Progress Notes | ||||||||
| Step 2.1b | hIl-6-HlyAs + YFP | hIl-6-HlyAs + YFP | Ant. | enzymes | Vector | Insert | Cloner | <a href="http://openwetware.org/images/5/50/2.1bw.png"> <img src="http://openwetware.org/images/5/50/2.1bw.png" alt="1.1b" width="100" height="40"></img></a> |
| Progress Notes | ||||||||
| Step 2.1c | RBS + TolC | RBS+ TolC | Ant. | enzymes | Vector | Insert | Cloner | <a href="http://openwetware.org/images/f/fc/2.1cw.png"><img src="http://openwetware.org/images/f/fc/2.1cw.png" alt="1.1c" width="100" height="40"></img></a> |
| Progress Notes | ||||||||
| Step 2.1d | Terminator + HlyR | TT + HlyR | Ant. | enzymes | Vector | Insert | Cloner | <a href="http://openwetware.org/images/d/d0/2.1dw.png"> <img src="http://openwetware.org/images/d/d0/2.1dw.png" alt="1.1d" width="100" height="40"></img></a> |
| Progress Notes | ||||||||
| Step 2.1e | Forward Flanking Sequence (FFS) + RBS | Hly-C + RBS | Ant. | enzymes | Vector | Insert | Cloner | <a href="http://openwetware.org/images/d/d8/2.1ew.png"><img src="http://openwetware.org/images/d/d8/2.1ew.png" alt="1.1e" width="100" height="40"></img></a> |
| Progress Notes | ||||||||
| Step 2.1f | Rear Flanking Sequence (RFS) + Terminator | HlyB,D + TT | Ant. | enzymes | Vector | Insert | Cloner | <a href="http://openwetware.org/images/a/ac/2.1fw.png"><img src="http://openwetware.org/images/a/ac/2.1fw.png" alt="1.1e" width="100" height="40"></img></a> |
| Progress Notes | ||||||||
| Step 2.2a | RBS + hIL-6-HlyAs (2.1a) | RBS + hIL-6-HlyAs-TT | Ant. | enzymes | Vector| | Insert | Cloner | <a href="http://openwetware.org/images/e/e1/2.2aw.png"> <img src="http://openwetware.org/images/e/e1/2.2aw.png" alt="1.2a" width="100" height="40"></img</a> |
| Progress Notes | ||||||||
| Step 2.2b | hIl-6-HlyAs-YFP(2.1b) + Terminator | hIl-6-HlyAs-YFP+ TT | Ant. | enzymes | Vector | Insert | Cloner | <a href="http://openwetware.org/images/f/f8/2.2bw.png"> <img src="http://openwetware.org/images/f/f8/2.2bw.png" alt="1.2b" width="100" height="40"></img></a> |
| Progress Notes | ||||||||
| Step 2.2c | TolC (2.1c) + HlyR Promoter Element (2.1d) | RBS-TolC + TT-HlyR | Ant. | enzymes | Vector | Insert | Cloner | <a href="http://openwetware.org/images/5/5e/2.2cw.png"> <img src="http://openwetware.org/images/5/5e/2.2cw.png" alt="1.2b" width="100" height="40"></img></a> |
| Progress Notes | ||||||||
| Step 2.2d | FFS-RBS (2.1e) + RFS-TT (2.1f) | FFS-RBS + RFS-TT | Ant. | enzymes | Vector | Insert | Cloner | <a href="http://openwetware.org/images/7/7f/2.2dw.png"> <img src="http://openwetware.org/images/7/7f/2.2dw.png" alt="1.2b" width="100" height="40"></img></a> |
| Progress Notes | ||||||||
| Step 2.3a | Trp Operator + hIL-6-HlyAs (2.2a) | Trp Op + RBS-hIL-6-HlyAs-TT | Ant. | enzymes | Vector | Insert | Cloner | <a href="http://openwetware.org/images/a/a1/2.3aw.png"> <img src="http://openwetware.org/images/a/a1/2.3aw.png" alt="1.3a" width="100" height="40"></img></a> |
| Progress Notes | ||||||||
