Difference between revisions of "Team:Edinburgh/Notebook/PMADetection"

PMA Detection

Week 1
Take Peroxidase out of the registry.
Part Conc (ng/µl) Peroxidase (BBa_K1336003) 1 71.3 Peroxidase (BBa_K1336003) 2 62.8

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