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

 
(24 intermediate revisions by 4 users not shown)
Line 148: Line 148:
 
<p>
 
<p>
 
  <ul>
 
  <ul>
−
   <li>Transformed fim H constructs (x10) into dh5alpha</li>
+
   <li>Transformed fim H constructs (x10) into DH5&#945;</li>
 
   <li>One pot: prepared for sequencing, made glycerol stocks, miniprep and qbit.</li>
 
   <li>One pot: prepared for sequencing, made glycerol stocks, miniprep and qbit.</li>
−
   <li>Dh5alpha overnights, made competent cells of top10.</li>
+
   <li>DH5&#945; overnights, made competent cells of top10.</li>
 
  </ul>
 
  </ul>
 
</p>
 
</p>
Line 174: Line 174:
 
  <ul>
 
  <ul>
 
   <li>Sequencing results confirm successful plasmid construction.</li>
 
   <li>Sequencing results confirm successful plasmid construction.</li>
−
   <li>Transformation of MoClo products into dh5alpha.</li>
+
   <li>Transformation of MoClo products into DH5&#945;.</li>
 
  </ul>
 
  </ul>
 
</p>
 
</p>
Line 197: Line 197:
 
<p>
 
<p>
 
  <ul>
 
  <ul>
−
   <li>Miniprep and Qubit of pRha+fimH+psB1A3 constructs.</li>
+
   <li>Miniprep and Qubit of P_Rha+fimH+psB1A3 constructs.</li>
−
   <li>Overnights cultures of pAra+fimop(pX3’6’0’0),  
+
   <li>Overnights cultures of P_Ara+fimop(pX3’6’0’0),  
−
J23100+fimop(pX3’6’0’0), J23100+fimop(PSB1A3)</li>
+
P_J23100+fimop(pX3’6’0’0), P_J23100+fimop(PSB1A3)</li>
 
  </ul>
 
  </ul>
 
</p>
 
</p>
Line 206: Line 206:
 
<p>
 
<p>
 
  <ul>
 
  <ul>
−
   <li>Prep of pRha+fimH miniprep DNA for sequencing.</li>
+
   <li>Prep of P_Rha+fimH miniprep DNA for sequencing.</li>
−
   <li>Qubit of overnight cultures: (J23100 x 2 + pAra) + fimH.</li>
+
   <li>Qubit of overnight cultures: (P_J23100 x 2 + P_Ara) + fimH.</li>
 
  </ul>
 
  </ul>
 
</p>
 
</p>
Line 214: Line 214:
 
<p>
 
<p>
 
  <ul>
 
  <ul>
−
   <li>Restriction digest of Fim operon in: pX3’6’0’0 with J23100 promoter,  
+
   <li>Restriction digest of Fim operon in: pX3’6’0’0 with P_J23100 promoter,  
−
pX3’6’0’0 with pAra, pSB1A3 with J23100.</li>
+
pX3’6’0’0 with P_Ara, pSB1A3 with P_J23100.</li>
 
   <li>The products of the restriction digest were ran on the electrophoresis
 
   <li>The products of the restriction digest were ran on the electrophoresis
 
  and only the pSB1A3 product was successful. Only two pSB1A3 plasmids were  
 
  and only the pSB1A3 product was successful. Only two pSB1A3 plasmids were  
 
sent for sequencing (5+6).</li>
 
sent for sequencing (5+6).</li>
−
   <li>The pRha+fimH+psB1A3 constructs were also sent for sequencing (13 constructs).</li>
+
   <li>The P_Rha+fimH+psB1A3 constructs were also sent for sequencing (13 constructs).</li>
 
  </ul>
 
  </ul>
 
</p>
 
</p>
Line 226: Line 226:
 
<p>
 
<p>
 
  <ul>
 
  <ul>
−
   <li>Poured CAM plates, Restriction digest of pAra_fimoperon_pSB1A3, J23100_fimoperon_
+
   <li>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  
 
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).</li>
 
of the DNA (the wrong gel bands were extracted, so Chloe did it again for us).</li>
−
   <li>Transformation of the FimH constructs in the pSB1C3 and pSB1A3 into DH5a.</li>
+
   <li>Transformation of the FimH constructs in the pSB1C3 and pSB1A3 into DH5&#945;.</li>
 +
  <li>Made a mannose serial dilution from 4% to 0.0625% and ran in the HPLC.</li>
 
