Team:Berkeley Wetlab/Passenger: Ag4 Peptide

From 2009.igem.org

(Difference between revisions)
 
(34 intermediate revisions not shown)
Line 1: Line 1:
-
==AgNO3 Reduction==
+
{{newtemplateBerkeley}}
 +
__NOTOC__
 +
==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'''<br>
 +
 
 +
[[Image:Ag4 peptide illustration.jpg|center]]<br>
 +
 
 +
'''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.
-
'''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:'''
'''constructs:'''
AG4 peptide (8)
AG4 peptide (8)
1363 negative control (1)
1363 negative control (1)
-
 
-
''experiment was done in triplicate''
 
''Initially verified: AgNO3 does not react with LB or TBS''
''Initially verified: AgNO3 does not react with LB or TBS''
-
===growing cells===
+
====growing cells====
#inoculate cells from stock into LB with the appropriate antibiotics and grow to saturation (12+ hours)
#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  
#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  
+
#pipet 100ul of cells to Costar V-bottom polystyrene plate and take OD  
====Wash cells and incubate in AgNO3====
====Wash cells and incubate in AgNO3====
#pipet 2mls of culture into a 24-well block
#pipet 2mls of culture into a 24-well block
-
#Pellet the saturated induced culture  
+
#pellet the saturated induced culture  
-
#Pour out the supernatant  
+
#pour out the supernatant  
-
#wash cells with 200ul of TBS 2X
+
#wash cells 2X with 200ul of TBS  
====Treating with Silver====
====Treating with Silver====
-
#Make 10mM stock of AgNO3.
+
#make 10mM stock of AgNO3
-
#Add 200uL of 0.1 mM  silver nitrate and resuspend the cells
+
#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 (24-48 hours) at room temperature
+
#incubate overnight at room temperature
-
#observe color change and precipitation of colored compound
+
#observe color change and precipitate formation
-
#Perform same procedure but vary concentration of AgNO3, cell density, and presence of arabinose (see group samples)
+
#take pictures with TEM
-
 
+
-
===Control Group - non-AG4 cells===
+
-
*note: do controls for each of the sample groups
+
-
 
+
-
====Picking and Incubating Colonies====
+
-
*Add 4mL of LB media with the appropriate antibiotics to a clean test tube
+
-
*Pick a well-isolated, round, and "normal" looking colony of control E coli with no silver binding peptide with a toothpick
+
-
*Drop it in the test tube
+
-
*Incubate at 37 overnight
+
-
 
+
-
====Cleaning====
+
-
#Pellet 1 mL of saturated culture by spinning full speed, 30 seconds.
+
-
#Dump supernatant, repeat to pellet another 1 mL (for a total of 3 mL)
+
-
#Pour out the supernatant (contains extracellular proteins, unneeded nutrients)
+
-
#Add  <font color="darkturquoise">1 mL </font>of TBS (pH 7.4) and resuspend
+
-
#Centrifuge the colony solution for 30 seconds
+
-
#Pour out the supernatant
+
-
#Add  200uL of TBS and resuspend
+
-
#Dilute <font color="darkturquoise">an aliquot of </font>the solution 10X. Measure OD of solution using spectrophotometer (at 600nm)
+
-
 
+
-
====Treating with Silver====
+
-
#Make 10mM stock of AgNO3
+
-
#Add  200uL of 0.1 mM silver nitrate and resuspend the cells  <font color="darkturquoise">JCA:  Ammend this step.  I think you want to say add 2uL of 10mM AgNO3 stock to 200uL of washed cells and vortext to mix (or something like that). </font>
+
-
#Incubate overnight (24-48 hours) at room temperature
+
-
#observe color change and precipitation of colored compound
+
-
#Perform same procedure but vary concentration of AgNO3, cell density, and presence of arabinose (see group samples)
+
-
 
