Difference between revisions of "BioE140LSpr09-Silver"

(New page: Silver Binding Assay – Characterization of AG4 composite parts Information Control: pBca9145-Bca1363 Composite Parts: 11: {Pbad.rbs.prepro.StrepTag}{<AG4>}{<CPG_L6!}{dblTerm} 12: {Pba...)
 
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Silver Binding Assay – Characterization of AG4 composite parts
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Silver Binding Assay – Characterization of AG4 composite parts <br>
  
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Information
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Information<br>
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Control: pBca9145-Bca1363
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Control: pBca9145-Bca1363<br>
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Composite Parts:
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Composite Parts: <br>
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11: {Pbad.rbs.prepro.StrepTag}{<AG4>}{<CPG_L6!}{dblTerm}
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11: {Pbad.rbs.prepro.StrepTag}{<AG4>}{<CPG_L6!}{dblTerm}<br>
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12: {Pbad.rbs.prepro.StrepTag}{<AG4>}{<eCPX!}{dblTerm}
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12: {Pbad.rbs.prepro.StrepTag}{<AG4>}{<eCPX!}{dblTerm}<br>
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13: {Pbad.rbs.prepro.StrepTag}{<AG4>}{<upaG_short!}{dblTerm}
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13: {Pbad.rbs.prepro.StrepTag}{<AG4>}{<upaG_short!}{dblTerm}<br>
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14: {Pbad.rbs.prepro.StrepTag}{<AG4>}{<Ag43_short!}{dblTerm}
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14: {Pbad.rbs.prepro.StrepTag}{<AG4>}{<Ag43_short!}{dblTerm}<br>
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15: {Pbad.rbs.prepro.StrepTag}{<AG4>}{<espP(beta)!}{dblTerm}
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15: {Pbad.rbs.prepro.StrepTag}{<AG4>}{<espP(beta)!}{dblTerm}<br>
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16: {Pbad.rbs.prepro.StrepTag}{<AG4>}{<ehaB!]{dblTerm}
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16: {Pbad.rbs.prepro.StrepTag}{<AG4>}{<ehaB!]{dblTerm}<br>
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17: {Pbad.rbs.prepro.StrepTag}{<AG4>}{<CPompX!}{dblTerm}
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17: {Pbad.rbs.prepro.StrepTag}{<AG4>}{<CPompX!}{dblTerm}<br>
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<br>
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LB: ampicillin and kanamycin<br>
  
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LB: ampicillin and kanamycin
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Conditions: pH 7.4 TBS. Ambient light. Constant agitation. Silver Nitrate (AgNO3) does not react with LB or TBS at dilute concentrations.<br>
  
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Conditions: pH 7.4 TBS. Ambient light. Constant agitation. Silver Nitrate (AgNO3) does not react with LB or TBS at dilute concentrations.
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Protocol<br>
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Protocol
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==Pick and Incubate Colonies==
 
==Pick and Incubate Colonies==
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1. Add 4mL of LB media with the appropriate antibiotics and arabinose  
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1. Add 4mL of LB media with the appropriate antibiotics and arabinose <br>
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2. Pick a well-isolated, round, and "normal" looking colony containing the silver binding peptide with a pipet tip
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2. Pick a well-isolated, round, and "normal" looking colony containing the silver binding peptide with a pipette tip<br>
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3. Drop it in the test tube
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3. Drop it in the test tube<br>
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4. Incubate at 37C overnight
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4. Incubate at 37C overnight<br>
 
==Wash cells and incubate in AgNO3==
 
==Wash cells and incubate in AgNO3==
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5. Pellet 1 mL of saturated culture by spinning full speed, 30 seconds.
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5. Pellet 1 mL of saturated culture by spinning full speed, 30 seconds.<br>
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6. Dump supernatant, repeat to pellet another 1 mL (for a total of 2 mL)
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6. Dump supernatant, repeat to pellet another 1 mL (for a total of 2 mL)<br>
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7. Pour out the supernatant (contains extracellular proteins, unneeded nutrients)
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7. Pour out the supernatant (contains extracellular proteins, unneeded nutrients)<br>
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8. Add  200uL of TBS (pH 7.4) and resuspend
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8. Add  200uL of TBS (pH 7.4) and resuspend<br>
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9. Centrifuge the colony solution for 30 seconds
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9. Centrifuge the colony solution for 30 seconds<br>
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10. Pour out the supernatant  
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10. Pour out the supernatant <br>
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11. Add TBS  and 0.1mM AgNO3, and resuspend
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11. Add TBS  and 0.1mM AgNO3, and resuspend<br>
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12. Incubate at room temperature for 24hours
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12. Incubate at room temperature for 24hours<br>
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13. Observe color
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13. Observe color<br>
 
==Measure Absorbance using Spectrophotomer==
 
==Measure Absorbance using Spectrophotomer==
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14. Pellet the cells  
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14. Pellet the cells <br>
 
15. Measure absorbance of supernatant
 
15. Measure absorbance of supernatant

Revision as of 00:56, 11 May 2009

Silver Binding Assay – Characterization of AG4 composite parts

Information
Control: pBca9145-Bca1363
Composite Parts:
11: {Pbad.rbs.prepro.StrepTag}{<AG4>}{<CPG_L6!}{dblTerm}
12: {Pbad.rbs.prepro.StrepTag}{<AG4>}{<eCPX!}{dblTerm}
13: {Pbad.rbs.prepro.StrepTag}{<AG4>}{<upaG_short!}{dblTerm}
14: {Pbad.rbs.prepro.StrepTag}{<AG4>}{<Ag43_short!}{dblTerm}
15: {Pbad.rbs.prepro.StrepTag}{<AG4>}{<espP(beta)!}{dblTerm}
16: {Pbad.rbs.prepro.StrepTag}{<AG4>}{<ehaB!]{dblTerm}
17: {Pbad.rbs.prepro.StrepTag}{<AG4>}{<CPompX!}{dblTerm}

LB: ampicillin and kanamycin

Conditions: pH 7.4 TBS. Ambient light. Constant agitation. Silver Nitrate (AgNO3) does not react with LB or TBS at dilute concentrations.

Protocol

Pick and Incubate Colonies

1. Add 4mL of LB media with the appropriate antibiotics and arabinose
2. Pick a well-isolated, round, and "normal" looking colony containing the silver binding peptide with a pipette tip
3. Drop it in the test tube
4. Incubate at 37C overnight

Wash cells and incubate in AgNO3

5. Pellet 1 mL of saturated culture by spinning full speed, 30 seconds.
6. Dump supernatant, repeat to pellet another 1 mL (for a total of 2 mL)
7. Pour out the supernatant (contains extracellular proteins, unneeded nutrients)
8. Add 200uL of TBS (pH 7.4) and resuspend
9. Centrifuge the colony solution for 30 seconds
10. Pour out the supernatant
11. Add TBS and 0.1mM AgNO3, and resuspend
12. Incubate at room temperature for 24hours
13. Observe color

Measure Absorbance using Spectrophotomer

14. Pellet the cells
15. Measure absorbance of supernatant