  </ul>
 
  </ul>
 
</p>
 
</p>
Line 238: Line 239:
 
   <li>Gel extraction of the bands (after that we found out the bands we extracted were
 
   <li>Gel extraction of the bands (after that we found out the bands we extracted were
 
  the wrong ones).</li>
 
  the wrong ones).</li>
−
   <li>Overnights of FimH constructs in the pSB1C3 and pSB1A3 into DH5a.</li>
+
   <li>Overnights of FimH constructs in the pSB1C3 and pSB1A3 into DH5&#945;.</li>
 +
  <li>Grew MG1655 on silica sand and NP-Bacto-Pellets.</li>
 
  </ul>
 
  </ul>
 
</p>
 
</p>
Line 247: Line 249:
 
   <li>Mini prep and Qubit from overnights of FimH constructs and transformation of  
 
   <li>Mini prep and Qubit from overnights of FimH constructs and transformation of  
 
the ligation from the gel extraction on CAM plates that Chloe did.</li>
 
the ligation from the gel extraction on CAM plates that Chloe did.</li>
 +
  <li>Prepared silica sand and NP-Bacto-Pellets samples for SEM imaging.</li>
 
  </ul>
 
  </ul>
 
</p>
 
</p>
Line 253: Line 256:
 
<p>
 
<p>
 
  <ul>
 
  <ul>
−
   <li>Miniprep, glycerol stocks and qubit of J23100 and pAra fim_op.</li>
+
   <li>Miniprep, glycerol stocks and qubit of P_J23100 and P_Ara fim_op.</li>
 
   <li>Diagnostic digest and electrophoresis of above - shows successful construction  
 
   <li>Diagnostic digest and electrophoresis of above - shows successful construction  
 
and transformation of plasmids. </li>
 
and transformation of plasmids. </li>
Line 272: Line 275:
 
  <ul>
 
  <ul>
 
   <li>Liquid overnights for previous transformations.</li>
 
   <li>Liquid overnights for previous transformations.</li>
 +
  <li> Grow overnight cultures of <i>E.coli</i> Top10 and <i>E.coli</i> Top10, transformed with chloramphenicol and ampicillin resistance genes for UV experiment. </li>
 +
  <li> Imaged silica sand and NP-Bacto-Pellets samples using SEM.</li>
 
  </ul>
 
  </ul>
 
</p>
 
</p>
Line 279: Line 284:
 
