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Team:Berkeley Wetlab/Passenger: Ag4 Peptide
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==Results== | ==Results== | ||
| + | |||
| + | ====Monday:==== | ||
| + | Grew more cells (in presence of arabinose) | ||
| + | |||
| + | ====Tuesday:==== | ||
| + | More incubations. TBS (4/24). fresh AgNO3. varied amount of cells and total volume of solution. <br> | ||
| + | |||
| + | constructs 11-14 (1ml and 2ml) = 0.1mM AgNO3 in 200ul <br> | ||
| + | constructs 15-17 and control (1ml) = 0.1mM AgNO3 in 1000ul <br> | ||
| + | constructs 15-17 and control (2ml) = 1.0mM AgNO3 in 1000ul <br> | ||
| + | |||
| + | incubate for 24 hours | ||
| + | |||
| + | ====Wednesday:==== | ||
| + | results: <br> | ||
| + | |||
| + | [[Image:050609results table.jpg]] <br> | ||
| + | note: the color that appeared were in solution, not in precipitate form. <br> Also, the lack of color from constructs 11-14 might be because of small volume since previous experiment had shown color and precipitate. | ||
| + | |||
| + | |||
| + | [[Image:Control2ml.JPG|250px]] [[Image:15 2ml.JPG|250px]] [[Image:16 2ml.JPG|250px]] [[Image:17 2ml.JPG|250px]] <br> | ||
| + | *2ml control, construct 15, construct 16, construct 17 <br> | ||
| + | |||
| + | |||
| + | [[Image:050609results Tecan1.jpg|700px]] <br> | ||
| + | <font color="darkblue">control</font> <br> | ||
| + | <font color="magenta">construct15</font> <br> | ||
| + | <font color="gold">construct16</font> <br> | ||
| + | <font color="turquoise">construct17</font> <br> | ||
| + | no noticeable peaks or differences between the constructs. perhaps scattering signal is too high because of the presence of cells. | ||
| + | |||
| + | |||
| + | |||
| + | [[Image:Pelletedcontrol.JPG|250px]] [[Image:Pelleted15.JPG|250px]] [[Image:Pelleted16.JPG|250px]] [[Image:Pelleted17.JPG|250px]] <br> | ||
| + | *pelleted 2ml control, construct 15, construct 16, construct 17 <br> | ||
| + | |||
| + | |||
| + | [[Image:050609results Tecan3.jpg|700px]] <br> | ||
| + | <font color="darkblue">control</font> <br> | ||
| + | <font color="magenta">construct15</font> <br> | ||
| + | <font color="gold">construct16</font> <br> | ||
| + | <font color="turquoise">construct17</font> <br> | ||
| + | noticeable increased absorbance around 440nm-500nm. To get rid of the scattering signal, cells were spun down, and this graph was generated using the respective supernatants. | ||
Revision as of 22:04, 18 October 2009
Contents |
Ag4 Peptide
The Ag4 peptide is a small 12 amino acid peptide (NPSSLFRYLPSD). This peptide is has been shown to bind to and reduce silver. The peptide has many interesting applications in areas of biomineralization such as the creation of new nanomaterials. The cell surface display of this silver binding peptide is advantageous because the cells can create a structure from which the silver binding peptides can synthesize and deposit silver to make these novel materials.
Functional Assay: AgNO3 Reduction
This assay tests for the presence of the AG4 silver binding peptide on the E. coli cell surface, and it's ability to bind and reduce silver
constructs: AG4 peptide (8) 1363 negative control (1)
experiment was done in triplicate
Initially verified: AgNO3 does not react with LB or TBS
growing cells
- inoculate cells from stock into LB with the appropriate antibiotics and grow to saturation (12+ hours)
- dilute culture 1:100 into media with arabinose and induce for 5-12hours
- pipet 100ul of cells to Costar V-bottom polystyrene plate and take OD
Wash cells and incubate in AgNO3
- pipet 2mls of culture into a 24-well block
- pellet the saturated induced culture
- pour out the supernatant
- wash cells 2X with 200ul of TBS
Treating with Silver
- make 10mM stock of AgNO3
- add 200uL, 1ml, and 2ml of 0.1 mM silver nitrate and resuspend the cells (add 180 ul of TBS to each pellet, resuspend cells, add 20 ul of 1 mM AgNO3)
- incubate overnight at room temperature
- observe color change and precipitate formation
- take pictures with TEM
Results
Monday:
Grew more cells (in presence of arabinose)
Tuesday:
More incubations. TBS (4/24). fresh AgNO3. varied amount of cells and total volume of solution.
constructs 11-14 (1ml and 2ml) = 0.1mM AgNO3 in 200ul
constructs 15-17 and control (1ml) = 0.1mM AgNO3 in 1000ul
constructs 15-17 and control (2ml) = 1.0mM AgNO3 in 1000ul
incubate for 24 hours
Wednesday:
results:
note: the color that appeared were in solution, not in precipitate form.
Also, the lack of color from constructs 11-14 might be because of small volume since previous experiment had shown color and precipitate.
- 2ml control, construct 15, construct 16, construct 17
700px
control
construct15
construct16
construct17
no noticeable peaks or differences between the constructs. perhaps scattering signal is too high because of the presence of cells.
- pelleted 2ml control, construct 15, construct 16, construct 17
control
construct15
construct16
construct17
noticeable increased absorbance around 440nm-500nm. To get rid of the scattering signal, cells were spun down, and this graph was generated using the respective supernatants.
