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

Line 272: Line 272:
 
  <ul>
 
  <ul>
 
   <li>Liquid overnights for previous transformations.</li>
 
   <li>Liquid overnights for previous transformations.</li>
 +
  <li> Grow overnight cultures of E.coli Top10 and E.coli Top10, transformed with chloramphenicol and ampicillin resistance genes for UV experiment. </li>
 
  </ul>
 
  </ul>
 
</p>
 
</p>
Line 279: Line 280:
 
  <ul>
 
  <ul>
 
   <li>First attempt at induction of fimH constructs.</li>
 
   <li>First attempt at induction of fimH constructs.</li>
 +
  <li>OD of overnight cultures set to one. Sample were irradiated, spread on plates and then incubated.</li>
 +
</ul>
 +
</p>
 +
 +
25/08/2017
 +
<p>
 +
<ul>
 +
  <li> Colonies on plates were counted, unexpected results. </li>
 
  </ul>
 
  </ul>
 
</p>
 
</p>
Line 285: Line 294:
 
<p>
 
<p>
 
<ul>
 
<ul>
−
<li>Overnights of transformed top10, WT top10 and WT MG1655</li>
+
<li>Overnights of transformed Top10, WT Top10 and WT MG1655</li>
 
</ul>
 
</ul>
 
</p>
 
</p>
Line 332: Line 341:
 
<li>Agglutination protocol</li>
 
<li>Agglutination protocol</li>
 
<li>Successful transformation of mouse metallothionein </li>
 
<li>Successful transformation of mouse metallothionein </li>
 +
<li>Overnight cultures of E.coli Top10 for UV </li>
 
</ul>
 
</ul>
 
</p>
 
</p>
Line 340: Line 350:
 
<li>Samples run through FACS and fluorescent cells sorted</li>
 
<li>Samples run through FACS and fluorescent cells sorted</li>
 
<li>Sorted cells plated on agar in 7x8 grid </li>
 
<li>Sorted cells plated on agar in 7x8 grid </li>
 +
<li>1 in 1000 dilution of overnight culture </li>
 +
<li>Cultures irradiated and plates spread </li>
 
</ul>
 
</ul>
 
</p>
 
</p>
Line 347: Line 359:
 
<ul>
 
<ul>
 
<li>Induction protocol</li>
 
<li>Induction protocol</li>
 +
<li> Colonies on plates counted, OD of culture too great.</li>
 
</ul>
 
</ul>
 
</p>
 
</p>
Line 375: Line 388:
 
<ul>
 
<ul>
 
<li>Induction of three FimH constructs at 0.1 and 0.2% rhamnose concentrations in BL21 and DH5alpha</li>
 
<li>Induction of three FimH constructs at 0.1 and 0.2% rhamnose concentrations in BL21 and DH5alpha</li>
 +
<li>Overnight cultures of E.coli Top10.</li>
 
</ul>
 
</ul>
 
</p>
 
</p>
Line 383: Line 397:
 
<li>TECAN reading of previous day's induced cultures.</li>
 
<li>TECAN reading of previous day's induced cultures.</li>
 
<li>Induction of three FimH constructs at 1 and 2% rhamnose concentrations.</li>
 
<li>Induction of three FimH constructs at 1 and 2% rhamnose concentrations.</li>
 +
<li>Culture irradiated using UV trans-illuminator</li>
 
</ul>
 
</ul>
 
</p>
 
</p>
Line 391: Line 406:
 
<ul>
 
<ul>
 
<li>TECAN reading of the previous day's induced cultures.</li>
 
<li>TECAN reading of the previous day's induced cultures.</li>
 +
<li>Colonies counted, positive results.</li>
 
</ul>
 
</ul>
 
</p>
 
</p>
Line 409: Line 425:
 
<li>TECAN reading of previous day's cultures for DoE</li>
 
<li>TECAN reading of previous day's cultures for DoE</li>
 
<li>Restriction digest, electrophoresis, gel extraction and ligation of pRha_FimH22sfGFP and pSB1A3.</li>
 
<li>Restriction digest, electrophoresis, gel extraction and ligation of pRha_FimH22sfGFP and pSB1A3.</li>
 +
<li> Overnight culture of E.coli Top10 </li>
 
</ul>
 
</ul>
 
</p>
 
</p>
Line 417: Line 434:
 
<ul>
 
<ul>
 
<li>TECAN reading of previous day's cultures for DoE</li>
 
<li>TECAN reading of previous day's cultures for DoE</li>
 +
<li>OD of overnight culture set to 1 and 1 in 1,000,000 applied</li>
 +
<li>Cultures irradiated</li>
 
</ul>
 
</ul>
 
</p>
 
</p>
Line 424: Line 443:
 
<ul>
 
<ul>
 
<li>Digestion and ligation of FimH_22sfGFP with pSB1A3 </li>
 
<li>Digestion and ligation of FimH_22sfGFP with pSB1A3 </li>
 +
<li>Colonies counted, results are as excepted.</li>
 
</ul>
 
</ul>
 
</p>
 
</p>

Revision as of 09:17, 30 September 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 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