Difference between revisions of "Team:Edinburgh/Notebook/PMADetection"
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<a href="https://2015.igem.org/Team:Edinburgh">Home</a></li> | <a href="https://2015.igem.org/Team:Edinburgh">Home</a></li> | ||
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| + | <a href="#" class="dropdown-toggle" data-toggle="dropdown" role="button" aria-expanded="false">About<span class="caret"></span></a> | ||
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| + | <li><a href="https://2015.igem.org/Team:Edinburgh/Team">Team</a></li> | ||
| + | <li><a href="https://2015.igem.org/Team:Edinburgh/Attributions">Attributions</a></li> | ||
| + | <li><a href="https://2015.igem.org/Team:Edinburgh/Collaborations">Collaboration</a></li> | ||
| + | <li><a href="https://2015.igem.org/Team:Edinburgh/Sponsors">Sponsors</a></li> | ||
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| − | <li><a href="https://2015.igem.org/Team:Edinburgh/Description"> | + | <li><a href="https://2015.igem.org/Team:Edinburgh/Description">Overview</a></li> |
| − | + | <!-- <li><a href="https://2015.igem.org/Team:Edinburgh/Experiments">Experiments</a> </li> --> | |
| − | <li><a href="https://2015.igem.org/Team:Edinburgh/ | + | <li><a href="https://2015.igem.org/Team:Edinburgh/HeroinBiosensor">Heroin Biosensor</a></li> |
| − | <li><a href="https://2015.igem.org/Team:Edinburgh/ | + | <li><a href="https://2015.igem.org/Team:Edinburgh/DNPBiosensor">DNP Biosensor</a></li> |
| − | <li><a href="https://2015.igem.org/Team:Edinburgh/ | + | <li><a href="https://2015.igem.org/Team:Edinburgh/PMABiosensor">PMA Biosensor</a></li> |
| + | <li><a href="https://2015.igem.org/Team:Edinburgh/CBD">Making it Stick</a></li> | ||
| + | <li><a href="https://2015.igem.org/Team:Edinburgh/Results">Limits of Detection</a></li> | ||
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<a href="#" class="dropdown-toggle" data-toggle="dropdown" role="button" aria-expanded="false">Parts<span class="caret"></span></a> | <a href="#" class="dropdown-toggle" data-toggle="dropdown" role="button" aria-expanded="false">Parts<span class="caret"></span></a> | ||
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<li><a href="https://2015.igem.org/Team:Edinburgh/Basic_Part">Basic Parts</a></li> | <li><a href="https://2015.igem.org/Team:Edinburgh/Basic_Part">Basic Parts</a></li> | ||
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| + | <li><a href="https://2015.igem.org/Team:Edinburgh/Improved_Part">Improved Parts</a></li> | ||
| + | <li><a href="https://2015.igem.org/Team:Edinburgh/Characterisation_Part">Improved Characterisation</a></li> | ||
| + | </ul> | ||
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| + | <a href="#" class="dropdown-toggle" data-toggle="dropdown" role="button" aria-expanded="false">Dry Lab<span class="caret"></span></a> | ||
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| + | <li><a href="https://2015.igem.org/Team:Edinburgh/Modeling">Modeling</a></li> | ||
| + | <li><a href="https://2015.igem.org/Team:Edinburgh/Design">Design</a></li> | ||
| + | <li><a href="https://2015.igem.org/Team:Edinburgh/Software">Software</a></li> | ||
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| − | <a href="#" class="dropdown-toggle" data-toggle="dropdown" role="button" aria-expanded="false"> | + | <a href="#" class="dropdown-toggle" data-toggle="dropdown" role="button" aria-expanded="false">Human Practices<span class="caret"></span></a> |
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<li><a href="https://2015.igem.org/Team:Edinburgh/Practices">Our Story</a></li> | <li><a href="https://2015.igem.org/Team:Edinburgh/Practices">Our Story</a></li> | ||
<li><a href="https://2015.igem.org/Team:Edinburgh/Safety">Safety</a> </li> | <li><a href="https://2015.igem.org/Team:Edinburgh/Safety">Safety</a> </li> | ||
<li><a href="https://2015.igem.org/Team:Edinburgh/Legality">Legality</a> </li> | <li><a href="https://2015.igem.org/Team:Edinburgh/Legality">Legality</a> </li> | ||
| + | <li><a href="https://2015.igem.org/Team:Edinburgh/Outreach">Outreach</a> </li> | ||
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<a href="#" class="dropdown-toggle" data-toggle="dropdown" role="button" aria-expanded="false">InterLab<span class="caret"></span></a> | <a href="#" class="dropdown-toggle" data-toggle="dropdown" role="button" aria-expanded="false">InterLab<span class="caret"></span></a> | ||
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<a href="#" class="dropdown-toggle" data-toggle="dropdown" role="button" aria-expanded="false">Notebook<span class="caret"></span></a> | <a href="#" class="dropdown-toggle" data-toggle="dropdown" role="button" aria-expanded="false">Notebook<span class="caret"></span></a> | ||
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| + | <li><a href="https://2015.igem.org/Team:Edinburgh/Project/Protocols">Protocols</a></li> | ||
<li><a href="https://2015.igem.org/Team:Edinburgh/Notebook/HeroinPurity">Heroin Purity</a></li> | <li><a href="https://2015.igem.org/Team:Edinburgh/Notebook/HeroinPurity">Heroin Purity</a></li> | ||
<li><a href="https://2015.igem.org/Team:Edinburgh/Notebook/PMADetection">PMA Detection</a> </li> | <li><a href="https://2015.igem.org/Team:Edinburgh/Notebook/PMADetection">PMA Detection</a> </li> | ||
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</ul> | </ul> | ||
</li> | </li> | ||
| − | + | <li><a href="https://2015.igem.org/Team:Edinburgh/MedalCriteria">Accomplishments</a></li> | |
| − | + | </ul> | |
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<h2>PMA Detection</h2> | <h2>PMA Detection</h2> | ||
<p> | <p> | ||
| + | <br><b>Week 1</b> | ||
| + | <br>Take Peroxidase out of the registry. | ||
| + | <br> | ||
| + | <img class="img-responsive" src="https://static.igem.org/mediawiki/2015/7/75/Edigem15_nb_1.png"> | ||
| + | <br> | ||
| + | <br>Order MaoA in two parts, N and C terminals. | ||
| + | <br> | ||
| + | <br><b>Week 2</b> | ||
| + | <br><b>16/06</b> | ||
| + | <br>Digest pSB1C3 (1.23O2), pSB1A3 (4.2H1) and pSB1K3 (4.6B1) with EcoRI HF and PstI. Digest MaoA N and MaoA C (EcoRI HF/PstI). | ||
| + | <br>Gel purify backbones, PCR purify inserts. Nanodrop. | ||
| + | <br> | ||
| + | <img class="img-responsive" src="https://static.igem.org/mediawiki/2015/f/fd/Edigem15_nb_2.png"> | ||
| + | <br> | ||
| + | <br><b>17/06</b> | ||
| + | <br>Ligate MaoA N 1 to pSB1C3, MaoA N 2 to pSB1K3, and MaoA C to pSB1A3. Transform. | ||
| + | <br> | ||
| + | <br><b>18/06</b> | ||
| + | <br>Growth appears on all plates (except for negative control). Less growth on the plates with backbone only which indicates that some of the insert ligated into the vector. | ||
| + | Culture 3 colonies from each plate. | ||
| + | <br> | ||
| + | <br><b>19/06</b> | ||
| + | <br>Miniprep cultures. Nanodrop. | ||
| + | <br> | ||
| + | <img class="img-responsive" src="https://static.igem.org/mediawiki/2015/2/23/Edigem15_nb_3.png"> | ||
| + | <br> | ||
| + | <br>Diagnostic digest to show if inserts are actually present. pSB1K3 seems to have false positives. | ||
