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Team:Columbia-Cooper-NYC/Columbia notebook 1
From 2012.igem.org
Contents |
Columbia Copper Experiment Notebook
June
Friday, June 22nd
Outline of activity today:
- Met with Jason, who showed us how to make A. ferrooxidans liquid media, prepared Copper 1 and 2 samples (with and without bacteria) and placed on shaker. Will be periodically checked until June 25 twice a day for activity.
- Showed us recipe for solid media, and made enough for two samples, 20 mL (one control with no bacteria). Copper samples were submerged in agarose (see Steven’s pictures). Also learned protocols listed below.
Starting Sample masses:
| Sample | Mass (g) |
|---|---|
| 1 | 0.0092 |
| 2 | 0.0107. |
| Sample | Mass (g) |
|---|---|
| 1 | 0.1318 |
| 2 | 0.1286. |
| Sample | Mass (g) |
|---|---|
| 1 | 0.1272 |
| 2 | 0.1282. |
Liquid media and flasks were placed in shaker at 11:35 am, 225 rpm, 30C.
Solid media and petri dishes were placed in incubator at 12:42 pm, 30C.
Saturday, June 23
- Checked the progress of all copper samples that included Copper 2 samples in solid media with and without bacteria, Copper 1 and 2 samples in liquid media each containing two samples of with and without bacteria.
- Observations were made for all samples. Optical density was measured for all of the samples submerged in liquid basal AFM low pH solution. Mass was weighed only copper 2 in liquid media with and without bacteria.
- Observations: Solid media mostly clear except for the outer edges of the Copper which appeared to be a light golden yellow color (note picture below). Copper turned to a dark brown color. No observable size change to the copper.
- Observations: Solid media turned from clear to a light golden yellow color (similar to that of the outer edges of the Copper from sample 1 mentioned above). Copper turned to a dark brown color. No observable size change to the copper.
- Observations: Noticeable 7+ small pieces of copper sample within liquid media (note picture below). Conclude to not take mass for this sample.
- Cuvette reading2: 0.003 A
- Current mass: N/A, Previous mass: 0.0092g, Initial mass: 0.0092g
- % Mass Lost: N/A
- Observations: No significant copper pieces visible in liquid media (note picture). Conclude to not take mass for this sample.
- Cuvette reading: 0.004 A
- Current mass reading: N/A, Previous mass: 0.0107g, Initial mass: 0.0107g
- % Mass lost: N/A
- Observations: No observable change (see photo below).
- Cuvette reading: .004A
- Current mass: 0.1063g, Previous mass: 0.1318g, Initial mass: 0.1318g
- % Mass Lost: 19.35%
- Observations: No observable change to copper (see picture below).
- Cuvette reading: 0.040 A**
- Cuvette reading: 0.018 A***
- Current mass reading: 0.0953g, Previous mass: 0.1286g, Initial mass: 0.1286g*
- % Mass lost: 25.89%
Sunday, June 24
- Took out all four samples for standard check of OD and mass of copper 2 samples. Solid media was also examined. All samples (liquid and solid) were photographed.
- No visible copper 1 samples present in either sample (see Observations)
- Observations: A tint of green color on copper surface, the immediate radius of the copper has brown color, outside of the previously mentioned radius is a yellow tinge to the solid media. The rest of the solid media was clear. No observable size change.
- Observations: A darker more present shade of green on copper, compared to that of sample 1 mentioned above. Outer edges of the copper appeared black. Media was a uniformed amber color. No observable size change.
- Observations: Liquid media copper brown, that may include ions that are not copper. No visible copper pieces, and conclude to not take mass for this sample. During procedure, centrifuged for another 1 minute to original 2 minutes at sane speed (due to the lack of expected visible pellets of some kind*)
- Cuvette reading1: 0.025 A
- Current mass: N/A, Previous mass: N/A, Initial mass: 0.0092g
- % Mass Lost: N/A
- Observations: Slight light green color to the media, and media appeared slightly cloudy. No significant copper pieces visible in liquid media. Conclude to not take mass for this sample.
