BioE140LSpr09-Silver

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 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