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From 2010.igem.org

General description of goal of this part of the project.

Contents 1 Oct 06, 2010; Making stock solutions 2 Oct 07, 2010; Bacterial growth assays, Cu ppt 3 Oct 12, 2010; Generating a standard curve, troubleshooting 3.1 Conclusion 4 Useful links

Oct 06, 2010; Making stock solutions

Generated the stock solutions for the Followed the standard curve protocol.

• Made a 100 mM stock solution of BCS.
• Made two CuSO4 dilution series, in LB and MM, from 10 mM Cu to 10 pM.

Oct 07, 2010; Bacterial growth assays, Cu ppt

#	Sample ID	Date and Time	1 (nm)	1 (Abs)
1	o/n culture	10/7/2010 11:16:10 AM	600	0.854
2	MM t0	10/7/2010 11:44:45 AM	600	0.002
3	LB t0	10/7/2010 11:31:06 AM	600	0.089
4	LB t1	10/7/2010 12:47:55 PM	600	0.189
5	MM t1	10/7/2010 12:45:13 PM	600	0.002
6	MM t2	10/7/2010 1:41:51 PM	600	0.004
7	LB t2	10/7/2010 1:43:38 PM	600	0.450
8	MM t3	10/7/2010 2:44:50 PM	600	0.001
9	LB t3	10/7/2010 2:46:09 PM	600	0.761
10	MM t4	10/7/2010 3:53:12 PM	600	0.005
11	LB t4	10/7/2010 3:54:36 PM	600	1.064

Oct 12, 2010; Generating a standard curve, troubleshooting

Checked Abs of a dilution series of CuSO4, from 100 mM to 1 nM, in LB.

To a cuvette, added:

1. 800 uL H2O
2. 200 uL LB + Cu
3. 2 uL 100 mM BCS

{insert data}

Saw no change between samples.

Revisited literature to see what we're missing.

Lebeau et al, 2000 used this method:

• Measurement of Cu(II) reduction. Seven hundred seventy μl of BDA stock solution (5.7 mM) BDA in 5.2 M ammonium acetate/10% ethanol was mixed with 720 μL of CuSO4 solution (1.25 mM) in water and 10 μL of test compounds (15 mM) except quercetin (1.5 mM) dissolved in ethanol [37]. The complex formation (BDA/Cu(I)) was followed by optical density measurements made at 484 nm. The amount of reduced copper was determined from a calibration curve constructed using solutions of Cu(II) (0–300 μM) reduced by ascorbate (1 mM) in the presence of BDA (2.7 mM). The percent of ethanol in water for the dosage does not exceed 5.6%.

Campos et al 2009 used this method:

• Different concentrations of copper sulfate (0.05–0.8 mM) and BCS (0.1–1.6 mM) were investigated by altering one variable at a time to optimize the assay. Final concentrations of 0.2 mM BCS and 0.1 mM CuSO4 gave enough absorption intensity at 490 nm with good linearity up to 2 mM trolox, as is shown below; therefore, they were established as optimal for the assay. EDTA (1.67 mM final concentration) is used as a stop reagent. When EDTA is added at this concentration before the sample, no color is developed (Fig. 1). On the other hand, we noticed that when a concentration of EDTA higher than 1.67 mM is used, a precipitate could appear in plasma samples.

Rapisarda et al used this method:

• Fig. 1. Fluorescence properties of BCS. (A) Excitation (solid line) and emission (dotted line) spectra of 1 μM BCS in 50 mM phosphate buffer, pH 7.5, with λem=770 nm and λex=580 nm, respectively. (B) Quenching of the BCS emission at 770 nm (λex=580 nm) by increasing concentrations of Cu(I) added as CuCl2 in the presence of 10 μM ascorbate. BCS concentration: 1 μM (closed circles) or 4 μM (open circles).

Conclusion

The BCS reagent only binds Cu(I). To convert our Cu(II)SO4 to Cu(I), add 1 mM ascorbate (Make 100 mM stock, dilute 100X). Hopefully this won't change the pH to outside the effective range of the reagent. If it does, add 1 M Tris pH to 50 mM final concentration (50X dilution). Dan has powdered ascorbate and a stock solution of Tris.

Useful links

LabNotebook

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