"
Team:Cambridge/LabBook/Week5
From 2010.igem.org
Monday
31. Experiment: Diagnostic gel to test for luciferase insertion (Theo & Anja)
Took overnight culture of TOP10 transformed with plasmid (potentially) containing promoter + rbs and luciferase
Plasmid DNA purification
following "QIAprep Spin Miniprep Kit using a Microcentrifuge" protocol
Gel electrophoresis
E-gel EX Agarose 1% mounted on transilluminator Nanodrop readings:
- Plasmid with promoter + rbs + luciferase: 18.5ng/μl => 5μl gives 100ng
- Plasmid with promoter + rbs only: 64.9ng/μl => 2μl gives 100ng
Gel loading mixture:
| promoter + rbs + luciferase | promoter + rbs only | |
|---|---|---|
| 6X orange loading Dye | 3μl | 3μl |
| plasmid DNA | 5μl | 2μl |
| deionised H2O | 12μl | 12μl |
Supercoiled DNA ladder loading mixture
- 1μl Supercoiled DNA ladder (NEB)
- 3μl 6X orange loading Dye
- 16μl deionised H2O
Gel was loaded following scheme below:
| Supercoiled DNA ladder | promoter + rbs only | promoter + rbs + luciferase |
| 20μl | 20μl | 20μl |
Empty wells were filled with 200μl deionised H2O. Gell was run and a clear band around 2kb was visible for promoter + rbs, a very faint band was observed around 4kb for promoter + rbs + luciferase.
Gel electrophoresis
was repeated with increased promoter + rbs + luciferase plasmid concentration
Gel loading mixture for promoter + rbs + luciferase
(others as above)
- 3μl 6X orange LD
- 15μl plasmid DNA
- 2μl deionised H2O
Gel was run and the same bands were observed as above, with the ~4kb promoter + rbs + luciferase more clearly than before
32. Experiment: In vitro assay for luciferase activity (Theo & Anja)
50μl of TOP10 cells transformed with plasmid containing promoter + rbs + luc were transferred to an Eppendorf tube and subjected to three rounds of freeze (-80°C) and thaw (37°C) treatement. 50μl of 2mg/ml luciferin were added tot he lysed bacteria and luminescence was tested using a CCD camera. The experiment was performed twice in a row and both times luminescence was observed with the CCD camera (though not by eye in the dark room).
Tuesday
33. Experiment: In vitro assay for luciferase activity using CHBT as substrate (Theo and Anja)
2ml of TOP10 with promoter, rbs and luc were spun at 13000rpm for 5 mins. Supernatant was discarded and cell pellet was resuspended in 500μl fresh LB to which 1 drop of chloroform was added. The cells were subjected to three rounds of freeze (-80°C) and thaw (37°C) treatment.
EXPERIMENT WAS CANCELLED!
34. Experiment: Preparation of Photobacterium Broth (Theo and Anja)
35.2g broth promoter was dissolved in 500ml water by warming/boiling. Broth was filter sterilised.
35. Experiment: Ampoule of Photobacterium (extracted by Theo and Peter)
Ampoule was broken, 500μl LB broth added and 100μl added to each of 5 falcons containing broth.
Wednesday
Theo added 5g agar to 250ml broth and placed for autoclaving.
After autoclaving the medium was turbid. It was poured into 9 empty, sterile dishes and left to set on the bench.
Thursday
36. Experiment: Renewed colonies of TOP10/pHK555 & pHK724 (Will)
- Inoculated 2x5ml LB with 1 colony for each
- Streaked out cells on Amp + Chl plate (*)
- Incubated both at 30°C overnight.
Results:
- 1 LB glow very faintly, other not at all
- Poor growth from streaking, but strong glow
Friday
36. Experiment: Transformation of TOP10 competent cells with luxR from registry (Bill)
- Followed transformation protocol from 02/08/10
- Transformed with BBa_I0462 from distribution from distribution Kit Plate 1 Well 80
- Plated out on 2 ampicillin plates the transformation, (amp is resistance marker of plasmid)
- Also cultured in 2 LB + Amp plates
Results (Hannah):
| Plate/Broth | Contains | Growth |
|---|---|---|
| Plate (Amp) | TOP10 LuxR | Yes |
| Plate (Amp) | TOP10 Only | No |
| Plate (No Amp) | TOP10 LuxR | Yes |
| Broth (LB + Amp) | TOP10 LuxR | No |
37. Experiment: Plating out of registry stabs (Theo)
Theo plated out BBa_K216007 and 08 on A plates
38. Experiment: Inoculated growth media for extraction of plasmid pHK555 for use in oligos (Will)
- 5ml SOB + 2.5μl Chloramphenicol + 1 colony Invitrogen 10 with pHK555 (from plate 5/8/10)
- 5ml SOB + 2.5μl Chloramphenicol + 10μl of glycerol stock of Slock10/pHK555 cells
- 5ml LB + 2.5μl Chloramphenicol + 1 colony Invitrogen 10 with pHK555 (from plate 5/8/10)
39. Experiment: Restreaked TOP10/pHK555+pHK724 (Will)
Using poorly grown strain from yesterday (*) restreaked onto LB agar+Chl+Amp (hope to select for strong growth).
40. Experiment: Streaked out Invitrogen 10 pHK555 (from plate 5/8/10) (Will)
On Chl plate, for colony PCR of pHK555 Plasmid
Saturday
41. Experiment: Culturing in broth of TOP10+LuxR from registry (Hannah)
- Took single colony from plate, added to LB+Amp broth
- 2 days later took them out and put in fridge
Sunday
