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 dh5alpha
• One pot: prepared for sequencing, made glycerol stocks, miniprep and qbit.
• Dh5alpha 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 dh5alpha.

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 pRha+fimH+psB1A3 constructs.
• Overnights cultures of pAra+fimop(pX3’6’0’0), J23100+fimop(pX3’6’0’0), J23100+fimop(PSB1A3)

11/08/2017

• Prep of pRha+fimH miniprep DNA for sequencing.
• Qubit of overnight cultures: (J23100 x 2 + pAra) + fimH.

14/08/2017

• Restriction digest of Fim operon in: pX3’6’0’0 with J23100 promoter, pX3’6’0’0 with pAra, pSB1A3 with 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 pRha+fimH+psB1A3 constructs were also sent for sequencing (13 constructs).

15/08/2017

• Poured CAM plates, Restriction digest of pAra_fimoperon_pSB1A3, 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 DH5a.

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 DH5a.

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.

21/08/2017

• Miniprep, glycerol stocks and qubit of J23100 and pAra 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.

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

30/08/2017

• Sand protocol
• First attempt at agglutination protocol

31/08/2017

• Induction protocol

01/09/2017

• Samples run through the TECAN

04/09/2017

• Overnight cultures of constructs

05/09/2017

• Induction protocol
• Agglutination protocol
• Successful transformation of mouse metallothionein
• Overnight cultures of E.coli Top10 for UV

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

12/09/2017

• Miniprep of pRha_GFP constructs for DoE

13/09/2017

• Induction of three FimH constructs at 0.1 and 0.2% rhamnose concentrations in BL21 and DH5alpha
• 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 pRha_FimH22sfGFP and with pSB1A3.

20/09/2017

• TECAN reading of previous day's cultures for DoE
• Restriction digest, electrophoresis, gel extraction and ligation of pRha_FimH22sfGFP and pSB1A3.
• Overnight culture of E.coli Top10

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

22/09/2017

• Digestion and ligation of FimH_22sfGFP with pSB1A3
• Colonies counted, results are as excepted.

25/09/2017

• Transformed FimH_22sfGFP_pSB1A3 into E.coli

28/09/2017

• TECAN reading for DoE
• Inoculated and induced cultures for DoE
• Diagnostic digestion of FimH_22sfGFP_pSB1A3 plasmid DNA

29/09/2017

• TECAN reading of previous day's cultures for DoE