Team:Newcastle/Notebook/LabBook

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Lab Book

All experiments and lab work are fully documented here. Click on the buttons below to filter the entries.

Month:

Aspect of the project:


April:


Week Commencing: 03/04/17

03/04/17:

    Synthetic Promoters:

  • Insert work here
  • Liquid Handling Robots Assembly Automation

  • Insert work here
  • General

  • Insert work here

04/04/17:

05/04/17:

    CFPS:

  • Transformed competent DH5α E. coli cells with BBa_J04450 in a pSB1AT3 plasmid backbone which will be used for the initial testing of E. coli BL21-based CFPS systems

06/04/17:

    CFPS:

  • Confirmed successful transformation of plasmid into cells by visualisation of red colonies on the LB agar AMP plate:

07/04/17:


Week Commencing: 10/04/17

10/04/17:

    CFPS:

  • Four Overnight cultures of J04450 in pSB1AT3 from transformation plates from 05/06/17
    • 5 mL LB broth with 100 μg/mL ampicilin, incubated overnight at 37oC and 250 RPM

11/04/17:

12/04/17:

    CFPS:

  • Measured concentration of minipreped pSB1AT3-J04450 plasmid DNA using a nanodrop
    • BB_MP1: 153.2 μg/mL
    • BB_MP2: 172.1 μg/mL
    • BB_MP3: 165.9 μg/mL
    • BB_MP4: 172.1 μg/mL

13/04/17:

14/04/17:


Week Commencing: 17/04/17

17/04/17:

18/04/17:

19/04/17:

20/04/17:

21/04/17:

    CFPS:

  • Tested S30 cell-free circular E. coli commercial kit from promega
      Reaction IDPlasmid DNA usedStock DNA concentration
      (ng/μL)
      DNA amount in reaction (μg)DNA volume in reaction (μL)
      Neg1N/a000
      Neg2N/a000
      Neg3N/a000
      Luc1pBEST-Luc (Promega)100022
      Luc2pBEST-Luc (Promega)100022
      RFPa1pSB1AT3-mRFP1 (BB_MP2)172.115.81
      RFPa2pSB1AT3-mRFP1 (BB_MP2)172.115.81
      RFPa3pSB1AT3-mRFP1 (BB_MP2)172.115.81
      RFPb1pSB1AT3-mRFP1 (BB_MP4)172.11.79.88
      RFPb2pSB1AT3-mRFP1 (BB_MP4)172.11.79.88
      RFPb3pSB1AT3-mRFP1 (BB_MP4)172.11.79.88
    • Reactions were prepared according to the manufactuer's protocol
    • Reactions were made up to 50 μL and pipetted into wells of a flat-bottom black 96 well plate
    • Reactions were incubated in a plate reader (BMG Fluostar Optima) for ~4 hours with fluorescence readings every 15 mins - gain was set at 900
    • For the Luc1 & 2 and Neg1 & 2 reactions, after four hours the plate was removed and 50 μL Luciferase assay reagent was added to the reactions before luminescence was measured by the plate reader
      • The gain was calculated as 40% of Luc1
    • Raw results can be accessed here
    • Time course of commercial S30 circular CFPS systems expressing pSB1AT3-mRFP1: Data points are averages of triplicate repeats and error bars show plus and minus standard error. Red circles are reactions with no plasmid DNA, black squares are reactions with 1 μg plasmid DNA, and blue triangles are reactions with 1.7 μg plasmid DNA.

      End point luminescence readings for commercial S30 cicular CFPS systems expressing pBEST-Luc: Data points are averages of duplicate reactions and error bars show plus and minus standard error. 'Neg' reactions contained no plasmid DNA. 'Luc' reactions contained 2 μg pBEST-Luc. Readings were taken after 3.75 hours incubation at 37oC. The Y axis is in log10.


