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

Device 1 Cloning Plan with Transposed Suffix

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<body> <table border CLASS="cell" width=95% height=20% align=center> <tr> <th> Cloning Step </th><th> Basic Parts </th><th> Detailed Parts </th><th> Antibiotics </th> <th> Enzymes </th><th> Vector & Size (kb) </th><th>Insert & Size (kb) </th><th>Current Cloner </th><th> Picture </th> </tr> <tr> <th>Step 1.1a </th> <td>SoxR/S Promoter + GFP tag </td><td> RBS-SoxR-TT-SoxS Promoter + RBS-GFP-TT </td><td> Ant. </td><td> enzymes</td><td> Vector </td><td> ----Insert---- </td><td> Cloner </td><td><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> </td> </tr> <tr> <td colspan="9"> Progress Notes </td> </tr> <tr> <th>Step 1.1b </th> <td>SoxR/S Promoter + Blh </td><td>RBS-SoxR-TT-SoxS Promoter + RBS-Blh </td><td> Ant. </td><td> enzymes</td><td> Vector </td><td> Insert </td><td>Cloner </td><td><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> </td> <tr> <td colspan="9"> Progress Notes </td> </tr> <tr> <th>Step 1.1c </th> <td> Blh + Terminator </td><td> RBS-Blh + TT </td><td> Ant. </td><td> enzymes</td><td> Vector </td><td> Insert</td><td> Cloner </td><td> <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> </td> <tr> <td colspan="9"> Progress Notes </td> </tr> <tr> <th>Step 1.1d </th> <td> Constitutive Promoter + Blh (1.1c) </td><td> Promoter +RBS-Blh-TT </td><td> Ant. </td><td> enzymes </td><td> Vector </td><td> Insert </td><td> Cloner </td><td><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> </td> <tr> <td colspan="9"> Progress Notes </td> </tr> <tr> <th>Step 1.1e </th> 

<td> Constitutive Promoter + Crt Cluster </td><td> Promoter + RBS-Crt </td><td> Ant. </td><td> enzymes </td><td> Vector </td><td> Insert </td><td> Cloner </td><td> <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> </td>

<tr> <td colspan="9"> Progress Notes </td> </tr> <tr> <th>Step 1.2a </th> <td> Constitutive Promoter + SoxR/s w/ GFP (1.1a) </td><td> Promoter+ RBS-SoxR-TT-SoxSP-RBS-GFP-TT </td><td> Ant. </td><td> enzymes </td><td> Vector| </td><td> Insert </td><td> Cloner </td><td><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> </td> <tr> <td colspan="9"> Progress Notes </td> </tr> <tr> <th>Step 1.2b </th> <td> SoxR/S-Blh (1.1b) + RALDH II </td><td> RBS-SoxR-TT-SoxSP-RBS-Blh + RBS-RALDH II-TT </td><td> Ant. </td><td> enzymes </td><td> Vector </td><td> Insert </td><td> Cloner </td><td><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> </td> <tr> <td colspan="9"> Progress Notes </td> </tr> <tr> <th>Step 1.3a </th> <td> Constitutive Promoter + SoxR/S w/ Blh and RaldhII </td><td> Promoter+ RBS-SoxR-TT-SoxSP-RBS-Blh-RBS-RALDH II-TT </td><td> Ant. </td><td> enzymes </td><td> Vector </td><td> Insert </td><td> Cloner </td><td><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> </td> <tr> <td colspan="9"> Progress Notes </td> </tr> <tr> <th>Controls W/ PQ (+) </th> <td> Constitutive Promoter + GFP tag </td><td> Promoter+ RBS-GFP-TT </td><td> Ant. </td><td> enzymes </td><td> Vector </td><td> Insert </td><td> Cloner </td><td> <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> </td> <tr> <td colspan="9"> Progress Notes </td> </tr> <tr> <th>Controls W/ PQ (-) </th> <td> GFP tag w/ No Promoter </td><td> RBS-GFP-TT</td><td> Ant. </td><td> enzymes </td><td> Vector </td><td> Insert </td><td> Cloner </td><td> <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> </td> <tr> <td colspan="9"> Progress Notes </td> </tr> <tr> <th>Controls Blh and Raldh II (+) </th> <td> Constitutive Promoter + Blh and RaldhII </td><td> Promoter+ RBS-Blh-TT-RBS-RALDH II-TT </td><td> Ant. </td><td> enzymes </td><td> Vector </td><td> Insert </td><td> Cloner </td><td> <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> </td> <tr> <td colspan="9"> Progress Notes </td> </tr> <tr> <th>Controls Blh and Raldh II (-) </th> <td> Blh and RaldhII w/ no Promoter</td><td> RBS-Blh-TT-RBS-RALDH II-TT </td><td> Ant. </td><td> enzymes </td><td> Vector </td><td> Insert </td><td> Cloner </td><td> <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> </td> <tr> <td colspan="9"> Progress Notes </td> </tr> </table>

