Difference between revisions of "Part:BBa K197023"

 
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<partinfo>BBa_K197023 short</partinfo>
 
<partinfo>BBa_K197023 short</partinfo>
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==Ag4 Peptide==
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Various peptides have been previously shown to mediate inorganic crystal growth and shape. It is hypothesized that these peptides interact with small metal clusters in solution to produce a chemically reducing environment for ion reduction. Moreover, peptide interactions lowers the surface energy of formed crystals to accelerate cluster formation.
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The Ag4 peptide is a small 12 amino acid peptide (NPSSLFRYLPSD) demonstrated to bind and reduce silver. The surface display of this peptide has the potential of inorganic material synthesis via mimicking the recognition and nucleation properties found in natural biomineralization processes. In cell surface display, there is the additional advantage of self-replicating cells that serve as both a continual factory of silver particle synthesis and the scaffold that supplies a matrix for the synthesized materials. 
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==Functional Assay: AgNO3 Reduction==
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'''This assay tests for the presence of the AG4 silver binding peptide on the E. coli cell surface, and its ability to bind and reduce silver'''<br>
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https://static.igem.org/mediawiki/2009/b/bb/Ag4_peptide_illustration.jpg<br>
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'''basic theory behind experiment''' -
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Incubating the cells expressing silver nitrate allows for reduction of the silver ions to silver. The reddish colorimetric change observed is the result of a size-dependent characteristic surface plasmon resonance band, which has an absorption at approximately 440nm. This phenotype is readily visible for qualitative analysis.
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'''constructs:'''
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AG4 peptide (8)
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1363 negative control (1)
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''Initially verified: AgNO3 does not react with LB or TBS''
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====growing cells====
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#inoculate cells from stock into LB with the appropriate antibiotics and grow to saturation (12+ hours)
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#dilute culture 1:100 into media with arabinose and induce for 5-12hours
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#pipet 100ul of cells to Costar V-bottom polystyrene plate and take OD
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====Wash cells and incubate in AgNO3====
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#pipet 2mls of culture into a 24-well block
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#pellet the saturated induced culture
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#pour out the supernatant
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#wash cells 2X with 200ul of TBS
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====Treating with Silver====
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#make 10mM stock of AgNO3
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#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)
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#incubate overnight at room temperature
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#observe color change and precipitate formation
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#take pictures with TEM
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[http://2009.igem.org/Recipes TBS recipe]
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==Results==
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https://static.igem.org/mediawiki/2009/4/45/050609results_table.jpg <br>
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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 volumes since previous experiment had shown color and precipitate.
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11 - {<CPG_L6!}<br>
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12 - {<eCPX!}<br>
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13 - {<upaG_short!}<br>
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14 - {<Ag43_short!}<br>
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15 - {<espP(beta)!}<br>
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16 - {<ehaB!]<br>
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17 - {<CPompX!}<br>
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[[Image:Negative control.jpg|200px]] [[Image:espP.jpg]] [[Image:ehaB.jpg]] [[Image:CPompX.jpg]]<br>
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negative control 1363, espP(beta) (15), ehaB (16), CPompX (17) <br>
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https://static.igem.org/mediawiki/2009/3/38/050609results_Tecan3.jpg <br>
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<font color="darkblue">control</font> <br>
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<font color="magenta">espP(beta)</font> <br>
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<font color="gold">ehaB</font> <br>
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<font color="turquoise">CPompX</font> <br>
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Absorbance around 440nm-500nm was observed most noticeably for espP(beta). To get rid of the scattering signal, cells were spun down, and this graph was generated using the respective supernatants. The formation of silver particles gives a characteristic surface plasmon resonsance around 440nm - 500nm. The visible colorimetric changes are confirmed by the UV-Spec readings.
  
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The Silver Binding peptide binds and reduces silver for biomineralization purposes.
 
  
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

Latest revision as of 04:46, 22 October 2009

Ag4

Ag4 Peptide

Various peptides have been previously shown to mediate inorganic crystal growth and shape. It is hypothesized that these peptides interact with small metal clusters in solution to produce a chemically reducing environment for ion reduction. Moreover, peptide interactions lowers the surface energy of formed crystals to accelerate cluster formation.

The Ag4 peptide is a small 12 amino acid peptide (NPSSLFRYLPSD) demonstrated to bind and reduce silver. The surface display of this peptide has the potential of inorganic material synthesis via mimicking the recognition and nucleation properties found in natural biomineralization processes. In cell surface display, there is the additional advantage of self-replicating cells that serve as both a continual factory of silver particle synthesis and the scaffold that supplies a matrix for the synthesized materials.


Functional Assay: AgNO3 Reduction

This assay tests for the presence of the AG4 silver binding peptide on the E. coli cell surface, and its ability to bind and reduce silver

Ag4_peptide_illustration.jpg

basic theory behind experiment - Incubating the cells expressing silver nitrate allows for reduction of the silver ions to silver. The reddish colorimetric change observed is the result of a size-dependent characteristic surface plasmon resonance band, which has an absorption at approximately 440nm. This phenotype is readily visible for qualitative analysis.


constructs: AG4 peptide (8) 1363 negative control (1)

Initially verified: AgNO3 does not react with LB or TBS

growing cells

  1. inoculate cells from stock into LB with the appropriate antibiotics and grow to saturation (12+ hours)
  2. dilute culture 1:100 into media with arabinose and induce for 5-12hours
  3. pipet 100ul of cells to Costar V-bottom polystyrene plate and take OD

Wash cells and incubate in AgNO3

  1. pipet 2mls of culture into a 24-well block
  2. pellet the saturated induced culture
  3. pour out the supernatant
  4. wash cells 2X with 200ul of TBS

Treating with Silver

  1. make 10mM stock of AgNO3
  2. 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)
  3. incubate overnight at room temperature
  4. observe color change and precipitate formation
  5. take pictures with TEM

[http://2009.igem.org/Recipes TBS recipe]

Results

050609results_table.jpg
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 volumes since previous experiment had shown color and precipitate.

11 - {<CPG_L6!}
12 - {<eCPX!}
13 - {<upaG_short!}
14 - {<Ag43_short!}
15 - {<espP(beta)!}
16 - {<ehaB!]
17 - {<CPompX!}

Negative control.jpg EspP.jpg EhaB.jpg CPompX.jpg
negative control 1363, espP(beta) (15), ehaB (16), CPompX (17)


050609results_Tecan3.jpg
control
espP(beta)
ehaB
CPompX
Absorbance around 440nm-500nm was observed most noticeably for espP(beta). To get rid of the scattering signal, cells were spun down, and this graph was generated using the respective supernatants. The formation of silver particles gives a characteristic surface plasmon resonsance around 440nm - 500nm. The visible colorimetric changes are confirmed by the UV-Spec readings.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]