Team:Virginia/Experiments
We had many options for the protecting group on leucine. After much research into the properties, environmental availability, and cost of each protected leucine option, we decided on three possibilities. These included proline-leucine (pro-leu), N-carbobenzyloxy-leucine (CBZ-leu), and N-methoxycarbonyl-leucine (N-moc-leu). Pro-leu and CBZ-leu were our first two choices, and N-moc-leu was added as an option after pro-leu was discounted for reasons mentioned below. Two experiments were conducted to determine whether the three protecting groups were appropriate for our purposes.
The first experiment was an uptake test to determine if the cell is able to take up protected leucine from the environment. XL1-Blue strain E. coli cells were incubated in Luria Broth (LB) containing leucine, pro-leu, or CBZ-leu for 24 hours. The cells were washed and lysed, and the purified lysate was analyzed using laser chromatography-mass spectrometry (LC-MS) to determine if the protected leucine was uptaken into the cell.
2mM, 1.5mM, 1mM, 500uM, and 200uM standards of leucine, pro-leu, and CBZ-leu were made in 0.1% formic acid. LC-MS conditions were as follows:
- Wavelength = Diode Array (Many wavelengths)
- Flow rate = 0.3 mL/min (between 0.2 mL/min and 0.4 mL/min)
- Column temperature: 30 deg C
- Injection volume: 10 uL
- Isocratic elution using 35% acetonitrile and 65% aqueous (0.1% v/v formic acid) solution for 13 minutes & wash the column with 80% acetonitrile
