GC-FID detection of isoprenol / isopentenol

Product identity
This method quantifies isoprenol, 3-methyl-3-buten-1-ol, also commonly called isopentenol in the isoprenoid-fuels literature.

Source notes
Original notes: HM Jensen with help from K George. Updated 2013-07-26; recovered from notes dated 2016-03-07.

Related isoprenol / isopentenol papers
George et al., 2015, Scientific Reports: high-yield C5 alcohol production in E. coli
Kang et al., 2016, Metabolic Engineering: IPP-bypass mevalonate pathways for isopentenol production
Sasaki, Eng et al., 2019, Biotechnology for Biofuels: C. glutamicum production from biomass hydrolysates
Luckie et al., 2024, Metabolic Engineering: C. glutamicum monoterpene platform
Carruthers et al., 2025, Nature Communications: automated ML optimization in P. putida
Menasalvas et al., 2025, Science Advances: biosensor-driven P. putida strain engineering

Purpose
Measure isoprenol / isopentenol production in engineered production strains. GC-FID provides a rapid way to quantify product concentration from extracted culture samples.

Critical notes

• Run isoprenol standards every time the GC is run.

• Whole-cell suspensions can be frozen at -20 C and extracted later, which is useful for time courses.

• Run an EtAc plus standard sample first, last, and every 10 samples to keep the lines clean.

• Internal standard: n-butanol in ethyl acetate, usually 20 mg/L. Be consistent across all standards and samples.

• Use an incubator rather than the warm room when possible, since temperature and humidity are less disrupted and temperature can be adjusted from 37 C to 30 C.

• Book Google Calendar for GCFID1-left. Estimate run time as t(hr) = n x 7.5 / 60, where n is the number of samples.

Materials

• Production strains adapted to the chosen production medium.

• Production medium plus appropriate antibiotics and IPTG.

• Sterile culture tubes.

• Ethyl acetate (EtAc).

• n-butanol.

• EtAc*: ethyl acetate spiked with 20 mg/L n-butanol.

• GC vials and caps.

Production culture

1. Thaw glycerol stock of adapted cells on ice.

2. Inoculate 5 mL production medium with antibiotics using 25 uL adapted cells.

3. Grow overnight at 37 C with 200-225 rpm shaking.

4. Dilute cells to OD600 about 0.05 in 5 mL production medium.

5. Grow cultures at 37 C with 200 rpm shaking until OD600 about 0.4-0.6. This is often about 5.5-6 h.

6. Induce with IPTG. Test a broad induction range first, for example 500 uM, 250 uM, 100 uM, 50 uM, and 10 uM, then narrow around the best condition.

7. Transfer cultures to 30 C and continue growth with 200 rpm shaking.

8. Take 200 uL aliquots at the planned time points, commonly 24, 48, and 72 h. Aliquots may be frozen at -20 C for later analysis.

Standards

1. Make standards fresh and run them every time.

2. Prepare 1000 mg/L isoprenol standard by spiking 5 mL growth medium with 5 mg isoprenol. Using density 0.853 g/mL at room temperature, this is about 5.86 uL in 5 mL medium.

3. Prepare 2-fold serial dilutions in the same medium to give 1000, 500, 250, 125, and 62.5 mg/L standards.

4. Extract and analyze standards exactly as samples are extracted and analyzed.

Extraction

1. Add 200 uL EtAc* to 200 uL whole-cell culture aliquot.

2. Vortex the 1:1 mixture at maximum speed for 10-15 min. Exact time matters less than consistency between standards and samples.

3. Dilute extracts 1:5 in EtAc* into a GC vial, for example 100 uL extract plus 400 uL EtAc*.

4. For time courses, early time points may need less dilution to keep the peak in the calibration range. If dilution differs, prepare matched standards.

GC-FID run

• Instrument: GCFID1-left.

• Run method: about 4 min per sample, with about 3 min 20 s downtime between runs.

• Run EtAc* blank first, last, and every 10 samples.

• Expected isoprenol peak: about 2.9 min. Confirm n-butanol retention time with current standards.

• Under Method / Advanced, double-check the output file path. Output should include retention time and area.

Before starting

• Check that the waste bottle is empty.

• Check that the EtAc bottle is filled.

• Press the double green arrow, sequence run, to start.

Waste

• In the hood, dump vials into the non-halogenated waste bin.

• Enter the approximate volume on the waste log around the hood.

Data processing

1. The data are saved as .area files. Open in Excel or another analysis tool.

2. Normalize isoprenol peak area to n-butanol peak area: IP area / n-BuOH area.

3. Plot the standard curve. It should be linear.

4. Calculate a best-fit linear equation with the intercept set to 0.

5. Use the slope to relate normalized peak area to mg/L.

6. For samples, divide normalized peak area by the calibration slope.

7. Apply any dilution correction only if the sample and standard dilution schemes differ.

Open questions / update before routine use

• Confirm current method name and retention times for isoprenol and n-butanol.

• Confirm whether whole-cell, supernatant, or paired measurements are most appropriate for the experiment.

• Record exact medium, carbon source, induction condition, sampling time, extraction dilution, and GC sequence position in the sample sheet.