Team:Edinburgh/Notebook/FluidDynamics

Fluid Dynamics

Week 1
Take CBDs out of the registry by transforming, culturing, and then miniprep. Nanodrop


Week 2
Order the sequence for the biobrick of RFP mcherry with the illegal sites for RFC25 removed as a gBlock.


Week 5
02/07
Digest the RFP gBlock for fusion into the pSB1C3 backbone. Digest RFP with EcoRI/SpeI. Ligate and transform into E.coli DH5α cells.

03/07
No growth of transformants.

Retry fusion of RFP into pSB1C3.Digest with EcoRI/PstI. Ligate and transform.

05/07
Inoculate transformants.

06/07
Miniprep cultures and nanodrop.


Diagnostic digest indicated that insert was present.
07/07
Sequence pSB1C3+RFP 1 and pSB1C3+RFP 2.


Week 6
08/07
Digest CBDs and RFP for both N and C terminal fusions.
CBDs: BBa_K1321339, BBa_K1321340, BBa_K1321003 and BBa_K1321002. For N terminal fusions use AgeI/SpeI and for C terminal fusions use XbaI/NgoMIV. Treat with Antarctic phosphatase.
For RFP N terminal fusions use NgoMIV/SpeI, for C terminal fusions use XbaI/AgeI.

PCR purify CBDs. The digest did not really work for the inserts so digest again over night.

09/07
Gel purify the overnight digest. Nanodrop.

Ligate each of the 4 CBDs to RFP for both N and C terminal fusions with controls of the CBDs without inserts.

10/07
Transform the ligations.
Plate bacteria for bacterial cellulose in liquid, shaking liquid, and solid at 22ºC.

11/07
Some of the ligations appeared to have worked.

12/07
Inoculate transformants that appeared to have successfully ligated.


Week 7
13/07
Make glycerol stocks of cultures. Miniprep and nanodrop.

Diagnostic digest.

Ligate RFP to CBDs. Transform ligations.

14/07
Sequence BBa_K1321339+RFP N1 and BBa_K1321340+RFP N1.

15/07
Inoculate successful transformations.

16/07
Make glycerols, miniprep and nanodrop cultures.


17/07
Diagnostic digest indicated presence of insert.

Week 8
20/07
Sequence BBa_K1321339+RFP C2, BBa_K1321339+RFP N4, BBa_K1321340+RFP N4, BBa_K1321002+RFP N2, BBa_K1321003+RFP N2, BBa_K1321003+RFP C1 and BBa_K1321002+RFP C1.

Inoculated cultures for BBa_K1321340+RFP C3 and 4.
21/07
Make glycerols, miniprep and nanodrop cultures.


Diagnostic digest.

Washed bacterial cellulose and transferred it to clean petri dishes to dry.

22/07
Sequence BBa_K1321340+RFP C3 and BBa_K1321340+RFP C4.

23/07
Fuse LacI to RFP. Digest the RFP gBlock with XbaI/PstI. Digest LacI with SpeI/PstI.
Fuse CBDCipA into pSB1C3. Digest CBDCipA gBlock with EcoRI/PstI, and digest linearised pSB1C3 with EcoRI/PstI.
Antarctic phosphatase treat the backbones. Ligate and transform.


Week 9
26/07
Inoculate successful transformants.

27/07
Make glycerols, miniprep and nanodrop cultures.


Diagnostic digest indicated there was no insert present.

29/07
Digest RFP-CBD fusions for insertion of LacI. Digest fusions with XbaI/PstI and digest LacI with SpeI/Pst1.
Retry fusion of CBDCipA into pSB1C3.
Antarctic phosphatase treat the backbones, ligate and transform.

Restreak pSB1A3+LacI 2 from glycerol.

30/07
Inoculate successful transformants.

31/07
Retry fusing LacI and RFP-CBD fusions. Digest fusions with XbaI/PstI and digest LacI with SpeI/PstI. Antarctic phosphatase, ligate and transform.

Make glycerols, miniprep and nanodrop cultures.


Diagnostic digest indicate the presence of insert.

02/08
No successful transformants.


Week 10
03/08
Fuse LacI to RFP, sfGFP and CBDs. Digest LacI with SpeI/PstI and digest enzymes and CBDs with XbaI/PstI. Phosphatase, ligate and transform.

Sequence pSB1C3+CBDCipA 1, pSB1C3+CBDCipA 2 and LacI+BBa_K1321340+RFP N1.

05/08
Inoculate successful transformants.

06/08
Make glycerols, miniprep and nanodrop.

Diagnostic digest indicated the presence of insert.

07/08
Sequence LacI+RFP 1, LacI+RFP 2, LacI+sfGFP 1, and LacI+sfGFP.


Week 11
11/08
Inoculate starter cultures for LacI+RFP and LacI+sfGFP.

Fuse LacI and RFP-CBD fusions. Digest LacI with EcoRI/SpeI and digest fusions with EcoRI/XbaI. Phosphatase treat, ligate and transform.

Measured the chads (chad measurement protocol)
CBD fused with GFP association experiment (association experiment protocol)

12/08
Inoculate successful transformants.

