To synthesize AuNP using the procedure developed here:
Bakshi, Mandeep S.; Kaur, Harpreet; Khullar, Poonam, Banipal, Tarlok, S.; Kaur, Gurinder; Singh, Narpinder Journal of Physical Chemistry C 2011, 115(7), 2982-2992
In addition, to repeat the experiment performed on 08/31/11 and 09/07/11.
To begin culturing bacteria to express inteins.
To transform DNA coding for mutant GFP (mutated on 09/20/11) into Novablue cells.
- Combine 10 mL of 50 mM acetate buffer (pH 5.46) with 0.974 mL of 0.0015 mM BSA and 0.581 mL of 0.25 mM HAuCl4 in a capped test tube at room temperature.
- Take UV-vis spectra (200 nm to 800 nm) of a sample of reaction mixture at 70°C every 30 min.
- Place reaction mixture in an oven at 80°C for 2.5 hr under thermostatic conditions.
- After 2.5 hr, remove from oven and cool to room temperature; leave sitting overnight.
Starter Culture Media, Protein Expression
- Make expression culture media.
- Prepare LB in a 250 mL Erlenmeyer flask.
- 0.875 g of LB
- 35 mL of water
- Cover the flask with foil.
- Allow flask to cool.
- Add 35 μL of 1000× ampicillin.
- Inoculate with the bacteria you are culturing by scraping frozen bacteria/glycerol mixture and adding to flask.
- Place the flask in a shaker/incubator and culture overnight at 37°C and 200 rpm.
- Digest non-methylated DNA with 1 μL Dpnl.
- Make LB/agar plates with ampicillin.
- Place sterile tube and DNA in ice bucket for 15 min.
- Add 5 μL of DNA and 40 μL of cells to the bottom of the tube.
- Incubate on ice for 30 min.
- Heat shock DNA/cells at 42°C for 30 s.
- Incubate on ice for 5 min.
- Add 250 μL of SOC media.
- Incubate in shaker at 37°C for 1 hr.
- Spread 100 μL of cells on LB/agar plate.
- Store plate (inverted) in 37°C oven overnight.
The absorbance values for the AuNP solution were subtracted from the absorbance values for a blank acetate buffer to create a set of corrected absorbance values. The corrected absorbance values at 550 nm were graphed against time. The graph shows a positive linear relationship, indicating that AuNPs were formed in solution.
In addition, a second graph showing corrected absorbance against wavelength was created, to show the absorbance levels across all of the wavelengths measured by the UV-vis.
A magnified version of this graph shows that absorbance values at around 550 nm increased over time, indicating the creation of AuNPs.
Plate did not appear to be successful; no cells grew.
The AuNP solution turned from clear to purple while in the oven, indicating that AuNPs were actually formed.