+
-
====other controls====
+
-
# Chemically reduce silver nitrate to precipitate silver (tells us silver nitrate is precipitating)
+
-
# Lyse cells over-expressing AG4 and run assay (tells us if AG4 is precipitating silver)
+
-
# Wash non-AG4 cells with silver nitrate and look for colour change (tells us if precipitation occurs without AG4)<font color="darkturquoise">
+
-
# Try growing any functional clones in cultures containing the AgNO3</font>
+
-
 
+
-
 
+
-
== Procedure carried out on 22 April 2009 ==
+
-
 
+
-
 
+
-
==== Preparation of Tris buffer solution ====
+
-
* Add 605 mg Tris and 876 mg NaCl to 80 ml H2O
+
[https://2009.igem.org/Recipes TBS recipe]
-
* Adjust pH to 7.4 by adding HCl and bring volume to 100 ml
+
-
==== Preparation of AgNO3 solution ====
+
==Results==
 +
[[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 volumes since previous experiment had shown color and precipitate.
-
* Weigh AgNO3 powder in an Eppendorf tube (weighed 0.013 grams)
+
11 - {<CPG_L6!}<br>
-
* AgNO3 has MW 169.87 g/mol - dilute 0.013g with 0.750ml for ~10 mM solution
+
12 - {<eCPX!}<br>
-
* Make 3ml of 1mM solutions by adding 100ul of 10mM AgNO3 into 900ul of H2O
+
13 - {<upaG_short!}<br>
 +
14 - {<Ag43_short!}<br>
 +
15 - {<espP(beta)!}<br>
 +
16 - {<ehaB!]<br>
 +
17 - {<CPompX!}<br>
-
==== Preparation of cells for assay ====
 
-
* Spin down 2ml of cells and throw away supernatant
+
[[Image:Control2ml.JPG|200px]] [[Image:15 2ml.JPG|200px]] [[Image:16 2ml.JPG|200px]] [[Image:17 2ml.JPG|200px]] <br>
-
* Add 200 ul of TBS and resuspend cells
+
negative control 1363, espP(beta) (15), ehaB (16), CPompX (17) <br>
-
* Spin down the cells and throw away supernatant
+
-
==== Preparing cells for growth in LBTAg media ====
 
-
* Dispense 3ml of LB media into 9 chambers in growth rack
+
[[Image:050609results Tecan3.jpg|700px]] <br>
-
* Add 200 ul of Tris buffer solution to the LB media
+
<font color="darkblue">control</font> <br>
-
* Add 32 ul of 10 mM AgNO3 to make 0.1 mM LBTAg media
+
<font color="magenta">espP(beta)</font> <br>
-
* Pick cells from each washed pellet (above) and add to corresponding growth chambers
+
<font color="gold">ehaB</font> <br>
-
** 11-17 + Control + another one of 11
+
<font color="turquoise">CPompX</font> <br>
-
* Add 3ul of 1000x Arabinose to the 9th chamber containing a second culture of cells 11
+
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.
-
* Cover the rack and incubate overnight at 37C
+
-
==== Running the assay on cells ====
 
-
* Add 180 ul of TBS to each pellet (prepared above)
+
===References===
-
* Resuspend cells
+
Rajesh, R. et al. Biomimetic synthesis and patterning of silver nanoparticles. Nature Materials. October 2002; (1): 169 - 172. Available Online: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2620822/ (Accessed: 20 October 2009).
-
* Add 20 ul of 1 mM AgNO3 to each TBS/cell-containing tube
+
-
* Incubate overnight at room temperature and with agitation
+

Latest revision as of 02:42, 22 October 2009

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

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!}


Control2ml.JPG 15 2ml.JPG 16 2ml.JPG 17 2ml.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.


References

Rajesh, R. et al. Biomimetic synthesis and patterning of silver nanoparticles. Nature Materials. October 2002; (1): 169 - 172. Available Online: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2620822/ (Accessed: 20 October 2009).

Retrieved from "http://2009.igem.org/Team:Berkeley_Wetlab/Passenger:_Ag4_Peptide"