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Device 1 Cloning Plan with Proper Sequences

Device 1 Cloning Plan with Proper Suffix

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<body> <table border CLASS="cell" width=95% height=20% align=center> <tr> <th> Cloning Step </th><th> Basic Parts </th><th> Detailed Parts </th><th> Antibiotics </th> <th> Enzymes </th><th> Vector & Size (kb) </th><th>Insert & Size (kb) </th><th>Current Cloner </th><th> Picture </th> </tr> <tr> <th>Step 1.1a </th> <td>SoxR/S Promoter + GFP tag </td><td> RBS-SoxR-TT-SoxS Promoter + RBS-GFP-TT </td><td> Ant. </td><td> enzymes</td><td> Vector </td><td> ----Insert---- </td><td> Cloner </td><td><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> </td> </tr> <tr> <td colspan="9"> Progress Notes </td> </tr> <tr> <th>Step 1.1b </th> <td>SoxR/S Promoter + Blh </td><td>RBS-SoxR-TT-SoxS Promoter + RBS-Blh </td><td> Ant. </td><td> enzymes</td><td> Vector </td><td> Insert </td><td>Cloner </td><td><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> </td> <tr> <td colspan="9"> Progress Notes </td> </tr> <tr> <th>Step 1.1c </th> <td> Blh + Terminator </td><td> RBS-Blh + TT </td><td> Ant. </td><td> enzymes</td><td> Vector </td><td> Insert</td><td> Cloner </td><td> <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> </td> <tr> <td colspan="9"> Progress Notes </td> </tr> <tr> <th>Step 1.1d </th> <td> Constitutive Promoter + Blh (1.1c) </td><td> Promoter +RBS-Blh-TT </td><td> Ant. </td><td> enzymes </td><td> Vector </td><td> Insert </td><td> Cloner </td><td><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> </td> <tr> <td colspan="9"> Progress Notes </td> </tr> <tr> <th>Step 1.1e </th> 

<td> Constitutive Promoter + Crt Cluster </td><td> Promoter + RBS-Crt </td><td> Ant. </td><td> enzymes </td><td> Vector </td><td> Insert </td><td> Cloner </td><td> <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> </td>

<tr> <td colspan="9"> Progress Notes </td> </tr> <tr> <th>Step 1.2a </th> <td> Constitutive Promoter + SoxR/s w/ GFP (1.1a) </td><td> Promoter+ RBS-SoxR-TT-SoxSP-RBS-GFP-TT </td><td> Ant. </td><td> enzymes </td><td> Vector| </td><td> Insert </td><td> Cloner </td><td><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> </td> <tr> <td colspan="9"> Progress Notes </td> </tr> <tr> <th>Step 1.2b </th> <td> SoxR/S-Blh (1.1b) + RALDH II </td><td> RBS-SoxR-TT-SoxSP-RBS-Blh + RBS-RALDH II-TT </td><td> Ant. </td><td> enzymes </td><td> Vector </td><td> Insert </td><td> Cloner </td><td><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> </td> <tr> <td colspan="9"> Progress Notes </td> </tr> <tr> <th>Step 1.3a </th> <td> Constitutive Promoter + SoxR/S w/ Blh and RaldhII </td><td> Promoter+ RBS-SoxR-TT-SoxSP-RBS-Blh-RBS-RALDH II-TT </td><td> Ant. </td><td> enzymes </td><td> Vector </td><td> Insert </td><td> Cloner </td><td><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> </td> <tr> <td colspan="9"> Progress Notes </td> </tr> <tr> <th>Controls W/ PQ (+) </th> <td> Constitutive Promoter + GFP tag </td><td> Promoter+ RBS-GFP-TT </td><td> Ant. </td><td> enzymes </td><td> Vector </td><td> Insert </td><td> Cloner </td><td> <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> </td> <tr> <td colspan="9"> Progress Notes </td> </tr> <tr> <th>Controls W/ PQ (-) </th> <td> GFP tag w/ No Promoter </td><td> RBS-GFP-TT</td><td> Ant. </td><td> enzymes </td><td> Vector </td><td> Insert </td><td> Cloner </td><td> <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> </td> <tr> <td colspan="9"> Progress Notes </td> </tr> <tr> <th>Controls Blh and Raldh II (+) </th> <td> Constitutive Promoter + Blh and RaldhII </td><td> Promoter+ RBS-Blh-TT-RBS-RALDH II-TT </td><td> Ant. </td><td> enzymes </td><td> Vector </td><td> Insert </td><td> Cloner </td><td> <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> </td> <tr> <td colspan="9"> Progress Notes </td> </tr> <tr> <th>Controls Blh and Raldh II (-) </th> <td> Blh and RaldhII w/ no Promoter</td><td> RBS-Blh-TT-RBS-RALDH II-TT </td><td> Ant. </td><td> enzymes </td><td> Vector </td><td> Insert </td><td> Cloner </td><td> <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> </td> <tr> <td colspan="9"> Progress Notes </td> </tr> </table>

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Device 2 Cloning Plan with Transposed Suffix

Device 2 Cloning Plan with Transposed Suffix

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<body> <table border CLASS="cell" width=95% height=20% align=center> <tr> <th> Cloning Step </th><th> Basic Parts </th><th> Detailed Parts </th><th> Antibiotics </th> <th> Enzymes </th><th> Vector & Size (kb) </th><th>Insert & Size (kb) </th><th>Current Cloner </th><th> Picture </th> </tr> <tr> <th>Step 2.1a </th> <td>hIl-6-HlyAs + Terminator </td><td> hIl-6-HlyAs + TT </td><td> Ant. </td><td> enzymes</td><td> Vector </td><td> ----Insert---- </td><td> Cloner </td><td><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> </td> </tr> <tr> <td colspan="9"> Progress Notes </td> </tr> <tr> <th>Step 2.1b </th> <td> hIl-6-HlyAs + YFP </td><td> hIl-6-HlyAs + YFP </td><td> Ant. </td><td> enzymes</td><td> Vector </td><td> Insert </td><td>Cloner </td><td><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> </td> <tr> <td colspan="9"> Progress Notes </td> </tr> <tr> <th>Step 2.1c </th> <td> RBS + TolC </td><td> RBS+ TolC </td><td> Ant. </td><td> enzymes</td><td> Vector </td><td> Insert</td><td> Cloner </td><td> <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> </td> <tr> <td colspan="9"> Progress Notes </td> </tr> <tr> <th>Step 2.1d </th> <td> Terminator + HlyR </td><td> TT + HlyR</td><td> Ant. </td><td> enzymes </td><td> Vector </td><td> Insert </td><td> Cloner </td><td><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> </td> <tr> <td colspan="9"> Progress Notes </td> </tr> <tr> <th>Step 2.1e </th> 

<td> Forward Flanking Sequence (FFS) + RBS </td><td> Hly-C + RBS</td><td> Ant. </td><td> enzymes </td><td> Vector </td><td> Insert </td><td> Cloner </td><td> <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> </td>

<tr> <td colspan="9"> Progress Notes </td> </tr> <tr> <th>Step 2.1f </th> 

<td> Rear Flanking Sequence (RFS) + Terminator </td><td> HlyB,D + TT</td><td> Ant. </td><td> enzymes </td><td> Vector </td><td> Insert </td><td> Cloner </td><td> <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> </td>

