Difference between revisions of "Team:Exeter/Notebook/Text"

Revision as of 18:36, 1 November 2017

10/07/2017

Extracted interlab devices from kit and made up spread plates.

11/07/2017

Streak plates of DH5α and MG1655.
Overnight cultures for interlab devices- 5ml + chloramphenicol + one colony.

12/07/2017

Overnight cultures for wild type (DH5α, MG1655) - 5ml LB + one colony.
Glycerol stocks (DH5α, MG1655).
Miniprep of interlab devices (and pBAD).
Qubit of devices (and pBAD).

13/07/2017

Glycerol stocks of wild types DH5α and MG1655.
Made 5x 200ml of LB (autoclaved)
Genomic DNA prep of DH5α and MG1655.
Qubit for wild types.

14/07/2017

Introduction to TECAN plate reader.
Competent cell preparation for DH5α (see protocol).
Measurements for interlab.

17/07/2017

Interlab device transformations (expt. 3)
Restriction digest for insertion of fim sections into plasmids (Expts. 1+2)
Stored fim plasmids.

18/07/2017

Liquid overnights for interlab (error expt.6)
PCR and electrophoresis (Taq polymerase) of fim genes (D,H and A) - confirms genes in DH5α (bad news) (expt.4)

19/07/2017

Transformed plasmids from expt.2 into cells (expt.5)
Liquid overnights for interlab.(expt.9)
Electrophoresis of fim operon from expt.4 (expt.7)
PCR (Phusion) of whole operon. (expt.8)

20/07/2017

Interlab measurements.(expt.10)
Miniprep and Qubit for fim constituents. (expt.11)
Competent cell prep (expt.12)
Genomic DNA extraction of Top10 strain (expt. 13)
Electrophoresis for PCR from expt.8 (expt.14)

21/07/2017

Prepared DNA from miniprep for sequencing (to confirm that transformation worked)
Competent cell prep (expt.12)
Genomic DNA extraction of Top10 strain (expt. 13)
Electrophoresis for PCR from expt.8 (expt.14)

24/07/2017

Interlab data analysis
Overnights for Top10

25/07/2017

Genomic extraction from Top10 (x2)
DNA extraction from gel band (MG1655 operon)
PCR of Top10 and MG1655 fim genes.

26/07/2017

Electrophoresis of Top10 and MG1655
One pot cloning of fimAIC, D, and FG with promoter.
Transformed fim containing plasmids into DH5ɑ
Streaked MG1655 and Top10 for Bioimaging.

27/07/2017

Overnights for MG1655 and Top10 for imaging.
Transformed MoClo products into DH5ɑ.

28/07/2017

Negative stain and transmission electron microscopy of MG1655 and Top10 samples.

31/07/2017

Overnights of MG1655, Top10 and MoClo products (x8) from restriction digest of fimH constructs and ligation into plasmids.

01/08/2017

Transformed fim H constructs (x10) into DH5α
One pot: prepared for sequencing, made glycerol stocks, miniprep and qbit.
DH5α overnights, made competent cells of top10.

02/08/2017

Restriction digest of operon MoClo products (for gel)
Prep. for fim operon sequencing

03/08/2017

MoClo of fimH constructs.

04/08/2017

Sequencing results confirm successful plasmid construction.
Transformation of MoClo products into DH5α.

07/08/2017

Transformation of fimH MoClos and preparation of fim operon for sequencing.

08/08/2017

Top 10 competent cells made

10/08/2017

Miniprep and Qubit of P_Rha+fimH+psB1A3 constructs.
Overnights cultures of P_Ara+fimop(pX3’6’0’0), P_J23100+fimop(pX3’6’0’0), P_J23100+fimop(PSB1A3)

11/08/2017

Prep of P_Rha+fimH miniprep DNA for sequencing.
Qubit of overnight cultures: (P_J23100 x 2 + P_Ara) + fimH.

14/08/2017

Restriction digest of Fim operon in: pX3’6’0’0 with P_J23100 promoter, pX3’6’0’0 with P_Ara, pSB1A3 with P_J23100.
The products of the restriction digest were ran on the electrophoresis and only the pSB1A3 product was successful. Only two pSB1A3 plasmids were sent for sequencing (5+6).
The P_Rha+fimH+psB1A3 constructs were also sent for sequencing (13 constructs).

15/08/2017

Poured CAM plates, Restriction digest of P_Ara_fimoperon_pSB1A3, P_J23100_fimoperon_ pSB1A3 and the pX3’6’0’0 plasmid, followed by a gel electrophoresis and gel extraction of the DNA (the wrong gel bands were extracted, so Chloe did it again for us).
Transformation of the FimH constructs in the pSB1C3 and pSB1A3 into DH5α.
Made a mannose serial dilution from 4% to 0.0625% and ran in the HPLC.

16/08/2017

Gel extraction of the bands (after that we found out the bands we extracted were the wrong ones).
Overnights of FimH constructs in the pSB1C3 and pSB1A3 into DH5α.
Grew MG1655 on silica sand and NP-Bacto-Pellets.

17/08/2017

Mini prep and Qubit from overnights of FimH constructs and transformation of the ligation from the gel extraction on CAM plates that Chloe did.
Prepared silica sand and NP-Bacto-Pellets samples for SEM imaging.

21/08/2017

Miniprep, glycerol stocks and qubit of P_J23100 and P_Ara fim_op.
Diagnostic digest and electrophoresis of above - shows successful construction and transformation of plasmids.

22/08/2017

Double transformations of two plasmids into Top10.
Transformation of fimH constructs into pEX1A3.
Transformation for UV experiments.

23/08/2017

Liquid overnights for previous transformations.
Grow overnight cultures of E.coli Top10 and E.coli Top10, transformed with chloramphenicol and ampicillin resistance genes for UV experiment.
Imaged silica sand and NP-Bacto-Pellets samples using SEM.

24/08/2017

First attempt at induction of fimH constructs.
OD of overnight cultures set to one. Sample were irradiated, spread on plates and then incubated.

25/08/2017

Colonies on plates were counted, unexpected results.

28/08/2017

Overnights of transformed Top10, WT Top10 and WT MG1655.

29/08/2017

Revised induction protocol.
Measured construct fluorescence in TECAN.
Grew MG1655 on silica sand and NP-Bacto-Pellets again following unexpected results previously.

30/08/2017

First attempt at agglutination protocol
Prepared and imaged silica sand and NP-Bacto-Pellets samples for SEM.
Investigated the flow rates that were achieved when polypropylene scaffold torus structures were inside the metal binding reactor.

31/08/2017

Induction protocol

01/09/2017

Samples run through the TECAN
Grew MG1655 on polypropylene scaffold torus structures, using a range of concentrations of a chlorhexidine gluconate surfactant.

04/09/2017

Overnight cultures of constructs
Examined the tori to observe which concentration of surfactant produced a biofilm on the surface.
Repeated the growing of MG1655 on polypropylene scaffold torus structures, using a range of concentrations of a chlorhexidine gluconate surfactant.

05/09/2017

Induction protocol
Agglutination protocol
Successful transformation of mouse metallothionein
Overnight cultures of E.coli Top10 for UV
Examined the tori to observe which concentration of surfactant produced a biofilm on the surface.

06/09/2017

Samples run through FACS and fluorescent cells sorted
Sorted cells plated on agar in 7x8 grid
1 in 1000 dilution of overnight culture
Cultures irradiated and plates spread

07/09/2017

Induction protocol
Colonies on plates counted, OD of culture too great.

08/09/2017

Dialysis trial run

11/09/2017

Induction protocol
Repeated the growing of MG1655 on polypropylene scaffold torus structures, using a range of concentrations of a chlorhexidine gluconate surfactant.

12/09/2017

Miniprep of P_Rha_GFP constructs for DoE
Ran the fluidising media reactor with tori at a range of concentrations of a chlorhexidine gluconate surfactant.
Imaged samples of the tori using SEM before and after being run through the metal binding reactor.
Ran HPLC for the mannose concentration of samples taken from the use of the metal binding reactor with the tori.