13/08
Fuse RFP to CBDCipA. Digest RFP with NgoMIV/PstI for M terminal fusion and with EcoRI/AgeI for the C terminal fusion. Digest CBDCipA with AgeI/PstI for N terminal fusions and with EcoRI/NgoMIV. Antarctic phosphatase backbones, ligate and transform.

Restreak pSB1A3+LacI 2.

Make glycerols, miniprep and nanodrop.

Diagnostic digest indicated the presence of insert.
RFP control association experiment
RFP dissociation experiment (dissociation experiment protocol), leave chads in RFP overnight in 4 °C

14/08
Dissociation experiment using chads incubated with RFP overnight
Dissociation experiment using chads saturating in RFP for 5 minutes


Week 12
16/08
Inoculate transformations.

17/08
Transform LacI-CBD-RFP fusions into E.coli BL21s for expression.

Make glycerols, miniprep and nanodrop cultures.



Diagnostic digest.
Dissociation experiment of GFP, GFP + 5.21M, RFP
18/08
Retry failed RFP+CBD fusions. Digest RFP 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 terminal fusions.
Fuse LacI into RFP+CBD fusions. Digest LacI with EcoRI/SpeI and digest fusions with EcoRI/XbaI.
Phosphatase backbones, ligate and transform.

Inoculate starter cultures for successful RFP fusions.

Dissociation experiment of GFP 5.20M using chads from Whatmann 54, unprocessed bacterial cellulose and processed bacterial cellulose

19/08
Inoculate successful transformants.

Amplify RFP and GFP using PCR. PCR purify and nanodrop.

Diagnostic digest confirmed the correct amplification.

20/08
Fuse LacI to remaining RFP fusions. Digest LacI with EcoRI/SpeI and digest fusions with EcoRI/XbaI. Phosphatase treat, ligate and transform.

Make glycerols, miniprep and nanodrop.

Diagnostic digest.

21/08
Retry RFP fusions. Digest RFP with NgoMIV/PstI for N terminal fusions and with EcoRI/AgeI for C terminal fusions. Digest CBDs with AgeI/Pst1 for N terminal fusions and with EcoRI/NgoMIV for C terminal fusions. Phosphatase treat backbones, ligate and transform.

Dissociation experiments of LacI+BBa_K1321339+RFP C, LacI+BBa_K1321002+RFP N, LacI+ BBa_K1321002 RFP C.


Week 13
23/08
Inoculate successful transformants.

24/08
Retry RFP fusion to CBDCipA. Digest RFP with NgoMIV/PstI for N terminal fusions and with EcoRI/AgeI for C terminal fusions. Digest CBDs with AgeI/Pst1 for N terminal fusions and with EcoRI/NgoMIV for C terminal fusions. Phosphatase treat backbones, ligate and transform.

Make glycerols, miniprep and nanodrop cultures.


Diagnostic digest indicated there was insert present.

Transform RFP fusions into BL21s.

25/08
Sequence LacI+BBa_K1321339+RFP N4 1, LacI+BBa_K1321339+RFP N4 2,
LacI+BBa_K1321003+RFP C1 1 and LacI+BBa_K1321003+RFP C1 2.

26/08
Retry RFP fusion to CBDCipA. Digest RFP with NgoMIV/PstI for N terminal fusions and with EcoRI/AgeI for C terminal fusions. Digest CBDs with AgeI/Pst1 for N terminal fusions and with EcoRI/NgoMIV for C terminal fusions.
Fuse LacI to CBDs. DIgest CBDs with EcoRI/XbaI and digest LacI with EcoRI/SpeI.
Phosphatase treat backbones, ligate and transform.

27/08
Inoculate successful cultures.

28/08
Make glycerols, miniprep and nanodrop cultures.

Diagnostic digest indicated the presence of some inserts.


Week 14
31/08
Transform LacI+BBa_K1321340+RFP N4 2 and LacI+BBa_K1321003+RFP N2 2 into BL21s.

01/09
Sequence LacI+BBa_K1321003 1, LacI+BBa_K1321003 2, CBDCipA+RFP N1, CBDCipA+RFP N2, CBDCipA+RFP C1 and CBDCipA+RFP C2.

Inoculate starter cultures for RFP fusions.


Week 15
07/09
Put LacI into RFP+CBD fusions and CBDs. Digest fusions/CBDs with EcoRI/XbaI and digest LacI with EcoRI/SpeI. Phosphatase backbones, ligate and transform.

08/09
Inoculate successful transformants.

09/09
Make glycerols, miniprep and nanodrop.

Diagnostic digest.

14/09
Sequence LacI+CBDCipA 1, LacI+CBDCipA 2, LacI+BBa_K1321339 1, LacI+BBa_K1321339 2, LacI+BBa_K1321002 1, LacI+BBa_K1321002 2, LacI+CBDCipA+RFP C1, LacI+CBDCipA+RFP C2, LacI+CBDCipA+RFP N1, LacI+CBDCipA+RFP N2, LacI+BBa_K1321003 1 and LacI+BBa_K1321003 2.

16/09
Characterised LacI+CBDCipA+RFP N, LacI+CBDCipA+RFP C using chad dissociation protocol.