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:
- Insert work here
- Insert work here
- Insert work here
Synthetic Promoters:
Liquid Handling Robots Assembly Automation
General
04/04/17:
05/04/17:
- 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
- Selected for plasmid by growth on ampicilin LB agar plates
- Transformation protocol
CFPS:
06/04/17:
- Confirmed successful transformation of plasmid into cells by visualisation of red colonies on the LB agar AMP plate:
CFPS:
07/04/17:
Week Commencing: 10/04/17
10/04/17:
- 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
CFPS:
11/04/17:
- Used 1 mL of overnight culture from 10/04/17 to prepare glycerol stock of DH5α with pSB1AT3-J04450 plasmid
- Used remaining overnight cultures for miniprep to extract pSB1AT3-J04450 plasmid DNA
CFPS:
12/04/17:
- 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
CFPS:
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:
- Tested S30 cell-free circular E. coli commercial kit from promega
- 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
Reaction ID Plasmid DNA used Stock DNA concentration
(ng/μL)DNA amount in reaction (μg) DNA volume in reaction (μL) Neg1 N/a 0 0 0 Neg2 N/a 0 0 0 Neg3 N/a 0 0 0 Luc1 pBEST-Luc (Promega) 1000 2 2 Luc2 pBEST-Luc (Promega) 1000 2 2 RFPa1 pSB1AT3-mRFP1 (BB_MP2) 172.1 1 5.81 RFPa2 pSB1AT3-mRFP1 (BB_MP2) 172.1 1 5.81 RFPa3 pSB1AT3-mRFP1 (BB_MP2) 172.1 1 5.81 RFPb1 pSB1AT3-mRFP1 (BB_MP4) 172.1 1.7 9.88 RFPb2 pSB1AT3-mRFP1 (BB_MP4) 172.1 1.7 9.88 RFPb3 pSB1AT3-mRFP1 (BB_MP4) 172.1 1.7 9.88 
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.
CFPS:
Week Commencing: 17/04/17
24/04/17:
- 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
CFPS:
25/04/17:
- 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
- The following amino acids were dissolved in 250 μL KOH (5 M):
CFPS:
26/04/17:
- 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 BL21 B. subtilis 168 0 0.043 0.034 60 0.109 0.045 90 0.305 0.068 120 0.636 0.136 150 1.090 0.282 180 1.575 0.677 210 1.887 1.198 245 2.228 1.601 270 2.372 1.843 300 2.592 2.099 330 2.687 2.114 
Growth curves for E. coli BL21 and B. subtilis 168
CFPS:
27/04/17:
28/04/17:
May:
Week Commencing: 01/05/17
01/05/17:
02/05/17:
03/05/17:
- prepared 400 mL of CFPS wash buffer (14 mM magnesium glutamate, 60 mM potassium glutamate, 10 mM TRIS-acetate)
CFPS:
04/05/17:
05/05/17:
Week Commencing: 08/05/17
08/05/17:
- prepared overnight cultures (5 mL LB broth) of E. coli BL21 and B. subtilis 168
CFPS:
09/05/17:
- 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
CFPS: |
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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:
