Template:Team:Edinburgh/Notebook/Week02

1) Sucrose hydrolase plates preparation
The agar for sucrose hydrolase plates was prepared as follows:
sterile water 187.5 ml
M9 base (x4) 62.5 ml
agar 3.75 g
yeast extract 50 mg
The prepared solution was left for autoclaving.
2) Liquid sucrose hydrolase culture preparation
The liquid sucrose hydrolase culture (5 ml final volume) was prepared as follows:
sterile water 3.40 ml
4xng 1.25 ml
20% sucrose 0.20 ml
10000 pps As 25 µl
Trace elements A 5 µl
Trace elements B 5 µl
yeast extract 10 µl
chloramphenicol 5 µl (CML) to half of the bottles
Half of the bottles were inoculated by cells transformed with sucrose hydrolase and half were inoculated with untransformed control cells. The resulting four bottles were:
Transformed cells – CML
Transformed cells + CML
Control cells –CML
Control cells + CML
The bottles were left overnight at 37ºC with agitation.
3) Liquid nitroreductase culture preparation (at higher metronidazole concentrations)
The growth of BS control and BS-nitred nitroreductase cultures were tested again in the presence of higher concentrations of metronidazole and in metronidazole free solution.
Each culture was inoculated into 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)
150 or 300 mg/l metronidazole (15 or 30 µl out of 50 g/l stock solution)
The resulting six bottles were:
BS control + 0 mg/l metronidazole
BS-nitred + 0 mg/l metronidazole
BS control +150 mg/l metronidazole
BS-nitred + 150 mg/l metronidazole
BS control + 300 mg/l metronidazole
BS-nitred + 300 mg/l metronidazole
The cultures were left overnight at 37ºC with agitation

1) Sucrose hydrolase plates preparation (continued from Monday 2nd July 2012)
The autoclaved agar was microwaved for 4+4 minutes. It was then left in pre-heated 55ºC water bath to cool down. After 10 minutes, trace elements A (250 µl) and trace elements B (250 µl) were added. 20% sucrose (0.5 ml) or 20 % glucose (0.5 ml) were added to each plated before the agar was poured to give 6 sucrose and 5 glucose plates. The plates were swirled to evenly distribute the sugars, left to solidify and stored in the fridge room.
2) Growth assessing of the nitroreductase cultures
OD600 of the two bacterial cultures prepared on the 2nd of July 2012 was measured at 1/5 dilution (0.2 ml culture + 0.8 ml water) with water blank:
BS control + 0 mg/l metronidazole: 0.275
BS-nitred + 0 mg/l metronidazole: 0.296
BS control +150 mg/l metronidazole: 0.146
BS-nitred + 150 mg/l metronidazole: 0.136
BS control + 300 mg/l metronidazole: 0.040
BS-nitred + 300 mg/l metronidazole: 0.074
3) Growth assessing of liquid sucrose hydrolase cultures
OD600 of the transformed and untransformed bacterial cultures prepared on the 2nd of July 2012 was measured with water blank:
Transformed cells – CML: 0.849
Transformed cells + CML: 0.199
Control cells –CML: 0.209
Control cells + CML: 0.080
4) Nitroreductase o/n culture preparation
Four bacterial cultures were grown for testing their nitroreductase activity.
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)
Each culture was inoculated in 2.5 ml pre-prepared LB containing:
100 mg/l carbenicillin (2.5 µl out of 100 g/l stock solution)
90 mg/l IPTG (2.5 µl out of 90 g/l stock solution)
The cultures were left overnight at 37ºC with agitation.

