Team:Edinburgh/Notebook/DNPDetection

DNP Detection

Week 1
03/06
Take laccases (BBa_K863006 and BBa_K863021) out of the distribution kit. Transform into DH5α.

04/06
Inoculate transformants.

05/06
Miniprep and nanodrop cultures.




Week 8
20/07
Ordered a Tranetes Versicolor laccase from IDT.


Week 9
30/07
Resuspend the laccase gBlock.
Fuse laccase into pSB1C3, LacI and CBDs. Digest laccase for pSB1C3 fusion and pSB1C3 with EcoR1/Pst1. Digest laccase for pSB1A3+LacI fusion with Xba1/Pst1. Digest pSB1A3+LacI with Spe1/Pst1. Digest the CBDs with Age1/Spe1 for N terminal fusion and Xba1/NgoMIV for C terminal fusions. Digest the laccase for N terminal CBD fusion with NgoMIV/Spe1 and with Xba1/Age1 for C terminal fusions. Antarctic phosphatase treat for 15 minutes, then ligate and transform into Top10s.

31/07
Fuse LacI into laccase. Digest LacI with Spe1/Pst1 and digest laccase with Xba1/Pst1. Phosphatase treat LacI, ligate and transform into Top10s.


Week 9
03/08
Inoculate successful transformants from 30/07 (no successful transformants from 31/07).

04/08
Make glycerols, miniprep and nanodrop cultures.




Diagnostic digest indicated insert only present in pSB1C3+laccase.

05/08
Sequence pSB1C3+laccase 1 and pSB1C3+laccase 2.
Retry laccase CBD fusions. Digest CBDs with Age1/Spe1 for N terminal fusions and with Xba1/Spe1 for C terminal fusions. Digest laccase with NgoMIV/Spe1 for N terminal fusions and with Xba1/Age1 for C terminal fusions. Phosphatase, ligate and transform into Top10s.

06/08
Inoculate successful transformants.

07/08
Make glycerols, miniprep and nanodrop the cultured cells.



Diagnostic digest showed insert was present in some cases.


Week 10
10/08
Sequence BBa_K1321339+laccase C1, BBa_K1321339+laccase C2, BBa_K1321340+laccase C1, BBa_K1321340+laccase C2, BBa_K1321002+laccase C1 and BBa_K1321002+laccase C2.

12/08
Fuse laccase to remaining CBDs. Digest CBDs with EcoR1/NgoMIV for C terminal fusions and with Age1/Pst1 for N terminal fusions. Digest laccase with EcoR1/Age1 for C terminal fusions and with NgoMIV/Pst1 for N terminal fusions. Phosphatase treat ligate and transform into Top10s.

13/08
Fuse laccase to CBDCipA.
Digest laccase with NgoMIV/Pst1 for N terminal fusions and with EcoR1/Age1 for C terminal fusions. Digest CBDCipA with Age1/Pst1 for N terminal fusions and with EcoR1/NgoMIV for C terminal fusions.
Fuse LacI into Laccase.
Digest laccase with EcoR1/Xba1 and digest LacI with EcoR1/Spe1.
All backbones were phosphatase treated. Then the digested fragments were ligated and transformed into E.coli Top10 cells.
Inoculate transformants from 12/08.

14/08
Retry fusing LacI into laccase. Digest LacI with EcoR1/Spe1 and laccase with EcoR1/Xba1.
Treat laccase with antarctic phosphatase, ligate and transform.
Make glycerols of cultures then miniprep and nanodrop.




16/08
Inoculate successful transformants from 13/08 and 14/08


Week 11
17/08
Diagnostic digest samples from 14/08 indicated insert present in four samples.
Make glycerols, miniprep and nanodrop cultures.



Diagnostic digest suggested insert present in one sample only.

18/08
Sequence BBa_K1321339+laccase N(2), BBa_K1321003+laccase C(1), BBa_K1321003+laccase N(1),BBa_K1321003+laccase N(2) and CBDCipA+laccase C(1).

Amplify pSB1C3 laccase (2) using PCR. PCR purify and run diagnostic digest to confirm the amplification of laccase.




19/08
Retry fusing laccase to CBDs. Digest laccase with NgoMIV/Pst1 for N terminal fusions and with EcoR1/Age1 for C terminal fusions. Digest CBDs with Age1/Pst1 for N terminal fusions and with EcoR1/NgoMIV for C terminal fusions. Antarctic phosphatase treat backbones, then ligate and transform.

20/08
Fuse LacI into laccase and laccase-CBD fusions. Digest LacI with EcoR1/Spe1 and digest backbones with EcoR1/Xba1. Phosphatase, ligate and transform.
Inoculate transformants from 19/08.

21/08
Retry laccase to CBDCipA fusion. Digest laccase with NgoMIV/Pst1 for N terminal fusions and with EcoR1/Age1 for N terminal fusions. Digest CBDCipA with Age1/Pst1 for N terminal fusions and with EcoR1/NgoMIV for C terminal fusions. Antarctic phosphatase treat CBDCipA then ligate and transform into E.coli Top10s.

Make glycerols, miniprep and nanodrop cultures.



Diagnostic digest indicated the presence of insert.

Inoculate successful transformants.


Week 12
24/08
Sequence BBa_K1321339+laccase C(1), BBa_K1321339+laccase C(2), BBa_K1321340+laccase C(1), BBa_K1321340+laccase C(2), BBa_K1321003+laccase C(2), BBa_K1321002+laccase C(1) and BBa_K1321002+laccase C(2).

Retry N terminal CBD fusions. Digest laccase with NgoMIV/Pst1 and digest CBDs with Age1/Pst1. Antarctic phosphatase treat CBDs, then ligate and transform.

Make glycerols, miniprep and nanodrop cultures from 21/08.



Diagnostic digest indicated the presence of LacI in some samples.

25/08
Sequence LacI+BBa_K1321339+laccase N2(1), Laccase+LacI (1) and laccase+LacI (2). Insert LacI into laccase-CBD fusions. Digest LacI with EcoR1/Spe1 and digest fusions with EcoR1/Xba1. Phosphatase, ligate and transform.

Inoculate transformants.

26/08
Retry failed CBD N terminal fusions. Digest laccase with NgoMIV/Pst1 and CBDs with Age1/Pst1.
Put LacI into laccase-CBD fusions. Digest LacI with EcoR1/Spe1 and digest the fusions with EcoR1/Xba1.
Phosphatase treat all backbones, then ligate and transform into E.coli Top10s.

Make glycerols, miniprep and nanodrop cultures.



Diagnostic digest indicated that no insert was present.

Inoculate transformants.

27/08
Fuse LacI to laccase and laccase fusions.. Digest LacI with EcoR1/Spe1 and fusions with EcoR1/Xba1.
Fuse laccase into LacI. Digest LacI for laccase with Spe1/Pst1 and laccase with Xba1/Pst1 Phosphatase treat, ligate and transform.

Make glycerols, miniprep and nanodrop cultures.



Diagnostic digest indicated presence of the insert.

28/08
Make glycerols, miniprep and nanodrop cultures.



Diagnostic digest indicated presence of the insert.


Week 13
31/08
Inoculate successful transformants.

01/09
Sequence LacI+CBDCipA+laccase C(2), CBDCipA+laccase N(1), CBDCipA+laccase N(2), BBa_K1321339+laccase N(1),BBa_K1321339+laccase N(2) and BBa_K1321003+laccase N(1).

Make glycerols, miniprep and nanodrop cultures.



Diagnostic digest indicated presence of insert.

02/09
Sequence pSB1C3 laccase+LacI (1), pSB1C3 laccase+LacI (3) and LacI+BBa_K1321340 +laccase C(2).

Week 14
07/09
Insert LacI into CBD+laccase fusions. Digest LacI with EcoR1/Spe1 and digest Fusions with EcoR1/Xba1. Phosphatase, ligate and transform.

08/09
Inoculate successful transformants.

Transform LacI+CBDCipA+Laccase C(2) into E.coli BL21 cells.

09/09
Make glycerols, miniprep and nanodrop cultures.



Diagnostic digest indicated the presence of insert.


Week 15
14/09
Sequence LacI+laccase (1), LacI+laccase (2) and LacI+BBa_K1321339+laccase N (1).