| + | <br> | ||
| + | <br><b>Week 4</b> | ||
| + | <br><b>22/06</b> | ||
| + | <br>Prepare miniprep products (MaoA N1 and MaoA C) for sequencing. | ||
| + | <br> | ||
| + | <br><b>Week 5</b> | ||
| + | <br><b>29/06</b> | ||
| + | <br>Digest MaoA N1 1 in pSB1C3 and MaoA C 1 in pSB1A3 for fusion. PCR purify pSB1C3+MaoA N, gel purify MaoA C. Nanodrop. | ||
| + | <br> | ||
| + | <img class="img-responsive" src="https://static.igem.org/mediawiki/2015/f/f1/Edigem15_nb_4.png"> | ||
| + | <br> | ||
| + | <br>Ligate pSB1C3+MaoA N to MaoA C overnight. | ||
| + | <br> | ||
| + | <br><b>30/06</b> | ||
| + | <br>Transform ligation reactions. | ||
| + | <br> | ||
| + | <br><b>01/07</b> | ||
| + | <br>All of the transformations appeared to have worked. Culture transformations | ||
| + | <br> | ||
| + | <br><b>02/07</b> | ||
| + | <br>Miniprep cultures. Nanodrop. | ||
| + | <br> | ||
| + | <img class="img-responsive" src="https://static.igem.org/mediawiki/2015/6/6c/Edigem15_nb_5.png"> | ||
| + | <br> | ||
| + | <br>Send pSB1C3+ MaoA N+MaoA C 1 and 2 for sequencing. | ||
| + | <br>Run a diagnostic digest on them. | ||
| + | <br>Diagnostic digest shows that MaoA N and C did not fuse, as together they should be around 2.3 kb and the insert is much smaller, closer to 1 kb which is the size of MaoA N alone. | ||
| + | <br>Culture 4 more colonies from pSB1C3+MaoA N+MaoA C to see if there was successful fusion in any of them. | ||
| + | <br> | ||
| + | <br><b>03/07</b> | ||
| + | <br>Miniprep the cultures. Nanodrop. | ||
| + | <br> | ||
| + | <img class="img-responsive" src="https://static.igem.org/mediawiki/2015/2/2d/Edigem15_nb_6.png"> | ||
| + | <br> | ||
| + | <br>Run diagnostic digest to see if the fusion worked. | ||
| + | <br> | ||
| + | <br>Diagnostic gel shows that the fusion did not work. | ||
| + | <br> | ||
| + | <br><b>Week 6</b> | ||
| + | <br><b>07/07</b> | ||
| + | <br>Retry fusing MaoA together. Digest pSB1C3 +MaoA N (AgeI/SpeI) and pSB1A3+MaoA C (NgoMIV/SpeI). Treat pSB1C3+ MaoA N with Antarctic phostphatase. | ||
| + | <br>Gel purify. Nanodrop. | ||
| + | <br> | ||
| + | <img class="img-responsive" src="https://static.igem.org/mediawiki/2015/3/30/Edigem15_nb_7.png"> | ||
| + | <br> | ||
| + | <br>Ligate MaoA C into pSB1C3+MaoA N. | ||
| + | <br> | ||
| + | <br><b>08/07</b> | ||
| + | <br>Transform ligation results to see if MaoA fused. | ||
| + | <br> | ||
| + | <br><b>09/07</b> | ||
| + | <br>No growth on plates which indicates that MaoA did not fuse. | ||
| + | <br>Try fusion again by going back to the gBlock. Digest pSB1C3+MaoA N (AgeI/SpeI) and the MaoA C gBlock (NgoMIV/SpeI). Treat pSB1C3+MaoA N with Antarctic Phosphatase. Ligate the two together. Transform the ligation. | ||
| + | <br> | ||
| + | <br><b>10/07</b> | ||
| + | <br>No colonies on control plates (backbone only), quite a few colonies on plates of backbone plus insert which indicates fusion of MaoA. | ||
| + | <br> | ||
| + | <br><b>12/07</b> | ||
| + | <br>Inoculate colonies of transformants. | ||
| + | <br> | ||
| + | <br><b>Week 7</b> | ||
| + | <br><b>13/07</b> | ||
| + | <br>Miniprep the two colonies with potential fusion of MaoA. Nanodrop. | ||
| + | <br> | ||
| + | <img class="img-responsive" src="https://static.igem.org/mediawiki/2015/2/2f/Edigem15_nb_8.png"> | ||
| + | <br> | ||
| + | <br>Diagnostic digest the plasmids to fully ensure fusion of MaoA. | ||
| + | <br> | ||
| + | <br><b>14/07</b> | ||
| + | <br>Sequence MaoA 1 and MaoA 2. | ||
| + | <br> | ||
| + | <br><b>Week 8</b> | ||
| + | <br><b>27/07</b> | ||