1) Growth assessing of liquid sucrose hydrolase cultures (prepared Monday 2nd July 2012)
The growth of liquid sucrose hydrolase cultures was assessed again by measuring OD600 with a water blank.
Transformed cells – CML: 1.676
Transformed cells + CML: 0.417
Control cells –CML: 0.399
Control cells + CML: 0.241
2) NADH-dependent nitroreductase activity assay and plating to check growth inhibition by DMSO, DNBA and metronidazole
The four nitroreductase liquid cultures (prepared Tuesday 3rd July 2012) were used for this assay.
3,5-dinitrobenzyl alcohol (23 mg) was dissolved in DMSO (460 µl) to give 50 mg/ml DNBA stock solution.
Nicotinamide adenine dinucleotide (reduced) (8 mg) was dissolved in PBS (0.5 ml) to give 16 mg/ml stock solution.
Carb so plates with added IPTG were inoculated with 100 µl of each of the cultures (Bluescript vector, BS-nitred, CFX-nitred, Nitred-6). Upon drying of the plates, 5 µl of DMSO, metronidazole and DNBA were added on three distinct spots on each of the four plates.
The rest of the liquid cultures were split into two eppendorf tubes and centrifuged for 5 minutes at 10000 rpm to pellet the cells. The supernatant was removed and the pellet was resuspended in PBS (250µl). The two eppendorf tubes for each culture (250 µl) were combined to give 500 µl resuspended bacterial cells. To each of these, 1 µl DTT (out of 1 M stock solution) was added to ensure that the cellular proteins are not oxidised once the cells are ruptured. The resultant solution was sonicated 6*(10s sonication + 20 s rest) to rupture the cells. The supernatant was further used for the NADH-dependent nitroreductase activity assay.
NADH (10 µl) and 10 µl of bacterial supernatant were added to PBS (1ml). The solution was mixed by inverting.
BS-nitred:
Background absorbance: OD340 changed from 1.271 to 1.265 for 1 minute.
Upon addition of DNBA (5 µl) and mixing, OD340 was monitored every 10 s.

BS-control:
Background absorbance: OD340 changed from 1.274 to 1.262 for 1 minute.
Upon addition of DNBA (5 µl) and mixing, OD340 was monitored every 10 s.

CFX-nitred:
Background absorbance: OD340 changed from 1.262 to 1.262 for 1 minute.
Upon addition of DNBA (5 µl) and mixing, OD340 was monitored every 10 s.

Nitred-6:
Background absorbance: OD340 changed from 1.244 to 1.226 for 1 minute.
Upon addition of DNBA (5 µl) and mixing, OD340 was monitored every 10 s.

The supernatants were left at 4ºC o/n.

1) NADH-dependent nitroreductase activity assay
The assay was repeated to check if the enzyme activity was lost o/n.
BS-nitred:
Background absorbance: OD340 changed from 1.206 to 1.201 for 1 minute.
Upon addition of DNBA (5 µl) and mixing, OD340 was monitored every 10 s.

BS-control:
Background absorbance: OD340 changed from 1.209 to 1.204 for 1 minute.
Upon addition of DNBA (5 µl) and mixing, OD340 was monitored every 10 s.

CFX-nitred:
Background absorbance: OD340 changed from 1.173 to 1.196 for 1 minute.
Upon addition of DNBA (5 µl) and mixing, OD340 was monitored every 10 s.

Nitred-6:
Background absorbance: OD340 changed from 1.209 to 1.185 for 1 minute.
Upon addition of DNBA (5 µl) and mixing, OD340 was monitored every 10 s.

Since the activity did not seem to be lost, triplicate measurements with DMSO (instead of DNBA) controls were made.
For these measurements, 5 µl NADH, 5 µl of supernatant and 5 µl of DNBA/DMSO were used.
Bs-nitred:

BS-control:

CFX-nitred:

Nitred-6:

2) Bradford protein assay
The protein concentration of each of the supernatants was estimated by protein assay. Standard curve was prepared by adding 0, 2, 4, 6, 8 µl ( out of 2 mg/ml Bovine Serum) to Bradford’s reagent (0.98 ml). Each supernatant (volume determined by comparison to standard curve) was added to Bradford’s reagent (0.98 ml).
Volume added for each supernatant:
BS-nitred 10 µl s/n
Nitred-6 5 µl s/n
CFX-nitred 20 µl s/n
BS-control 10 µl s/n
Each s/n sample was prepared in duplicate and the absorbance was measured at 595 nm.