Week Commencing: 17/04/17

24/04/17:

    CFPS:

  • Streak plate of E. coli BL21 cells from glycerol stock onto plain LB agar plate
  • Obtainined a streak plate of B. subtilis 168 from a lab member (Dr. Wendy Smith)
  • Cells will be used to prepare CFPS cell extract

25/04/17:

    CFPS:

  • Overnight cultures of E. coli BL21 and B.subtilis 168 from streak plates (24/04/17) were prepared
    • 5 mL LB agar broth, incubated overnight at 37oC and 250 RPM
  • Prepared amino acid stock solutions
    • The following amino acids were dissolved in 250 μL KOH (5 M):
      • Aspartic Acid: 125 mg
      • Cysteine: 74.5 mg
      • Glutamic acid: 134.5 mg
      • Glutamine: 91.5 mg
      • Glycine: 79 mg
      • Histidine: 127.5 mg
      • Isoleucine: 123.5 mg
      • Leucine: 83.5 mg
      • Lysine: 87.5 mg
      • Methionine: 93 mg
      • Phenylalanine: 71 mg
      • Proline: 112 mg
      • Serine: 104.5 mg
      • Threonine: 114.5 mg
      • Tryptophan: 85 mg
      • Tyrosine: 108.5 mg
      • Valine: 76 mg
    • The following amino acids were dissolved in 500 μL KOH (5 M):
      • Alanine: 182 mg
      • Arginine: 202 mg
      • Asparagine-monohydrate: 282 mg
    • Note that some amino acids may not fully dissolve and stay in solution
    • Stock solutions were stored at -20oC

26/04/17:

    CFPS:

  • Used 1 mL of overnight culture from 25/04/17 to prepare glycerol stocks of E. coli BL21 and B. subtilis 168
  • Growth curves for E. coli BL21 and B. subtilis 168
    • Inoculated 2 * 200 mL LB broth in a sterile 2 L conical flask with 2 mL of E. coli BL21 overnight culture in one flask, and B. subtilis 168 in the other
    • Incubated at 37oC and 250 RPM
    • OD600 readings with a Fenway spectrophotometer - plain LB broth used as a blank
      • Readings at 0 mins, 60 mins, then every 30 mins
    • Time (mins)Strain
      E. coli BL21B. subtilis 168
      00.0430.034
      600.1090.045
      900.3050.068
      1200.6360.136
      1501.0900.282
      1801.5750.677
      2101.8871.198
      2452.2281.601
      2702.3721.843
      3002.5922.099
      3302.6872.114

      Growth curves for E. coli BL21 and B. subtilis 168

27/04/17:

28/04/17:


May:


Week Commencing: 01/05/17

01/05/17:

02/05/17:

03/05/17:

04/05/17:

05/05/17:


Week Commencing: 08/05/17

08/05/17:

    CFPS:

  • prepared overnight cultures (5 mL LB broth) of E. coli BL21 and B. subtilis 168

09/05/17:

    CFPS:

  • CFPS cell extract preparation
    • Inoculated 200 mL LB broth in a 2 L conical flask with 2 mL of E. coli culture (from 09/09/17)
    • Inoculated another 200 mL LB broth in a 2 L conical flask with 2 mL of B. subtilis culture
    • Shake incubated the flasks at 37oC until late exponential phase was reached (OD600 ~ 2.5 for E. coli and ~2.0 for B. subtilis
    • Cells were harvested by centrifugation at 4,500 RPM and 4oC for 20 mins

10/05/17:

11/05/17:

12/05/17:


Week Commencing: 15/05/17

15/05/17:

16/05/17:

17/05/17:

18/05/17:

19/05/17:


Week Commencing: 22/05/17

22/05/17:

23/05/17:

24/05/17:

25/05/17:

26/05/17:


Week Commencing: 29/05/17

29/05/17:

30/05/17:

31/05/17:


June:


Week Commencing: 29/05/17

01/06/17:

02/06/17:


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