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 liquid 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° C
- Injection volume: 10 μL
- Isocratic elution using 35% acetonitrile and 65% aqueous (0.1% v/v formic acid) solution for 13 minutes & wash the column with 80% acetonitrile
Figure 1 shows the LC-MS identifying peaks for each standard.
The positive control leucine uptake condition showed a peak at 91 m/z at 1 minute (Figure 2). The pro-leu condition did not have the indicative peaks at m/z 227 and 455 around the 2 minute mark (Figure 3). At 13 mins, CBZ-leu had the identifying spikes at m/z 264 and 310, but also had a large spike at 91 and 215 (Figure 4). These data indicate that pro-leu was not uptaken by E. coli while CBZ-leu was tentatively uptaken.
The second experiment was a growth test to assay whether leucine auxotrophic E. coli can survive on protected leucine. Growth indicated the organism has an intrinsic cleavage enzyme to cleave the protected leucine, and a lack of growth indicated there is no intrinsic enzyme. We wanted to avoid selecting a protecting group that can be cleaved by the cell’s own mechanisms to prevent cleavage of the protecting group prior to charging of the leucyl-tRNA. JW5807-2 leucine auxotrophic strain was grown in five different conditions:
- Negative control with only minimal media
- Positive control of minimal media + leucine
- Minimal media + 3mM pro-leu
- Minimal media + 3mM CBZ-leu
- Minimal media + 8mM CBZ-leu
The cells were grown for 47 hours and the concentration of cells were measured at OD600 in a Nanodrop at 12, 18, 24, and 47 hour timepoints. The cells in the positive control with leucine all grew, but there was little to no growth in the CBZ-leu and pro-leu conditions (Figure 5).
However, after conducting the liquid growth experiments, we realized the experimental method left significant room for error because each concentration measurement was an opportunity for contamination of the culture. Furthermore, we realized at this point that pro-leu is not a good choice for a protecting group because of its availability in the environment. For our biocontainment system to be effective, the organism must not be able to scavenge protected leucine from the environment, and as a dipeptide, pro-leu can be easily accessed from the breakdown of peptides. Intrinsic proteases can likely cleave pro-leu, too. Additional research also revealed that pro-leu is toxic to E. coli cells, so E. coli have evolved a mechanism to exocytose pro-leu. Thus, we abandoned the use of pro-leu for future experiments and tested N-methoxy-leu instead.
To confirm the results of the growth test using better experimental design, we decided to streak JW5807-2 cells on minimal medium agar plates of 5 different conditions:
