User:Mary Mendoza/Notebook/CHEM 571 Experimental Biological Chemistry I/2012/09/18

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Preparation of LB Broth

  • Following the protocols, starter culture media and expression culture media, the LB (1.0% Tryptone, 0.5% yeast extract, and 1.0% sodium chloride) broth were prepared in the beginnning of laboratory period to allot time for autoclave period.
  • For the starter culture media, four measurements of 0.875 g of yellow, granular powder of LB broth were weighed to be dissolved in four volumes of 25 mL of water. However, not exact weights were acquired. The different weights are listed below in the table. The starter culture media were contained in 250 mL Erlenmeyer flasks.
  • The expression culture media requires 25 g of LB broth to be dissolved in 1 L of water. Not exact measurements were made and the actual amount dissolved in water are listed below in the table. The expression culture media were contained in 1 L Fernbach flasks.
  • After the dissolution of the yellow, granular powder of LB broth powder in water, the broths were capped with aluminum foil.
  • The broths must be sterilized through an autoclave. This is to ensure that there are no other microbes from the surrounding environment that would compete with the desired bacteria of which the media will be inoculated with. Boiling the broths can sanitize the solution. However, the pressure inside the autoclave is preferable to bring the broths into a sterilized state.
  • The parameters of the autoclave were set to 121°C. Time is dependent whether the autoclave has been used beforehand or not. If the autoclave has not been used prior to the procedure, as indicated on the instruction of the balance room, time is set to 60 min. If it has been used, time is set to 50 min. or less dependent on the requirements of the procedure.
  • There is limited amount of space in the autoclave. Therefore, the LB broths were autoclaved in two batches. Two of the starter culture media (broths 1 and 2) and two of the expression culture media (broths 1 and 2) were autoclaved at 1:05 PM. This batch was autoclaved for 60 min. as specified by the the instructions posted on the balance room.
  • In addition to the 60 min. given for the sterilization of the LB broths in the autoclave, 30 min. is allotted to allow the pressure to decrease back to zero.
  • Before the removal of broths from the autoclave, the psi meter was verified to be at zero. At 2:38 PM, batch 1 was taken out of the autoclave and were replaced with broths 3 and 4 to autoclave both the starter culture and expression media. The time set for this batch was 50 min.
broth number weight of LB Broth dissolved in 35 mL of water (g) weight of LB Broth dissolved in 1 L of water (g)

Inoculation of Starter Culture Media

  • At 3:58 PM, batch 2 (consisting broths 3 and 4 of both starter and expression culture media) was taken out from the autoclave. The solutions were allowed to cool. Broths 1 and 2 of the starter culture media were already cool at this point while broths 1 and 2 of expression culture media are still above room temperature.
  • While waiting for the autoclave, calculations were made to verify the amount of antibiotic, Kanamycin, to be added to the media. The antibiotic used was prepared by faculty, Dr. Miller, on 09/17/2012. The antibiotic will eradicate any other undesirable microbes that will out compete the transformed bacteria. The transformed bacteria is resistant to Kanamycin.

1. Converted the concentration of the Kanamycin solution into μg/mL

50\frac{mg}{mL} of stock solution of Kanamycin × \frac{1 \mu g}{.001 mg} = 50,000\frac{\mu g}{mL} of Kanamycin

2. Used the dilution equation, M1V1 = M2V2, to obtain the required volume to be added to the starter culture media.

VKanamycin = \frac{50\mu g/mL * 35 mL}{50000\mu g/mL } = .035 mL of Kanamycin

  • The antibiotic was stored inside the freezer to prevent it from breaking down. Before allocating 35 μL of the antibiotic to the starter culture media, the antibiotic was thawed to room temperature.
  • Following aseptic techniques, broths 1 and 2 of the starter culture media were inoculated 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 forceps (tweezers) were flamed before and after use. For a detailed procedure of the inoculation process refer to the table below.
  • Upon the completion of the inoculation of broths 1 and 2, broths 3 and 4 were still warm and not at room temperature. Broths 3 and 4 were cooled inside a refrigerator for approximately 4 min.
  • When broths 3 and 4 were cool to room temperature, the broths were inoculated with the same method and aseptic techniques as applied to broths 1 and 2.
  • When inoculation was done for all broths, the broths were placed inside the incubator orbital shaker at 37°C, 237 rpm overnight. One notable feature among the broths is that broth 1 exhibited a slightly different opacity in terms of the color of the media. Broth 1 was slightly murky while broths 2, 3, and 4 had a stronger yellow tint to it.
  • The broths for the expression culture media were placed at room temperature over the bench top.
Steps followed during the inoculation of the starter culture media
1) Aluminum foil cap was removed from the Erlenmeyer. The cap was sterilized by passing it through the flame.
2) The mouth of the Erlenmeyer and metal forceps were also flamed.
3) Using the flamed metal forceps, a sterilized, cylinrical, wooden splint was obtained. The splint (now held with the metal forceps) were used to take an isolated colony from a plate of transformed E. coli. Be mindful not to create any scrapings or depression on the surface of the agar plate (This is to ensure there is no debris from the agar that will be transferred to the new media). A simple swift touch of the isolated colony is sufficient. This can be verified by observing a smudge on the plate which indicates that the colony is already on the splint or any inoculation tool used.
4) After obtaining the colony, the medium (broth) is stabbed with the splint (held with the metal forceps). Some swirling of the splint was applied to ensure the bacteria is in the medium.
5) The mouth of the Erlenmeyer was flamed so as the aluminum foil cap. After the flaming of both, the Erlenmeyer was recapped with the flamed aluminum foil cap. The splint was disposed to the biological waste bin and the metal forceps was flamed.
6) Steps 1-5 were repeated for broths 2, 3, and 4.

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