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 Luc1-3 and Neg1-3 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 Luc3 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 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.
CFPS:
Week Commencing: 17/04/17
24/04/17:
25/04/17:
26/04/17:
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:
