Team:Exeter/Notebook/Text

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)