| + | <br>Inoculate more colonies from the plate with pSB1C3+MaoA fusion. | ||
| + | <br> | ||
| + | <br><b>28/07</b> | ||
| + | <br>Make glycerols, miniprep and nanodrop cultures. | ||
| + | <br> | ||
| + | <img class="img-responsive" src="https://static.igem.org/mediawiki/2015/4/47/Edigem15_nb_9.png"> | ||
| + | <br> | ||
| + | <br>Diagnostic digest. | ||
| + | <br> | ||
| + | <br><b>29/07</b> | ||
| + | <br>Sequence pSB1C3+MaoA 3, pSB1C3+MaoA 4, pSB1C3+MaoA 5 and pSB1C3+MaoA 6. | ||
| + | <br> | ||
| + | <br><b>Week 9</b> | ||
| + | <br><b>07/08</b> | ||
| + | <br>Retransform pSB1C3+MaoA 4 into E.coli Top10 cells. | ||
| + | <br> | ||
| + | <br><b>09/08</b> | ||
| + | <br>Inoculate transformants. | ||
| + | <br> | ||
| + | <br><b>Week 10</b> | ||
| + | <br><b>10/08</b> | ||
| + | <br>Make glycerols, miniprep and nanodrop cultures. | ||
| + | <br> | ||
| + | <img class="img-responsive" src="https://static.igem.org/mediawiki/2015/0/03/Edigem15_nb_10.png"> | ||
| + | <br> | ||
| + | <br>Diagnostic digest. | ||
| + | <br> | ||
| + | <br><b>11/08</b> | ||
| + | <br>Sequence MaoA 4(1) and MaoA 4(2). | ||
| + | <br> | ||
| + | <br><b>14/08</b> | ||
| + | <br>Insert LacI into pSB1C3+MaoA 4. Digest LacI with EcoRI/SpeI, and MaoA with EcoRI/XbaI. Phosphatase treat, ligate and transform into E.coli Top10s. | ||
| + | <br> | ||
| + | <br><b>16/08</b> | ||
| + | <br>Inoculate successful transformants. | ||
| + | <br> | ||
| + | <br><b>Week 11</b> | ||
| + | <br><b>17/08</b> | ||
| + | Make glycerols, miniprep and nanodrop cultures. | ||
| + | <br> | ||
| + | <img class="img-responsive" src="https://static.igem.org/mediawiki/2015/8/8f/Edigem15_nb_11.png"> | ||
| + | <br> | ||
| + | <br>Diagnostic digest indicated the presence of insert. | ||
| + | <br> | ||
| + | <br><b>18/08</b> | ||
| + | <br>Sequence MaoA 4+LacI 1. | ||
| + | <br> | ||
| + | <br>Amplify pSB1C3+MoaA 4 (1) using PCR. PCR purify the products, nanodrop and run a diagnostic digest to confirm the correct sequence was amplified. | ||
| + | <br> | ||
| + | <img class="img-responsive" src="https://static.igem.org/mediawiki/2015/0/08/Edigem15_nb_12.png"> | ||
| + | <br> | ||
| + | <br><b>19/08</b> | ||
| + | <br>Fuse MaoA into each of the CBDs. Digest the MaoA for N terminal fusions with NgoMIV/PstI and digest with EcoRI/AgeI for C terminal fusions. Digest the CBDs for N terminal fusions with AgeI/PstI and for C terminal fusions digest with EcoRI/NgoMIV. Treat with antarctic phosphatase, ligate and transform. | ||
| + | <br> | ||
| + | <br><b>20/08</b> | ||
| + | <br>Inoculate successful transformants. | ||
| + | <br> | ||
| + | <br><b>21/08</b> | ||
| + | <br>Fuse MaoA to CBDCipA. Digest MaoA with NgoMIV/PstI for N terminal fusions and with EcoRI/AgeI for C terminal fusions. Digest CBDCipA with AgeI/PstI for N terminal fusions and with EcoRI/NgoMIV for C terminal fusions. Treat with antarctic phosphatase, ligate and transform into E.coli Top10s. | ||
| + | <br> | ||
| + | <br>Make glycerols, miniprep and nanodrop cultures. | ||
| + | <br> | ||
| + | <img class="img-responsive" src="https://static.igem.org/mediawiki/2015/b/be/Edigem15_nb_13.png"> | ||
| + | <br> | ||
| + | <br>Diagnostic digest indicated the presence of insert. | ||
| + | <br> | ||
| + | <br>Transform LacI+MaoA into E.coli BL21 cells. | ||
| + | <br> | ||
| + | <br><b>Week 12</b> | ||
| + | <br><b>24/08</b> | ||
| + | <br>Sequence BBa_K1321339+MaoA C1, BBa_K1321339+MaoA C2, BBa_K1321340+MaoA C1, BBa_K1321340+MaoA C2, BBa_K1321003+MaoA C1, BBa_K1321003+MaoA C2, BBa_K1321002+MaoA C1 and BBa_K1321002+MaoA C2. | ||