- Cuvette reading: 0.033 A
- Current mass reading: N/A, Previous mass: N/A, Initial mass: 0.0107g
- % Mass lost: N/A
- Observations: Visible brown copper color to liquid media. Pellets formed when the media was centrifuged. Many fluctuations in mass was seen, possible sources of error maybe water droplets or cleanness of the tweezers.
- Cuvette reading: .667A
- Current mass 1: 0.0550g, Current mass 2: 0.0388g, Current mass 3: 0.0285g
- Previous mass: 0.1063g, Initial mass: 0.1318g
- % Mass Lost 1: 48.26%, % Mass Lost 2: 63.50%, % Mass Lost 3: 73.19%
- Observations: Copper is visible in faint blue-green liquid media. No observable change to copper. Observable pellets after centrifuging (Unknown origin). Note the varied measurements in the masses, although not of a significant drop as in Copper 2 liquid media sample 1.
- Cuvette reading: 0.040 A
- Current mass reading1: 0.0907g, Current mass reading2: .0842g
- Previous mass: 0.0953g, Initial mass: 0.1286g
- % Mass lost 1: 4.83%, % Mass lost 2: 11.65%
Monday, June 25
- Took out all four samples for standard check of OD and mass of copper samples. Solid media was also examined. All samples (liquid and solid) were photographed.
- Only sample 2 of copper 2 in liquid media was able to be measured for the other samples were either etched by bacteria or physically torn apart by shaking or by the AMF media.
Copper 2- Sample 1 (without bacteria)
Removal: 9:51, Return: 9:55
Observations: Green copper sample on surface. No clear media visible. (see photo below).
Copper 2- Sample 2 (with bacteria) Removal: 9:51, Return: 9:55 Observations: A light shade of green on copper. Outer edges of the copper appeared black. Media was a uniformed amber color. No observable size change. (see photo below).
Note: One or both of the solid media samples dried up, therefore requiring another set of experiments.
Copper 2- Sample 1 (with bacteria)
Removal1: 9:56, Return1: 10:23, Removal2: 10:41, Return2: 10:54*
Observations: Visible brown and murky copper color to liquid media. Pellets formed when the media was centrifuged. Copper appeared to turn into little powder, pellet form, and conclude mass is difficult to measure and may alter data. (see photo below).
Cuvette reading1: .034A, Cuvette reading2: .057A*
Today’s mass (fourth day): N/A
Third day’s mass1: 0.0550g, Third day’s mass2: 0.0388g, Third day’s mass3: 0.0285g
Second day’s mass: 0.1063g, Initial mass: 0.1318g
% Mass Lost: N/A (All of the copper was etched)
- Note: Since the spectrophotometer reading was significantly lower than that of yesterday (.667 A), the sample’s OD was read twice
Copper 2- Sample 2 (without bacteria) Removal: 10:24, Return: 10:58 Observations: Copper is visible in muddy but faint blue-green liquid media. No observable change to copper (see picture below). Observable pellets after centrifuging (Unknown origin again like yesterday). Note the varied measurements in the masses, although not of a significant drop as in Copper 2 liquid media sample 1 which turned to nothing. Cuvette reading: 0.118 A Current (fourth day) mass1: .0400g Current (fourth day) mass2: .0324g Third day’s mass reading1: 0.0907g, Third day’s mass reading2: .0842g Second day’s mass: 0.0953g, Initial mass: 0.1286g
- %Mass change1: 55.90%, % Mass change2: 64.28%
- %Mass change1: 52.49%, % Mass change2: 61.52%
- Note: Based on first reading from yesterday
- Note: Based on second reading from yesterday
Copper 1- Sample 1 (with bacteria) Removal1: 10:55, Return: 11:04 Observations: Liquid media copper brown, that may include ions that are not copper (note picture below). Some visible tiny fragments of original sample, but conclude to not take mass for this sample. After centrifuging there were visible pellets (bacteria). Cuvette reading1: 0.094 A Current mass: N/A, Previous mass: N/A, Initial mass: 0.0092g % Mass Lost: N/A
Copper 1- Sample 2 (without bateria) Removal: 10:24, Return: 11:19 Observations: Slight light green color to the media, and media appeared slightly cloudy. No significant copper pieces visible in liquid media (note picture). Conclude to not take mass for this sample. Cuvette reading: 0.022 A Current mass reading: N/A, Previous mass: N/A, Initial mass: 0.0107g % Mass lost: N/A
July
August
July 29 - August 4
After a failed attempt at setting up solid media copper etching experiments to test for control and efficiency, we were able to correct our mistakes by adjusting for the firmness of our solid media solutions and proceed with experimental setup.