| Step 2.3b | RBS + hIL-6-HlyAs-YFP-TT (2.2b) | RBS + hIL-6-HlyAs-YFP-TT | Ant. | enzymes | Vector | Insert | Cloner | <a href="http://openwetware.org/images/1/19/2.3bw.png"> <img src="http://openwetware.org/images/1/19/2.3bw.png" alt="1.3a" width="100" height="40"></img></a> |
| Progress Notes | ||||||||
| Step 2.3c | TolC-HlyR (2.2c) + FFS-RFS (2.2d) | RBS-TolC-TT-HlyR + FFS-RBS-RFS-TT | Ant. | enzymes | Vector | Insert | Cloner | <a href="http://openwetware.org/images/c/c7/2.3cw.png"> <img src="http://openwetware.org/images/c/c7/2.3cw.png" alt="1.3a" width="100" height="40"></img></a> |
| Progress Notes | ||||||||
| Step 2.4a | Trp Operator + HIl-6-HlyAs-YFP (2.3b) | Trp Op + RBS-HIl-6-HlyAs-YFP-TT | Ant. | enzymes | Vector | Insert | Cloner | <a href="http://openwetware.org/images/d/d7/2.4aw.png"> <img src="http://openwetware.org/images/d/d7/2.4aw.png" alt="1.3a" width="100" height="40"></img></a> |
| Progress Notes | ||||||||
| Step 2.4b | Const. Promoter + TolC-HlyR-FFS-RFS (2.3c) | Const. Promoter + RBS-TolC-TT-HlyR-FFS-RBS-RFS-TT | Ant. | enzymes | Vector | Insert | Cloner | <a href="http://openwetware.org/images/3/3c/2.4bw.png"> <img src="http://openwetware.org/images/3/3c/2.4bw.png" alt="1.3a" width="100" height="40"></img></a> |
| Progress Notes | ||||||||
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Device 2 Cloning Plan with Proper Sequences
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| Cloning Step | Basic Parts | Detailed Parts | Antibiotics | Enzymes | Vector & Size (kb) | Insert & Size (kb) | Current Cloner | Picture |
|---|---|---|---|---|---|---|---|---|
| Step 2.1a | hIl-6-HlyAs + Terminator | hIl-6-HlyAs + TT | Ant. | enzymes | Vector | ----Insert---- | Cloner | <a href="http://openwetware.org/images/9/99/2.1aw.png"><img src="http://openwetware.org/images/9/99/2.1aw.png" alt="1.1a" width="100" height="40"></img></a> |
| Progress Notes | ||||||||
| Step 2.1b | hIl-6-HlyAs + YFP | hIl-6-HlyAs + YFP | Ant. | enzymes | Vector | Insert | Cloner | <a href="http://openwetware.org/images/5/50/2.1bw.png"> <img src="http://openwetware.org/images/5/50/2.1bw.png" alt="1.1b" width="100" height="40"></img></a> |
| Progress Notes | ||||||||
| Step 2.1c | RBS + TolC | RBS+ TolC | Ant. | enzymes | Vector | Insert | Cloner | <a href="http://openwetware.org/images/f/fc/2.1cw.png"><img src="http://openwetware.org/images/f/fc/2.1cw.png" alt="1.1c" width="100" height="40"></img></a> |
| Progress Notes | ||||||||
| Step 2.1d | Terminator + HlyR | TT + HlyR | Ant. | enzymes | Vector | Insert | Cloner | <a href="http://openwetware.org/images/d/d0/2.1dw.png"> <img src="http://openwetware.org/images/d/d0/2.1dw.png" alt="1.1d" width="100" height="40"></img></a> |
| Progress Notes | ||||||||
| Step 2.1e | Forward Flanking Sequence (FFS) + RBS | Hly-C + RBS | Ant. | enzymes | Vector | Insert | Cloner | <a href="http://openwetware.org/images/d/d8/2.1ew.png"><img src="http://openwetware.org/images/d/d8/2.1ew.png" alt="1.1e" width="100" height="40"></img></a> |
| Progress Notes | ||||||||
