Team:Edinburgh/Notebook

1)Transformation of Citrobacter with various replicons (results)

These results show that all but one of the plasmids have successfully been transformed into both E. coli and Cf, therefore these replicons are compatible with Cf. Cf cells with the multi-host plasmid did not grow at all, and there can be several reasons why: the codon usage in Cf differs from E. coli and Bacillus; the promoter does not work properly in Cf or Cf is less resistant to chloramphenicol. Bacteria transformed with ptg262 will be plated onto plates with differing concentrations of chloramphenicol to assess whether this plasmid is compatible at all with Cf.
The pSB2K3 transforms were plated onto plates containing X-gal in order to assess whether the Cf cells were lacZ positive (had a lacZ gene on their chromosome). Since the colonies turned blue, this means that the Cf cells are lacZ positive (as the plasmid contained no lacZ minigene) whereas the E. coli cells are lacZ negative since the colonies were red. The RFP was expressed in both cells, as evidenced by red fluorescence under a blue light.

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 (according to standard protocol) and then plated these cells onto plates containing IPTG and the relevant antibiotic (to which the plasmid confers resistance) and incubated them at 37 C until Monday. The plate containing pSB2K3 also had X-gal on it.
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

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:

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) 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) 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) 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)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

1) results from week 1 day 4 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) results from Week 1 Day 2 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 Day 2 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 Day 2 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) 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 Day 3 - experiment 1 and 3

Plate with glucose resulted in some minor growth (both in transformed and control cells)
Plate with sucrose resulted in no growth
Plate with no sugars resulted in no growth
Plate with sucrose and arsenate with colonies from Xgal blue/white selection showed some growth where blue colonies were plated (transformed with high efficiency promoter regulated by arsenate)

2) Results from day 3 - experiment 2

No growth - we conclude that shewanella strain used for plating is not viable.

3) results from day 3 - experiment 4

All plates showed noticeable growth and will be used for nitroreductase tests next week

4) Gel analysis of sucrose hydrolase plasmid

agarose gel was prepared following OpenWetWare protocol (using 0,5x TAE) [http://openwetware.org/wiki/Cfrench:AGE (protocol)] and used to analyse fusion proteins

1) Results from day 1 - experiment 2

The plates continued to show no growth, neither the control nor the sample grew so we theorize that something has gone wrong with the experimental setup. The experiment was repeated by plating out test and control bacteria on NO SUGAR, SUCROSE and GLUCOSE plates. A small amount of yeast extract was also added to all three plates to supply vitamins and trace elements.

2) Results from day 2 - experiment 2

The Shewanella plate showed no sign of growth, it was left to incubate for a further day to see whether it is at all viable.

3) Results from day 2 - experiment 4

The bacteria on the xgal + arsenic plates grew as expected, white colonies could be seen where the controls had been plated (that lacked the promoter and the lacZ fragment), while blue colonies grew where the samples containing the arsenic promoter and lacZ fragment were plated.

White and blue colonies were picked from this plate and plated onto fresh medium containing sucrose as the sole carbon source, along with some yeast extract and arsenic to see how well the gene works.

4) Transformation of 3 different nitroreductase plasmids into E. coli

We are trying to see whether plasmids containing the nitroreductase gene (prepared in 1997, the plasmids probably differ in what promoter is attached to the gene) still function. If they do, we will proceed to BioBrick-ify this gene. The plasmids also contain a Carbenicillin resistance gene.

E. coli cells were transformed according to the protocol described at 'Day 1' and plated onto plates containing carbenicillin, agar, phosphate and ammonium chloride and then incubated at 37 C overnight.

