Team:Edinburgh/Notebook

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.