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Operon Fusion

Methods & Materials
Data & Analysis

Methods & Materials

  • Chloroform was used to sterilize the lysate and MC Buffer was used to was and suspend the lysate.
  • E. coli strain MAL103, density ~2.0 E8 cells/mL was infected with the lysate.
  • 11 sterile microfuge tubes were used to make dilutions of the lysate and MC4100 E. coli.
  • LBC Top Agar was used to make five LBC plates.
  • In the assay, four LacMac + Amp plates, three AraMac+ Amp plates, and one AraMac – Amp plates were used.
  • The lysate of MuD1 and MuCts was created using 5 mL of strain MAL103 E. Coli. After incubation at 37˚C, the bacterial cell debris was sterilized with chloroform, vortexed and centrifuged. The unwanted cell debris formed a pellet which could be separated from the supernatant fluid which contained the desired newly-formed phages.
  • This lysate was diluted in combination with MC buffer to create a 10-1, 10-2, 10-3, 10-5, 10-6 and 10-7 dilution.
  • Phage and cell controls were put aside, which respectively contained lysate or MC4100 only.
  • The 10-5, 10-6 and 10-7 dilutions were infected with lysate and plated on three LBC plates. These plates were incubated at 30˚C for twenty-four hours. Data was recorded the following day. Quantitative data such as the number of plaques on each plate and turbidity vs. clearness of the plate was recorded.
  • The 10-1, 10-2 and 10-3 dilutions were used to infect a quantitiy of MC4100 and plated to three LacMac+Amp plates. Uninfected MC4100 was also plated, to serve as a control. These plates were incubated for twenty-four hours at 30˚C and examined the following day. The colonies were counted and scored for color.
  • The 10-1 and 10-2 were allowed to infect MC4100 and plated to three AraMac + Amp plates. A 10-1 diluted but uninfected amount of MC4100 was plated to one other AraMac + Amp plate. This plate would determine the frequency of reverse araD mutations. All four plates were incubated for twenty-four hours at 30˚C. Colonies counted and scored for color the following day.
  • From the AraMac + Amp plates, twenty colonies were chosen to be patched to both AmpLacMac + Amp and AraLacMac plates. These plates were allowed to incubate for twenty-four hours at 30˚C.

Steric Hinderance - Sec 002 - BIOL 302L - FA09