Team:Edinburgh/Notebook/PMADetection
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