<tr> <td colspan="9"> Progress Notes </td> </tr> <tr> <th>Step 2.2a </th> <td> RBS + hIL-6-HlyAs (2.1a) </td><td> RBS + hIL-6-HlyAs-TT</td><td> Ant. </td><td> enzymes </td><td> Vector| </td><td> Insert </td><td> Cloner </td><td><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> </td> <tr> <td colspan="9"> Progress Notes </td> </tr> <tr> <th>Step 2.2b </th> <td> hIl-6-HlyAs-YFP(2.1b) + Terminator </td><td> hIl-6-HlyAs-YFP+ TT </td><td> Ant. </td><td> enzymes </td><td> Vector </td><td> Insert </td><td> Cloner </td><td><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> </td> <tr> <tr> <td colspan="9"> Progress Notes </td> </tr> <th>Step 2.2c </th> <td> TolC (2.1c) + HlyR Promoter Element (2.1d)</td><td> RBS-TolC + TT-HlyR </td><td> Ant. </td><td> enzymes </td><td> Vector </td><td> Insert </td><td> Cloner </td><td><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> </td> <tr> <tr> <td colspan="9"> Progress Notes </td> </tr> <th>Step 2.2d </th> <td> FFS-RBS (2.1e) + RFS-TT (2.1f)</td><td> FFS-RBS + RFS-TT </td><td> Ant. </td><td> enzymes </td><td> Vector </td><td> Insert </td><td> Cloner </td><td><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> </td> <tr> <td colspan="9"> Progress Notes </td> </tr> <tr> <th>Step 2.3a </th> <td> Trp Operator + hIL-6-HlyAs (2.2a) </td><td> Trp Op + RBS-hIL-6-HlyAs-TT </td><td> Ant. </td><td> enzymes </td><td> Vector </td><td> Insert </td><td> Cloner </td><td><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> </td> <tr> <td colspan="9"> Progress Notes </td> </tr> <tr> <th>Step 2.3b </th> <td> RBS + hIL-6-HlyAs-YFP-TT (2.2b) </td><td> RBS + hIL-6-HlyAs-YFP-TT </td><td> Ant. </td><td> enzymes </td><td> Vector </td><td> Insert </td><td> Cloner </td><td><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> </td> <tr> <td colspan="9"> Progress Notes </td> </tr> <tr> <th>Step 2.3c </th> <td> TolC-HlyR (2.2c) + FFS-RFS (2.2d)</td><td> RBS-TolC-TT-HlyR + FFS-RBS-RFS-TT </td><td> Ant. </td><td> enzymes </td><td> Vector </td><td> Insert </td><td> Cloner </td><td><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> </td> <tr> <td colspan="9"> Progress Notes </td> </tr> <tr> <th>Step 2.4a </th> <td> Trp Operator + HIl-6-HlyAs-YFP (2.3b)</td><td> Trp Op + RBS-HIl-6-HlyAs-YFP-TT </td><td> Ant. </td><td> enzymes </td><td> Vector </td><td> Insert </td><td> Cloner </td><td><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> </td> <tr> <td colspan="9"> Progress Notes </td> </tr> <tr> <th>Step 2.4b </th> <td> Const. Promoter + TolC-HlyR-FFS-RFS (2.3c) </td><td> Const. Promoter + RBS-TolC-TT-HlyR-FFS-RBS-RFS-TT </td><td> Ant. </td><td> enzymes </td><td> Vector </td><td> Insert </td><td> Cloner </td><td><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> </td> <tr> <td colspan="9"> Progress Notes </td> </tr> </table>

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Device 2 Cloning Plan with Proper Sequences

Device 2 Cloning Plan with Proper Suffix

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<body> <table border CLASS="cell" width=95% height=20% align=center> <tr> <th> Cloning Step </th><th> Basic Parts </th><th> Detailed Parts </th><th> Antibiotics </th> <th> Enzymes </th><th> Vector & Size (kb) </th><th>Insert & Size (kb) </th><th>Current Cloner </th><th> Picture </th> </tr> <tr> <th>Step 2.1a </th> <td>hIl-6-HlyAs + Terminator </td><td> hIl-6-HlyAs + TT </td><td> Ant. </td><td> enzymes</td><td> Vector </td><td> ----Insert---- </td><td> Cloner </td><td><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> </td> </tr> <tr> <td colspan="9"> Progress Notes </td> </tr> <tr> <th>Step 2.1b </th> <td> hIl-6-HlyAs + YFP </td><td> hIl-6-HlyAs + YFP </td><td> Ant. </td><td> enzymes</td><td> Vector </td><td> Insert </td><td>Cloner </td><td><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> </td> <tr> <td colspan="9"> Progress Notes </td> </tr> <tr> <th>Step 2.1c </th> <td> RBS + TolC </td><td> RBS+ TolC </td><td> Ant. </td><td> enzymes</td><td> Vector </td><td> Insert</td><td> Cloner </td><td> <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> </td> <tr> <td colspan="9"> Progress Notes </td> </tr> <tr> <th>Step 2.1d </th> <td> Terminator + HlyR </td><td> TT + HlyR</td><td> Ant. </td><td> enzymes </td><td> Vector </td><td> Insert </td><td> Cloner </td><td><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> </td> <tr> <td colspan="9"> Progress Notes </td> </tr> <tr> <th>Step 2.1e </th> 

<td> Forward Flanking Sequence (FFS) + RBS </td><td> Hly-C + RBS</td><td> Ant. </td><td> enzymes </td><td> Vector </td><td> Insert </td><td> Cloner </td><td> <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> </td>

<tr> <td colspan="9"> Progress Notes </td> </tr> <tr> <th>Step 2.1f </th> 

<td> Rear Flanking Sequence (RFS) + Terminator </td><td> HlyB,D + TT</td><td> Ant. </td><td> enzymes </td><td> Vector </td><td> Insert </td><td> Cloner </td><td> <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> </td>

<tr> <td colspan="9"> Progress Notes </td> </tr> <tr> <th>Step 2.2a </th> <td> RBS + hIL-6-HlyAs (2.1a) </td><td> RBS + hIL-6-HlyAs-TT</td><td> Ant. </td><td> enzymes </td><td> Vector| </td><td> Insert </td><td> Cloner </td><td><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> </td> <tr> <td colspan="9"> Progress Notes </td> </tr> <tr> <th>Step 2.2b </th> <td> hIl-6-HlyAs-YFP(2.1b) + Terminator </td><td> hIl-6-HlyAs-YFP+ TT </td><td> Ant. </td><td> enzymes </td><td> Vector </td><td> Insert </td><td> Cloner </td><td><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> </td> <tr> <tr> <td colspan="9"> Progress Notes </td> </tr> <th>Step 2.2c </th> <td> TolC (2.1c) + HlyR Promoter Element (2.1d)</td><td> RBS-TolC + TT-HlyR </td><td> Ant. </td><td> enzymes </td><td> Vector </td><td> Insert </td><td> Cloner </td><td><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> </td> <tr> <tr> <td colspan="9"> Progress Notes </td> </tr> <th>Step 2.2d </th> <td> FFS-RBS (2.1e) + RFS-TT (2.1f)</td><td> FFS-RBS + RFS-TT </td><td> Ant. </td><td> enzymes </td><td> Vector </td><td> Insert </td><td> Cloner </td><td><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> </td> <tr> <td colspan="9"> Progress Notes </td> </tr> <tr> <th>Step 2.3a </th> <td> Trp Operator + hIL-6-HlyAs (2.2a) </td><td> Trp Op + RBS-hIL-6-HlyAs-TT </td><td> Ant. </td><td> enzymes </td><td> Vector </td><td> Insert </td><td> Cloner </td><td><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> </td> <tr> <td colspan="9"> Progress Notes </td> </tr> <tr> <th>Step 2.3b </th> <td> RBS + hIL-6-HlyAs-YFP-TT (2.2b) </td><td> RBS + hIL-6-HlyAs-YFP-TT </td><td> Ant. </td><td> enzymes </td><td> Vector </td><td> Insert </td><td> Cloner </td><td><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> </td> <tr> <td colspan="9"> Progress Notes </td> </tr> <tr> <th>Step 2.3c </th> <td> TolC-HlyR (2.2c) + FFS-RFS (2.2d)</td><td> RBS-TolC-TT-HlyR + FFS-RBS-RFS-TT </td><td> Ant. </td><td> enzymes </td><td> Vector </td><td> Insert </td><td> Cloner </td><td><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> </td> <tr> <td colspan="9"> Progress Notes </td> </tr> <tr> <th>Step 2.4a </th> <td> Trp Operator + HIl-6-HlyAs-YFP (2.3b)</td><td> Trp Op + RBS-HIl-6-HlyAs-YFP-TT </td><td> Ant. </td><td> enzymes </td><td> Vector </td><td> Insert </td><td> Cloner </td><td><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> </td> <tr> <td colspan="9"> Progress Notes </td> </tr> <tr> <th>Step 2.4b </th> <td> Const. Promoter + TolC-HlyR-FFS-RFS (2.3c) </td><td> Const. Promoter + RBS-TolC-TT-HlyR-FFS-RBS-RFS-TT </td><td> Ant. </td><td> enzymes </td><td> Vector </td><td> Insert </td><td> Cloner </td><td><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> </td> <tr> <td colspan="9"> Progress Notes </td> </tr> </table>

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