July 31
A. ferroxidans were left to grow overnight and Solution C (pH 2.35 and 2 w% iron, or ~10g/500 mL) was made.Aug 2
The A. ferroxidans pellets were separated from its supernatant using a centrifuge. Solution D, an agar solution necessary for making the solid media, was made to have a concentration of 1%. The following experiments were set up twice in solid media (unless otherwise stated)--one dish for each experiment contained cells, and one dish did not:
- One 5x5 cm copper piece, with cardstock placed on top of the solid media
- Four 2x2 cm copper pieces with nail polish (one fully painted on both sides, one half painted on both sides, one half painted on one side, and one not painted) with cells on each copper for the petri dish containing cells
- Four 2x2 cm in 15 mL of solid media with 15 mL of AFM on top
- Solid media enclosing copper in liquid media in shaker
Aug 3
The agar concentration was found to be too low for the experiments set up on 8/2, as the solid media did not solidify overnight. The experiments were scrapped and restarted with solid media of a higher agar concentration (4%).Aug 4
4% agar was a more successful alternative. The experiments listed above for August 2nd were set up once again.
August 5 - August 11
Observations were made of the experiments set up the past week. Firmer solid media, greater % of agar in solution, may have also contributed to the reduced oxidation we saw in the copper pieces. The bacteria in most of our setups were also consistently etching more copper in the same period of time compared to the dishes that did not contain bacteria.
Aug 5
The copper pieces were massed and more experiments were set up.
Aug 7
Observations were taken of the flasks filled with liquid media containing the copper piece in a solid media "shell." The solution in flasks containing no bacteria appeared unchanged. Copper in solid media in these flasks appeared darker compared to Day 1. In the flasks containing bacteria, the solutions turned a deep amber color. The solid media was still intact. The flask labelled Bac 1 developed a yellow residue and its solution was more opaque than flasks Bac 2 and 3. The yellow residue also seemed to be coating solid media.
Aug 8
The copper pieces in the flasks were massed and it was determined that the average mass of copper etched away by the flask containing copper was greater than the average mass of copper etched in the case where there was no bacteria (11% mass loss versus 1%, respectively). Biological etching was considered a success.
Aug 9
Observations were made of the petri dish setups.
- Big Copper with Cardstock:
- w/ bacteria: bacteria seems to promote oxidation of Cu (Copper turned green) w/o bacteria: copper turned a very dark brown. solid media in bac and no bac have turned yellow.
- Nail Polished Copper:
no visible etching seem through layer of nail polish for both bacteria and no bacteria. - etching where Cu is exposed is still occurring.
- Solid and Liquid Media:
- w/ bacteria: liquid media turned orange-brown and there is some oxidation on copper pieces - w/o bac: copper pieces not shiny anymore and turned dark brown. Liquid media is still transparent.
Aug 10
The copper pieces were removed from all the petri dish experiments. It was determined that more copper was etched from the petri dishes containing bacteria in all cases.
