User:Puja Mody/Notebook/Chem 571: Gold Nanoparticles/2012/10/24

Transformation, protein expression, lysozyme Uv-Vis

Goals

• Transform PCR product into E.Coli Cells
• Induce ADA protein expression
• Run Uv-Vis of the lysozyme and AuNP solution

Procedure

• The PCR product was transformed by following the Transformation Protocol

The only changes that were made are as follows:

1. Of the two cell solutions made, 200μL of LB media was added to one of the solutions while 200 μL of SOC media was added to the other solution of cells. The two solutions were then shaken at 275 rpm for 1 hr.
2. 25 μL of transformed cells was plated on one LB Petri dish, while 200 μL of transformed cells was plated on the other. For K110A mutation, a total of 4 plates were made. 2 for K110A(f) and 1 for K110A (r).

• ADA protein was expressed in the same manner as on 2012/09/19
  • This morning, Michael came in around 9am to centrifuge the starter culture media and resuspended the pellets in 4mL of fresh LB. These resuspended cells were then redivided into the expression culture media which was then inoculated in the shaker at 160rpm and 37°C. Finally, the IPTG was added at around 10am.
  • 5mL of .4M IPTG solution was made and 1mL was placed into each of the 4 fernbach flasks.
  • .005L x.4M= .002 moles x 238.31g/mol= .477g IPTG in 5mL. Actual= .4754g in 5mL of water.

• The 4 flasks were then shaken for 4 hours.

• For information regarding the Lysozyme please see either Mikes notebook or Marys notebook. The lysozyme did create purple gold nanoparticle fibers upon removal from the oven.