Template:BioE140LSpr09-Silver

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Silver Binding Assay

Silver Binding Assay – Characterization of AG4 composite parts

Information

Control: pBca9145-Bca1363

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

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 pipet 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.The supernatant was tested for absorbance using spectroscopy on a TECAN set to measure the wavelength range 350nm-750nm at 10nm intervals. Silverbinding.jpg