Standard curve:

From the standard curve and the OD595, the protein content of each supernatant was calculated :
Protein concentration (mg/ml or g/l)
BS-nitred 0.954333
BS-control 2.285262
CFX-nitred 0.277431
Nitred-6 1.041443
The change of NADH concentration was estimated by the change of absorbance per minute, background substracted and specific activity calculated.
Specific activity ( micro mol NADH oxidised per min per mg protein)
BS-nitred 0.025775131
BS-control 0.001711888
CFX-nitred 0.006760859
Nitred-6 0.013430536
3) The four nitroreductase strain plates (prepared on 4th July 2012) were examined to determine the size of the zone of clearance (by measuring the diameter).
DMSO showed no zone of clearance in all of the plates.

The plates were returned to the incubator o/n at 37ºC.
3)Sucrose hydrolase plasmid loss
Four colonies from the plate labelled CF 25.6.12 CMM (Chris’ minimal medium, which is like M9 but with half the ionic strength, designed for growing bacteria) + Sucrose DNA(+) were streaked onto three different plates: 1 sucrose plate, 1 glucose plate and 1 sucrose plate spread with 15uL chloramphenicol in 100uL water (and left to get absorbed by the agar for an hour). After streaking, the plates were incubated at 37C.

1)The four nitroreductase strain plates (prepared on 4th July 2012) were examined again. The diameter of the zone of clearance was measured.

DMSO did not show any zones of clearance.
CFX-nitred and BS-nitred are growing on individual colonies rather than a smear.
2) LA agar plates with different metronidazole and DNBA concentrations were prepared.
250ml LA agar was heated until boiling and left to cool for 20 min on preheated (55ºC) water bath. Carbanacillin (200 µl) was added upon cooling of the agar.
The DNBA/metronidazole final concentrations were:
0.02 mg/ml (10 µl out of 50 mg/ml stock)
0.05 mg/ml (25 µl out of 50 mg/ml stock)
0.1 mg/ml (50 µl out of 50 mg/ml stock)
0.2 mg/ml (100 µl out of 50 mg/ml stock)
4 plates with DNBA at these final concentrations, 4 plates with these final concentrations of metronidazole and 2 plates with no DNBA and metronidazole were prepared.
All plates were inoculated with the 4 nitroreductase strains.
3) Sucrose hydrolase plasmid loss (results)
All 3 of the plates showed some growth and they were left to incubate over the weekend. On Monday 9th of July, all three plates showed lots of growth, meaning that the plasmid was well-retained in the cells and that growth on sucrose medium was not just due to cells cross-feeding from only a few that were capable of metabolizing sucrose.
4) Transformation of Citrobacter with various replicons
We started the characterization of Citrobacter freundii (Cf) by testing whether different replicons of plasmids deposited in the registry work in Cf. We revived the relevant parts and have transformed them into E. coli and Cf cells following Cfrench:compcellprep1 protocol and then plated these cells onto plates containing IPTG and the relevant antibiotic (kanamycin for pSB2K3 and chloramphenicol for other plasmids) and incubated them at 37 C until Monday. The plate containing pSB2K3 also had X-gal on it.

parts revival: 15 ul of water were added to individual wells of iGEM plate with appropriate parts:
pSB3C5, pSB 4C5, pSB2K3
revived parts were then transferred to separate tubes.

Replicons we have tested in Cf:
F’ and P1 lytic: pSB2K3
pSC101: pSB4C5
p15A: pSB3C5
pMB1: pSB1C3
repA: Multi-host plasmid (ptg262) – Edinburgh had a stock of these

5) Cytochrome expression assay
Growth medium (2 x 100 ml) was prepared:
Per 100 ml of medium:
Water 65 ml
4xM9 25 ml
LB 10 ml
50% glycerol 0,8 ml
Sodium Fumarate 640 mg (40 mM)
One of:
TMAO 222 mg
sodium nitrate (NaNO3) 170 mg

media were aliquoted into 20 ml sealed bottles and Inoculated with S. oneidensis (TMAO, aerobic and anaerobic culture) or E. coli (TMAO and NaNO3, both in aerobic and anaerobic culture)
Cultures were left to incubate over the weekend at 37 oC, aerobic ones were shaken, anaerobic were left still