13/09/2017

Induction of three FimH constructs at 0.1 and 0.2% rhamnose concentrations in BL21 and DH5α
Overnight cultures of E. coli Top10.

14/09/2017

TECAN reading of previous day's induced cultures.
Induction of three FimH constructs at 1 and 2% rhamnose concentrations.
Culture irradiated using UV trans-illuminator

15/09/2017

TECAN reading of the previous day's induced cultures.
Colonies counted, positive results.

19/09/2017

DoE: the first block
Restriction digest and ligation of P_Rha_FimH_1sfGFP and with pSB1A3.

20/09/2017

TECAN reading of previous day's cultures for DoE
Restriction digest, electrophoresis, gel extraction and ligation of P_Rha_FimH_1sfGFP and pSB1A3.
Overnight culture of E. coli Top10
Grew MG1655 on polypropylene scaffold torus structures, for runs 1-3 for design of experiment.

21/09/2017

TECAN reading of previous day's cultures for DoE
OD of overnight culture set to 1 and 1 in 1,000,000 applied
Cultures irradiated
Runs 1-3 for the design of experiment in the metal binding reactor.
Grew MG1655 on polypropylene scaffold torus structures, for runs 4-5 for design of experiment.

22/09/2017

Digestion and ligation of FimH_1sfGFP with pSB1A3
Colonies counted, results are as expected.
Runs 4-5 for the design of experiment in the metal binding reactor.

25/09/2017

Transformed FimH_1sfGFP_pSB1A3 into E.coli

26/09/2017

Grew MG1655 on polypropylene scaffold torus structures, for runs 6-8 for design of experiment.

27/09/2017

Runs 6-8 for the design of experiment in the metal binding reactor.
Grew MG1655 on polypropylene scaffold torus structures, for runs 9, 10 and 1 for design of experiment.

28/09/2017

TECAN reading for DoE
Inoculated and induced cultures for DoE
Diagnostic digestion of FimH_1sfGFP_pSB1A3 plasmid DNA
Runs 9, 10 and 1 for the design of experiment in the metal binding reactor.

29/09/2017

Transformation for Newcastle collaboration

02/10/2017

Transformation of T7_FimH_225sfGFP into BL21

04/10/2017

Induction protocol

09/10/2017

Ran Newcastle samples through FACS, analysed the data and sent it off.

11/10/2017

Prepared samples for subsequent SDS page analysis of fim operon in top10

12/10/2017

Induction protocol

13/10/2017

Restriction digest of all parts in pSB1C3 and diagnostic electrophoresis gel showed successful construction
Preparation of all parts for sequencing

16/10/2017

Qubit of all parts in pSB1C3

17/10/2017

SDS-PAGE and Western blot of FimH_1His constructs and wild types in four E.coli strains

18/10/2017

Western blot and SDS-PAGE results showed successful expression and binding of FimH_1His pili with no bands for the wild types.

20/10/2017

Preparation of DNA parts for submission

23/10/2017

Transformation of FimB KO with fim operon (under both promoters) with FimH, FimH_1His, FimH_1SynMT, FimH_1MouseMT

24/10/2017

DNA parts sent off

25/10/2017

Inoculate 50ml cultures with FimB KO with either P_Ara-Fim_Operon or PJ23100-Fim_Operon and FimH, FimH_1_His, FimH_1_SynMT, FimH_1_MouseMT
TECAN readings for T7-FimH_225_GFP in BL21(DE3) after induction as well as BL21(DE3) wild type (similar treatment)

26/10/2017

Dialysis: FimB KO of PJ23100 and PRha_FimH/PRha_FimH_1_His/PRha_FimH_1_SynMT/PRha_FimH_1_MouseMT by adding either CuCl₂ and/or NiSO₄

31/10/2017

SDS page and Western blot of T7-FimH_225_GFP in BL21(DE3) after induction as well as BL21(DE3) wild type (similar treatment)

@@ Line 595: / Line 595: @@
 <li>TECAN readings for T7-FimH_225_GFP in BL21(DE3) after induction as well as BL21(DE3) wild type (similar treatment)</li>
 </ul>
+</p>
 /10/2017