User:Puja Mody/Notebook/Chem 571: Gold Nanoparticles/2012/09/18

Day 5

• Protein Expressions and Starters

Goals

• Grow E.Coli cells and have them express ADA protein.
• Follow Protocols for making starter and expression cultures of the cells.

Procedure

• Following the Starter Culture media protocol and the expression culture media protocol, we created LB broth for growing cells. The Starter culture media was used mainly today and was eventually combined with the expression culture media.

• Started Culture Protocol
  • add 35 mL H₂Oto each to 4 250mL erlenmeyer flask
  • LB was added to each flask.
    • Flask 1=.8793g
    • Flask 2=.8800g
    • Flask 3=.8748g
    • Flask 4=.8757g

(note that while there were the amounts measured out- the amount of LB added to the flasks was actually less due to loss of sample in transferring it from the weigh dish to the flask)

• Expression Culture Media
  • 1L of distilled water were added to 4.8L Fernbach flasks
  • LB was added to each flask
    • Flask 1= 25.0289g
    • Flask 2= 25.0069g
    • Flask 3= 25.0020g
    • Flask 4= 25.0036g

(note that while there were the amounts measured out- the amount of LB added to the flasks was actually less due to loss of sample in transferring it from the weigh dish to the flask)

• Next, two of the erlenmeyer flasks and two of the Fernbach flasks were capped with aluminum foil and placed in the Autoclave at a temperature of 121°C for 60minutes followed by a 30 minute cool down period. The autoclave is necessary in order to be sterilized and to ensure that no microbes from the surrounding environment could compete with the bacteria. After the first four samples were removed from the autoclave, the remaining 4 were placed in the autoclave for 50 minutes at the same temperature and were cooled down for 30 minutes as well. The samples had to be autoclaved separately because of lack of room in the autoclave. The autoclave is used in order to kill all other living organisms so that they do not react with the LB because we want to only grow/culture the E.Coli cells.

• While autoclaving the samples, the amount of antibiotic Kanamycin that would be added in was calculated. The stock solution of the antibiotic was 50mg/mL. The desired concentration of kanamycin for the flasks was 50μg/mL.

Using the formula M₁V₁=M₂V₂ The volume or amount of kanamycin that would need to go into each of the flasks was 50 μg/mL x 35mL ÷50,000 μg = .035mL Kanamycin.

• The bacteria was inoculated by retrieving a single colony from the petri dish and dropping it into the flask.
  • After all the starter culture media broths cooled down, they were inoculate with transformed Escherichia coli. Aseptic techniques are implemented to prevent any undesirable microbes from being included in the inoculation of the media.
• E. coli were transformed by the insertion of plasmid encoding for Adenosine Deaminase (ADA).

• Aseptic techniques were applied by executing the inoculation process near the proximity of a Bunsen burner. The aluminum foil tops of the Erlenmeyer containing the broths were flamed in between uncapping and capping. The metal tweezers were flamed before and after use. Sterile wood sticks were used to capture a colony of the E.Coli

• The starter culture media flasks were then placed in a shaker incubator at 37°C at 237rpm overnight.

Preparation of Tris Buffer

• pH 10- this buffer would be used with the AuNP/BSA made at a concentration of 70 last week

the desired concentration of the stock was .1 M tris and a total of 25mL of stock was made.

1 mol/ 1L x .025 L= .025 mol

.025 mol x 121.14 grams/1 mol = 3.0285 grams of tris in 25mL of water

* in actuality, 3.080 grams of tris was added. 

Calculations for amount of water and tris needed to achieve desired concentrations:

(The tris and water were not combined with the AuNP/BSa solutions today)