Template:Team:Edinburgh/Notebook/Week01

1) Shewanella oneidensis growth
We managed to grow viable colonies of S. oneidensis

2) csc sucrose hydrolase experiment
a) Cell transformation
Competent E coli cells J15 were taken from the freezer. They were transformed with plasmid DNA following OpenWetWare protocol Cfrench:compcellprep1 ([http://openwetware.org/wiki/Cfrench:compcellprep1 protocol]).
100 ul of J15 cells were transfered to a clean microfuge tube labelled KK 25.6.12 No DNA contr.
1,5 ul of plasmid DNA was added to the remaining 100 ul of J15 cells

b) Plating cells
Following cell transformation, 100 ul of transformed and untransformed J15 cells were plated on the following plates (all contain M9 minimal growth agar, plates with sugars also contain arsenic for activation of arsenic promoter):

chloramphenicol (labelled CF 26.6.12 cml 10 + ipt990 DNA (+ or -)),
glucose (labelled CF 25.6.12 cnn + glucose DNA (+ or -),
sucrose (labelled CF 25.6.12 cnn + sucrose DNA (+ or -),
no sugars (labelled CF 25.6.12 cnn + no sug DNA (+ or -)

1) results from Week 1, 26.06.2012, Tuesday experiment 2b
Cells transformed with DNA grew well on chloramphenicol but showed no growth on sugar medium.

2) Incubation of sucrose hydrolase transformed cells in liquid medium
We prepared 4 glass tubes with minimal liquid medium and sucrose to test for growth of transformed and untransformed cells in liquid medium. Following ingredients were added to each of the 4 tubes:
3,4 ml sterilised water
1,25 ml M9 x4 medium
0,2 ml 20% sucrose
25 ul 10000 ppb sodium arsenate
5 ul trace elements A
5 ul trace elements B
10 ul yeast extract
Moreover, 5 ul chloramphenicol were added to 2 of the tubes The tubes were inoculated with 50 ul either transformed or untransformed cells.
Finally 4 tubes contained: cells with DNA, cells without DNA, chlorampheniocol + cell with DNA, chlorampheniocol + cell without DNA

1) results from Week 1, 26.06.2012, Tuesday Experiment 2
Minimal growth was present in all bottles except bottle with chloramphenicol and cells transformed with sucrose hydrolase genes.

2) Incubation of sucrose hydrolase transformed cells in liquid medium spread on a water agar plate.
Water agar plates were prepared by heating up 100 ml of 0,6 water agar and pouring it on plates (about 25ml of agar per plate) (plates were marked KK 28.6.12 H20 agar +M9 suc liq)
Next, M9 liquid medium from Week 1, 26.06.2012, Tuesday Experiment 2 control samples (with or without chloramphenicol) were poured on plates and inoculated with 25 ul transformed or untransformed cells. Resulting 4 plates contained:
+DNA+CML: 2,5 ml of M9 liquid medium with chloramphenicol and 25 ul of J15 transformed cells
-DNA+CML: 2,5 ml of M9 liquid medium with chloramphenicol and 25 ul of J15 untransformed cells
+DNA-CML: 2,5 ml of M9 liquid medium without chloramphenicol and 25 ul of J15 transformed cells
-DNA-CML: 2,5 ml of M9 liquid medium without chloramphenicol and 25 ul of J15 untransformed cells
Plates were left to incubate overnight.

1) results from Week 1, 28.06.2012, Thursday experiment 2
No growth was present on the plates
2) Microscopic examination of Shewanella oneidensis plate
Plate with revived Shewanella oneidensis strain contained two different kinds of colonies. These colonies were subcultured, gram stained and examined under the microscope. Both colonies appeared as gram-negative rods
3) Nitroreductase culture setup
Four bacterial cultures were tested for growth inhibition by metronidazole:
Bluescript vector (control)
BS-nitred (Bluescript vector with nitroreductase gene)
CFX-nitred (CF’s expression vector with nitroreductase gene)
Nitred-6 (Expression vector with nitroreductase gene)
Metronidazole (23 mg) was dissolved in DMSO (460 µl) to give 50 g/l stock solution.
Each culture was inoculated in 5 ml pre-prepared LB containing:
100 mg/l carbenicillin (5 µl carbenicillin out of 100 g/l stock solution)
90 mg/l IPTG (5 µl out of 90 g/l stock solution)
7.5 mg/l metronidazole (7.5 µl out of 50 g/l stock solution)
The cultures were left overnight at 37ºC with agitation.

1)Growth assessing of nitroreductase cultures
OD600 of the four bacterial cultures prepared on the 29th of June 2012 was measured at 1/5 dilution (0.2 ml culture + 0.8 ml water) with water blank:
Bluescript vector (control): 0.114
BS-nitred (Bluescript vector with nitroreductase gene): 0.047
CFX-nitred (CF’s expression vector with nitroreductase gene): 0.137
Nitred-6 (Expression vector with nitroreductase gene):0.138