  <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>
 
  </ul>
 +
</p>
 +
 +
25/08/2017
 +
<p>
 +
<ul>
 +
  <li> Colonies on plates were counted, unexpected results. </li>
 +
</ul>
 +
</p>
 +
 +
28/08/2017
 +
<p>
 +
<ul>
 +
<li>Overnights of transformed Top10, WT Top10 and WT MG1655.</li>
 +
</ul>
 +
</p>
 +
 +
29/08/2017
 +
<p>
 +
<ul>
 +
<li>Revised induction protocol.</li>
 +
<li>Measured construct fluorescence in TECAN.</li>
 +
<li>Grew MG1655 on silica sand and NP-Bacto-Pellets again following unexpected results previously.</li>
 +
</ul>
 +
</p>
 +
 +
30/08/2017
 +
<p>
 +
<ul>
 +
<li>First attempt at agglutination protocol</li>
 +
<li>Prepared and imaged silica sand and NP-Bacto-Pellets samples for SEM.</li>
 +
<li>Investigated the flow rates that were achieved when polypropylene scaffold torus structures were inside the metal binding reactor.</li>
 +
</ul>
 +
</p>
 +
 +
31/08/2017
 +
<p>
 +
<ul>
 +
<li>Induction protocol</li>
 +
</ul>
 +
</p>
 +
 +
01/09/2017
 +
<p>
 +
<ul>
 +
<li>Samples run through the TECAN</li>
 +
<li>Grew MG1655 on polypropylene scaffold torus structures, using a range of concentrations of a chlorhexidine gluconate surfactant.</li>
 +
</ul>
 +
</p>
 +
 +
04/09/2017
 +
<p>
 +
<ul>
 +
<li>Overnight cultures of constructs</li>
 +
<li>Examined the tori to observe which concentration of surfactant produced a biofilm on the surface.</li>
 +
<li>Repeated the growing of MG1655 on polypropylene scaffold torus structures, using a range of concentrations of a chlorhexidine gluconate surfactant.</li>
 +
</ul>
 +
</p>
 +
 +
05/09/2017
 +
<p>
 +
<ul>
 +
<li>Induction protocol</li>
 +
<li>Agglutination protocol</li>
 +
<li>Successful transformation of mouse metallothionein </li>
 +
<li>Overnight cultures of <i>E.coli</i> Top10 for UV </li>
 +
<li>Examined the tori to observe which concentration of surfactant produced a biofilm on the surface.</li>
 +
</ul>
 +
</p>
 +
 +
06/09/2017
 +
<p>
 +
<ul>
 +
<li>Samples run through FACS and fluorescent cells sorted</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>
 +
</p>
 +
 +
07/09/2017
 +
<p>
 +
<ul>
 +
<li>Induction protocol</li>
 +
<li> Colonies on plates counted, OD of culture too great.</li>
 +
</ul>
 +
</p>
 +
 +
08/09/2017
 +
<p>
 +
<ul>
 +
<li>Dialysis trial run</li>
 +
</ul>
 +
</p>
 +
 +
11/09/2017
 +
<p>
 +
<ul>
 +
<li>Induction protocol</li>
 +
<li>Repeated the growing of MG1655 on polypropylene scaffold torus structures, using a range of concentrations of a chlorhexidine gluconate surfactant.</li>
 +
</ul>
 +
</p>
 +
 +
12/09/2017
 +
<p>
 +
<ul>
 +
<li>Miniprep of P_Rha_GFP constructs for DoE</li>
 +
<li>Ran the fluidising media reactor with tori at a range of concentrations of a chlorhexidine gluconate surfactant.</li>
 +
<li>Imaged samples of the tori using SEM before and after being run through the metal binding reactor.</li>
 +
<li>Ran HPLC for the mannose concentration of samples taken from the use of the metal binding reactor with the tori.</li>
 +
</ul>
 +
</p>
 +
 +
13/09/2017
 +
<p>
 +
<ul>
 +
<li>Induction of three FimH constructs at 0.1 and 0.2% rhamnose concentrations in BL21 and DH5&#945;</li>
 +
<li>Overnight cultures of <i>E. coli</i> Top10.</li>
 +
</ul>
 +
</p>
 +
 +
14/09/2017
 +
<p>
 +
<ul>
 +
<li>TECAN reading of previous day's induced cultures.</li>
 +
<li>Induction of three FimH constructs at 1 and 2% rhamnose concentrations.</li>
 +
<li>Culture irradiated using UV trans-illuminator</li>
 +
</ul>
 +
</p>
 +
 +
 +
15/09/2017
 +
<p>
 +
<ul>
 +
<li>TECAN reading of the previous day's induced cultures.</li>
 +
<li>Colonies counted, positive results.</li>
 +
</ul>
 +
</p>
 +
 +
 +
19/09/2017
 +
<p>
 +
<ul>
 +
<li>DoE: the first block</li>
 +
<li>Restriction digest and ligation of P_Rha_FimH_1sfGFP and with pSB1A3.</li>
 +
</ul>
 +
</p>
 +
 +
 +
20/09/2017
 +
<p>
 +
<ul>
 +
<li>TECAN reading of previous day's cultures for DoE</li>
 +
<li>Restriction digest, electrophoresis, gel extraction and ligation of P_Rha_FimH_1sfGFP and pSB1A3.</li>
 +
<li> Overnight culture of <i>E. coli</i> Top10 </li>
 +
<li>Grew MG1655 on polypropylene scaffold torus structures, for runs 1-3 for design of experiment.</li>
 +
</ul>
 +
</p>
 +
 +
 +
21/09/2017
 +
<p>
 +
<ul>
 +
<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>
 +
<li>Runs 1-3 for the design of experiment in the metal binding reactor.</li>
 +
<li>Grew MG1655 on polypropylene scaffold torus structures, for runs 4-5 for design of experiment.</li>
 +
</ul>
 +
</p>
 +
 +
22/09/2017
 +
<p>
 +
<ul>
 +
<li>Digestion and ligation of FimH_1sfGFP with pSB1A3 </li>
 +
<li>Colonies counted, results are as expected.</li>
 +
<li>Runs 4-5 for the design of experiment in the metal binding reactor.</li>
 +
</ul>
 +
</p>
 +
 +
25/09/2017
 +
<p>
 +
<ul>
 +
<li>Transformed FimH_1sfGFP_pSB1A3 into <i>E.coli</i></li>
 +
</ul>
 +
</p>
 +
 +
26/09/2017
 +
<p>
 +
<ul>
 +
<li>Grew MG1655 on polypropylene scaffold torus structures, for runs 6-8 for design of experiment.</li>
 +
</ul>
 +
</p>
 +
 +
27/09/2017
 +
<p>
 +
<ul>
 +
<li>Runs 6-8 for the design of experiment in the metal binding reactor.</li>
 +
<li>Grew MG1655 on polypropylene scaffold torus structures, for runs 9, 10 and 1 for design of experiment.</li>
 +
</ul>
 +
</p>
 +
 +
28/09/2017
 +
<p>
 +
<ul>
 +
<li>TECAN reading for DoE</li>
 +
<li>Inoculated and induced cultures for DoE </li>
 +
<li>Diagnostic digestion of FimH_1sfGFP_pSB1A3 plasmid DNA </li>
 +
<li>Runs 9, 10 and 1 for the design of experiment in the metal binding reactor.</li>
 +
</ul>
 +
</p>
 +
 +
29/09/2017
 +
<p>
 +
<ul>
 +
<li>Transformation for Newcastle collaboration</li>
 +
</ul>
 +
</p>
 +
 +
02/10/2017
 +
<p>
 +
<ul>
 +
<li> Transformation of T7_FimH_225sfGFP into BL21 </li>
 +
</ul>
 +
</p>
 +
 +
04/10/2017
 +
<p>
 +
<ul>
 +
<li> Induction protocol </li>
 +
</ul>
 +
</p>
 +
 +
09/10/2017
 +
<p>
 +
<ul>
 +
<li>Ran Newcastle samples through FACS, analysed the data and sent it off. </li>
 +
</ul>
 +
</p>
 +
 +
11/10/2017
 +
<p>
 +
<ul>
 +
<li>Prepared samples for subsequent SDS page analysis of fim operon in top10 </li>
 +
</ul>
 +
</p>
 +
 +
12/10/2017
 +
<p>
 +
<ul>
 +
<li>Induction protocol </li>
 +
</ul>
 +
</p>
 +
 +
13/10/2017
 +
<p>
 +
<ul>
 +
<li> Restriction digest of all parts in pSB1C3 and diagnostic electrophoresis gel showed successful construction </li>
 +
<li> Preparation of all parts for sequencing </li>
 +
</ul>
 +
</p>
 +
 +
16/10/2017
 +
<p>
 +
<ul>
 +
<li>Qubit of all parts in pSB1C3 </li>
 +
</ul>
 +
</p>
 +
 +
17/10/2017
 +
<p>
 +
<ul>
 +
<li>SDS-PAGE and Western blot of FimH_1His constructs and wild types in four <i>E.coli</i> strains </li>
 +
</ul>
 +
</p>
 +
 +
18/10/2017
 +
<p>
 +
<ul>
 +
<li>Western blot and SDS-PAGE results showed successful expression and binding of FimH_1His pili with no bands for the wild types. </li>
 +
</ul>
 +
</p>
 +
 +
20/10/2017
 +
<p>
 +
<ul>
 +
<li>Preparation of DNA parts for submission </li>
 +
</ul>
 +
</p>
 +
 +
23/10/2017
 +
<p>
 +
<ul>
 +
<li>Transformation of FimB KO with fim operon (under both promoters) with FimH, FimH_1His, FimH_1SynMT, FimH_1MouseMT</li>
 +
</ul>
 +
</p>
 +
 +
24/10/2017
 +
<p>
 +
<ul>
 +
<li>DNA parts sent off</li>
 +
</ul>
 +
</p>
 +
 +
25/10/2017
 +
<p>
 +
<ul>
 +
<li>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</li>
 +
<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>
 +
 +
26/10/2017
 +
<p>
 +
<ul>
 +
<li>Dialysis: FimB KO of PJ23100 and PRha_FimH / PRha_FimH_1_His / PRha_FimH_1_SynMT / PRha_FimH_1_MouseMT by adding either CuCl<sub>2</sub> and/or NiSO<sub>4</sub></li>
 +
</ul>
 +
</p>
 +
 +
31/10/2017
 +
<p>
 +
<ul>
 +
<li>SDS page and Western blot of T7-FimH_225_GFP in BL21(DE3) after induction as well as BL21(DE3) wild type (similar treatment) </li>
 +
</ul>
 
</p>
 
</p>

Latest revision as of 18:40, 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)