1) Results from day 1 - experiment 2

CML plate with transformed cells ( +DNA sample) resulted in multiple colonies while CML plate with control cells ( NO DNA sample) didnt result in growth, indicating that the cells used for experiment are competent and readily transform.
GLUCOSE plates (both +DNA and NO DNA) showed little growth while SUCROSE and NO SUGAR plates resulted in no growth. Plates were returned for further incubation. Lack of growth may be attributed to low metabolic rate with single sugars as sole carbon source.
2) Shewanella oneidensis MR-1 cell revival

A sample of S. oneidensis MR-1 culture was plated on nutrient agar and incubated at room temperature in order to revive the cells and examine whether the culture is still viable.

3) Magic Dust (fluorescent fusion protein, project unrelated to iGEM 2012) plasmid DNA preparation

Plasmid DNA minipreps were prepared following OpenWetWare minipreps1 protocol by Dr Chris French.

4) Xgal white/blue selection for sucrose hydrolase with arsenate-induced promoter

Sample blue colonies from day 1 Xgal plate were transfered to a fresh Xgal plate. Arsenate was added at the edge of the plate to create a gradient of arenate concentration. White colonies were also plated as control.

Experiment 1: Gel analysis of Magic Dust (fluorescent fusion proteins, project unrelated to iGEM 2012)
Agarose gel was prepared following OpenWetWare AGE protocol by Dr Chris French. Prepared TAE buffer was of 1x strength (as suggested by protocol) rather than 0,5x strength commonly used in the lab; further preparations should use 0,5x strength buffer in order to allow electrophoresis at higher speed. 2 ul of AGE buffer were added to samples 1E,2E,3E,4E and E (10 ul each). The samples were briefly centrifuged (short pulse spin at max speed) in order to mix the content. The samples were then loaded on the gel along with molecular marker and electrophoresis was performed at 100V and 50mA for 30 minutes. Following the electrophoresis, the gel was incubated with SYBR-green stain for 30 minutes. Following the incubation, gel was visualised with blue light. Gel was then destained in order to obtain high quality photo with results that will be posted along with this report at later date.
Experiment 2: Transformation of competent cells and sugar degradation selective marker test
a) Agar plates preparation
At first M9 base agar was prepared by adding 250 ul of trace elements (buffer with Mg and Ca ions) to molten agar (containing phosphate buffer and NH4Cl as nitrogen source). The base agar was then poured into petri dishes to form 12 plates labelled iGEM 19.6.12 M9 base. 6 of these plates were taken for further use and the rest was sealed using parafilm.
Following plates were prepared: 2 NO SUGAR plates - control with no sugars, 2 GLUCOSE plates - plates with 200 mg glucose solubilised in water, 2 SUCROSE plates - 200 mg sucrose solubilised in water, 2 CML plates - with chloramphenicol and 2 XGAL plates for white-blue selection.
b) Competent cells transformation
Samples J7 and J9 (with competent cells prepared on 18.6.12) were takend from the freezer and transfered to ice water to thaw.
15 ul DNA sample were added to sample J9
100 ul of J7 sample were taken into a fresh tube marker control cell
5 ul of LIGN DNA were added to remaining 100 ul of J7 sample.
following 30 minutes ice incubation all three tubes were subjected to heat shock by 90 seconds water bath at 40 degrees C. Following the heat shock, cells were returned to ice for 90 seconds and then incubated in order to let them express resistance to chloramphenicol. It was noted that with non-antibiotic resistance selection, this incubation step will no longer be necessary, expediting the process.
After incubation, LB (from 18.6.12) was added to all three samples to reach 1ml volume.
c) Plating of transformed cells
The samples were plated as follows (using spreader sterilised with alcohol):
100 ul of J9 was plated on plates NO SUGAR DNA+, SUCROSE DNA+, GLUCOSE DNA+ and CML DNA+;
100 ul of control cell sample was plated on plates NO SUGAR NO DNA, SUCROSE NO DNA, GLUCOSE NO DNA and CML NO DNA;
100 ul of J7 was plated on first XGAL plate, then the rest of J7 was centrifuged; the pellet was resuspended in 100 ul LB and plated on second XGAL plate. The plates were left overnight for incubation.