| + | <br> | ||
| + | <br>Retry fusing MaoA into CBDs. Digest MaoA with NgoMIV/PstI for N terminal fusions and with EcoRI/AgeI for C terminal fusions. Digest CBDs with AgeI/PstI for N terminal fusions and with EcoRI/NgoMIV for C terminal fusions. Antarctic phosphatase treat the CBD backbones, then ligate and transform. | ||
| + | <br> | ||
| + | <br><b>25/08</b> | ||
| + | <br>Inoculate successful transformants. | ||
| + | <br> | ||
| + | <br><b>26/08</b> | ||
| + | <br>Retry the failed MaoA to CBD fusions. Digest MaoA with NgoMIV/PstI for N terminal fusions and with EcoRI/AgeI for C terminal fusions. Digest the CBDs with AgeI/PstI for N terminal fusions and with EcoRI/NgoMIV for C terminal fusions. Phosphatase treat the CBDs, then ligate and transform. | ||
| + | <br>Make glycerols, miniprep and nanodrop the cultures. | ||
| + | <br> | ||
| + | <img class="img-responsive" src="https://static.igem.org/mediawiki/2015/2/2f/Edigem15_nb_14.png"> | ||
| + | <br> | ||
| + | <br>Diagnostic digest indicated there was no insert present. | ||
| + | <br> | ||
| + | <br><b>27/08</b> | ||
| + | <br>Insert LacI into MaoA+CBD fusions. Digest LacI with EcoRI/SpeI and digest the backbones with EcoRI/XbaI. Antarctic phosphatase treat the backbones, ligate and transform. | ||
| + | <br> | ||
| + | <br>Inoculate successful transformants. | ||
| + | <br> | ||
| + | <br><b>28/08</b> | ||
| + | <br>Make glycerols, miniprep and nanodrop cultures. | ||
| + | <br> | ||
| + | <img class="img-responsive" src="https://static.igem.org/mediawiki/2015/9/97/Edigem15_nb_15.png"> | ||
| + | <br> | ||
| + | <br>Diagnostic digest indicated the presence of insert. | ||
| + | <br> | ||
| + | <br><b>Week 13</b> | ||
| + | <br><b>31/08</b> | ||
| + | <br>Inoculate successful transformants. | ||
| + | <br> | ||
| + | <br>Transform LacI+MaoA 4 into E.coli BL21 cells. | ||
| + | <br> | ||
| + | <br><b>01/09</b> | ||
| + | <br>Sequence CBDCipA+MaoA N(2), CBDCipA+MaoA C(1), CBDCipA+MaoA C(2), 5.16O+MaoA N(1) and 5.16O+MaoA N(2). | ||
| + | <br> | ||
| + | <br>Make glycerols, miniprep and nanodrop cultures. | ||
| + | <br> | ||
| + | <img class="img-responsive" src="https://static.igem.org/mediawiki/2015/9/9f/Edigem15_nb_16.png"> | ||
| + | <br> | ||
| + | <br>Diagnostic digest indicated there was no insert present. | ||
| + | <br> | ||
| + | <br><b>Week 14</b> | ||
| + | <br><b>07/09</b> | ||
| + | <br>Insert LacI into MaoA+CBD fusions. Digest LacI with EcoRI/SpeI and digest the backbones with EcoRI/XbaI. Antarctic phosphatase treat the backbones, ligate and transform into E.coli Top10 cells. | ||
| + | <br> | ||
| + | <br><b>08/09</b> | ||
| + | <br>Inoculate successful transformants. | ||
| + | <br> | ||
| + | <br><b>09/09</b> | ||
| + | <br>Make glycerols, miniprep and nanodrop cultures. | ||
| + | <br> | ||
| + | <img class="img-responsive" src="https://static.igem.org/mediawiki/2015/c/c2/Edigem15_nb_17.png"> | ||
| + | <br> | ||
| + | <br>Diagnostic digest indicated there may be insert present. | ||
| + | <br> | ||
| + | <br><b>Week 15</b> | ||
| + | <br><b>14/09</b> | ||
| + | <br>Sequence LacI+BBa_K1321339+MaoA N (1). | ||
| + | <br> | ||
| + | <br>*Ran the Monoamine oxidase A assay | ||
| + | <br> | ||
| + | <br><b>16/09</b> | ||
| + | <br> | ||
| + | <br>*Retried the Monoamine oxidase A assay | ||
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</p> | </p> | ||
Latest revision as of 18:59, 20 November 2015
PMA Detection
Week 1
Take Peroxidase out of the registry.
Order MaoA in two parts, N and C terminals.
Week 2
16/06
Digest pSB1C3 (1.23O2), pSB1A3 (4.2H1) and pSB1K3 (4.6B1) with EcoRI HF and PstI. Digest MaoA N and MaoA C (EcoRI HF/PstI).
Gel purify backbones, PCR purify inserts. Nanodrop.
17/06
Ligate MaoA N 1 to pSB1C3, MaoA N 2 to pSB1K3, and MaoA C to pSB1A3. Transform.
18/06
Growth appears on all plates (except for negative control). Less growth on the plates with backbone only which indicates that some of the insert ligated into the vector.
Culture 3 colonies from each plate.
19/06
Miniprep cultures. Nanodrop.
Diagnostic digest to show if inserts are actually present. pSB1K3 seems to have false positives.
Week 4
22/06
Prepare miniprep products (MaoA N1 and MaoA C) for sequencing.
Week 5
29/06
Digest MaoA N1 1 in pSB1C3 and MaoA C 1 in pSB1A3 for fusion. PCR purify pSB1C3+MaoA N, gel purify MaoA C. Nanodrop.
Ligate pSB1C3+MaoA N to MaoA C overnight.
30/06
Transform ligation reactions.
01/07
All of the transformations appeared to have worked. Culture transformations
02/07
Miniprep cultures. Nanodrop.
Send pSB1C3+ MaoA N+MaoA C 1 and 2 for sequencing.
Run a diagnostic digest on them.
Diagnostic digest shows that MaoA N and C did not fuse, as together they should be around 2.3 kb and the insert is much smaller, closer to 1 kb which is the size of MaoA N alone.
Culture 4 more colonies from pSB1C3+MaoA N+MaoA C to see if there was successful fusion in any of them.
03/07
Miniprep the cultures. Nanodrop.
Run diagnostic digest to see if the fusion worked.
Diagnostic gel shows that the fusion did not work.
Week 6
07/07
Retry fusing MaoA together. Digest pSB1C3 +MaoA N (AgeI/SpeI) and pSB1A3+MaoA C (NgoMIV/SpeI). Treat pSB1C3+ MaoA N with Antarctic phostphatase.
Gel purify. Nanodrop.
Ligate MaoA C into pSB1C3+MaoA N.
08/07
Transform ligation results to see if MaoA fused.
09/07
No growth on plates which indicates that MaoA did not fuse.
Try fusion again by going back to the gBlock. Digest pSB1C3+MaoA N (AgeI/SpeI) and the MaoA C gBlock (NgoMIV/SpeI). Treat pSB1C3+MaoA N with Antarctic Phosphatase. Ligate the two together. Transform the ligation.
10/07
No colonies on control plates (backbone only), quite a few colonies on plates of backbone plus insert which indicates fusion of MaoA.
12/07
Inoculate colonies of transformants.
Week 7
13/07
Miniprep the two colonies with potential fusion of MaoA. Nanodrop.
Diagnostic digest the plasmids to fully ensure fusion of MaoA.
14/07
Sequence MaoA 1 and MaoA 2.
Week 8
27/07
Inoculate more colonies from the plate with pSB1C3+MaoA fusion.
28/07
Make glycerols, miniprep and nanodrop cultures.
Diagnostic digest.
29/07
Sequence pSB1C3+MaoA 3, pSB1C3+MaoA 4, pSB1C3+MaoA 5 and pSB1C3+MaoA 6.
Week 9
07/08
Retransform pSB1C3+MaoA 4 into E.coli Top10 cells.
09/08
Inoculate transformants.
Week 10
10/08
Make glycerols, miniprep and nanodrop cultures.
Diagnostic digest.
11/08
Sequence MaoA 4(1) and MaoA 4(2).
14/08
Insert LacI into pSB1C3+MaoA 4. Digest LacI with EcoRI/SpeI, and MaoA with EcoRI/XbaI. Phosphatase treat, ligate and transform into E.coli Top10s.
16/08
Inoculate successful transformants.
Week 11
17/08
Make glycerols, miniprep and nanodrop cultures.
Diagnostic digest indicated the presence of insert.
18/08
Sequence MaoA 4+LacI 1.
Amplify pSB1C3+MoaA 4 (1) using PCR. PCR purify the products, nanodrop and run a diagnostic digest to confirm the correct sequence was amplified.
19/08
Fuse MaoA into each of the CBDs. Digest the MaoA for N terminal fusions with NgoMIV/PstI and digest with EcoRI/AgeI for C terminal fusions. Digest the CBDs for N terminal fusions with AgeI/PstI and for C terminal fusions digest with EcoRI/NgoMIV. Treat with antarctic phosphatase, ligate and transform.
20/08
Inoculate successful transformants.
21/08
Fuse MaoA to CBDCipA. Digest MaoA with NgoMIV/PstI for N terminal fusions and with EcoRI/AgeI for C terminal fusions. Digest CBDCipA with AgeI/PstI for N terminal fusions and with EcoRI/NgoMIV for C terminal fusions. Treat with antarctic phosphatase, ligate and transform into E.coli Top10s.
Make glycerols, miniprep and nanodrop cultures.
Diagnostic digest indicated the presence of insert.
Transform LacI+MaoA into E.coli BL21 cells.
Week 12
24/08
Sequence BBa_K1321339+MaoA C1, BBa_K1321339+MaoA C2, BBa_K1321340+MaoA C1, BBa_K1321340+MaoA C2, BBa_K1321003+MaoA C1, BBa_K1321003+MaoA C2, BBa_K1321002+MaoA C1 and BBa_K1321002+MaoA C2.
Retry fusing MaoA into CBDs. Digest MaoA with NgoMIV/PstI for N terminal fusions and with EcoRI/AgeI for C terminal fusions. Digest CBDs with AgeI/PstI for N terminal fusions and with EcoRI/NgoMIV for C terminal fusions. Antarctic phosphatase treat the CBD backbones, then ligate and transform.
25/08
Inoculate successful transformants.
26/08
Retry the failed MaoA to CBD fusions. Digest MaoA with NgoMIV/PstI for N terminal fusions and with EcoRI/AgeI for C terminal fusions. Digest the CBDs with AgeI/PstI for N terminal fusions and with EcoRI/NgoMIV for C terminal fusions. Phosphatase treat the CBDs, then ligate and transform.
Make glycerols, miniprep and nanodrop the cultures.
Diagnostic digest indicated there was no insert present.
27/08
Insert LacI into MaoA+CBD fusions. Digest LacI with EcoRI/SpeI and digest the backbones with EcoRI/XbaI. Antarctic phosphatase treat the backbones, ligate and transform.
Inoculate successful transformants.
28/08
Make glycerols, miniprep and nanodrop cultures.
Diagnostic digest indicated the presence of insert.
Week 13
31/08
Inoculate successful transformants.
Transform LacI+MaoA 4 into E.coli BL21 cells.
01/09
Sequence CBDCipA+MaoA N(2), CBDCipA+MaoA C(1), CBDCipA+MaoA C(2), 5.16O+MaoA N(1) and 5.16O+MaoA N(2).
Make glycerols, miniprep and nanodrop cultures.
Diagnostic digest indicated there was no insert present.
Week 14
07/09
Insert LacI into MaoA+CBD fusions. Digest LacI with EcoRI/SpeI and digest the backbones with EcoRI/XbaI. Antarctic phosphatase treat the backbones, ligate and transform into E.coli Top10 cells.
08/09
Inoculate successful transformants.
09/09
Make glycerols, miniprep and nanodrop cultures.
Diagnostic digest indicated there may be insert present.
Week 15
14/09
Sequence LacI+BBa_K1321339+MaoA N (1).
*Ran the Monoamine oxidase A assay
16/09
*Retried the Monoamine oxidase A assay