| Step 2.1f | Rear Flanking Sequence (RFS) + Terminator | HlyB,D + TT | Ant. | enzymes | Vector | Insert | Cloner | <a href="http://openwetware.org/images/a/ac/2.1fw.png"><img src="http://openwetware.org/images/a/ac/2.1fw.png" alt="1.1e" width="100" height="40"></img></a> |
| Progress Notes | ||||||||
| Step 2.2a | RBS + hIL-6-HlyAs (2.1a) | RBS + hIL-6-HlyAs-TT | Ant. | enzymes | Vector| | Insert | Cloner | <a href="http://openwetware.org/images/e/e1/2.2aw.png"> <img src="http://openwetware.org/images/e/e1/2.2aw.png" alt="1.2a" width="100" height="40"></img</a> |
| Progress Notes | ||||||||
| Step 2.2b | hIl-6-HlyAs-YFP(2.1b) + Terminator | hIl-6-HlyAs-YFP+ TT | Ant. | enzymes | Vector | Insert | Cloner | <a href="http://openwetware.org/images/f/f8/2.2bw.png"> <img src="http://openwetware.org/images/f/f8/2.2bw.png" alt="1.2b" width="100" height="40"></img></a> |
| Progress Notes | ||||||||
| Step 2.2c | TolC (2.1c) + HlyR Promoter Element (2.1d) | RBS-TolC + TT-HlyR | Ant. | enzymes | Vector | Insert | Cloner | <a href="http://openwetware.org/images/5/5e/2.2cw.png"> <img src="http://openwetware.org/images/5/5e/2.2cw.png" alt="1.2b" width="100" height="40"></img></a> |
| Progress Notes | ||||||||
| Step 2.2d | FFS-RBS (2.1e) + RFS-TT (2.1f) | FFS-RBS + RFS-TT | Ant. | enzymes | Vector | Insert | Cloner | <a href="http://openwetware.org/images/7/7f/2.2dw.png"> <img src="http://openwetware.org/images/7/7f/2.2dw.png" alt="1.2b" width="100" height="40"></img></a> |
| Progress Notes | ||||||||
| Step 2.3a | Trp Operator + hIL-6-HlyAs (2.2a) | Trp Op + RBS-hIL-6-HlyAs-TT | Ant. | enzymes | Vector | Insert | Cloner | <a href="http://openwetware.org/images/a/a1/2.3aw.png"> <img src="http://openwetware.org/images/a/a1/2.3aw.png" alt="1.3a" width="100" height="40"></img></a> |
| Progress Notes | ||||||||
| Step 2.3b | RBS + hIL-6-HlyAs-YFP-TT (2.2b) | RBS + hIL-6-HlyAs-YFP-TT | Ant. | enzymes | Vector | Insert | Cloner | <a href="http://openwetware.org/images/1/19/2.3bw.png"> <img src="http://openwetware.org/images/1/19/2.3bw.png" alt="1.3a" width="100" height="40"></img></a> |
| Progress Notes | ||||||||
| Step 2.3c | TolC-HlyR (2.2c) + FFS-RFS (2.2d) | RBS-TolC-TT-HlyR + FFS-RBS-RFS-TT | Ant. | enzymes | Vector | Insert | Cloner | <a href="http://openwetware.org/images/c/c7/2.3cw.png"> <img src="http://openwetware.org/images/c/c7/2.3cw.png" alt="1.3a" width="100" height="40"></img></a> |
| Progress Notes | ||||||||
| Step 2.4a | Trp Operator + HIl-6-HlyAs-YFP (2.3b) | Trp Op + RBS-HIl-6-HlyAs-YFP-TT | Ant. | enzymes | Vector | Insert | Cloner | <a href="http://openwetware.org/images/d/d7/2.4aw.png"> <img src="http://openwetware.org/images/d/d7/2.4aw.png" alt="1.3a" width="100" height="40"></img></a> |
| Progress Notes | ||||||||
| Step 2.4b | Const. Promoter + TolC-HlyR-FFS-RFS (2.3c) | Const. Promoter + RBS-TolC-TT-HlyR-FFS-RBS-RFS-TT | Ant. | enzymes | Vector | Insert | Cloner | <a href="http://openwetware.org/images/3/3c/2.4bw.png"> <img src="http://openwetware.org/images/3/3c/2.4bw.png" alt="1.3a" width="100" height="40"></img></a> |
| Progress Notes | ||||||||
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