Team:Hong Kong HKUST/Project/module1

Cell Line

For our project we made use of two mammalian cell lines: HepG2 human hepatoma cells, and HEK293FT human embryonic kidney cells. HepG2 cells was used for characterizing inducible promoters and the glyoxylate systems. To take advantage of their higher transfection efficiency, the characterizations of mitochondria leader sequence and constitutive promoter were conducted using HEK293FT cells.

Cell Culture

HepG2 and HEK293FT cells were grown in DMEM supplemented with 10% heat-inactivated FBS, 50ug/mL penicillin and 50ug/mL streptomycin. The cells were incubated at 37℃ in a humidified atmosphere containing 5% CO2. Cells were transfected in petri dishes and multi-well plates with Lipofectamine 2000 (Invitrogen; Carlsbard, CA) transfection reagent according to the manufacturer’s protocols. GFP signals were observed under fluorescent microscope or under confocal microscope if necessary.

Fatty Acid Quantification

Two fatty acid quantification methods were investigated to measure fatty acid uptake rate of our constitutive and inducible glyoxylate systems: 1) Gas Chromatography-Mass Spectrophotometry (GC-MS), and 2) fatty acid quantification kit (Sigma-Aldrich; St. Louis, MO). While we managed to measure the fatty acid quantity in cell culture medium using GC-MS, we were not able to try the fatty acid quantification kit due to time limitations.

Fatty Acid Treatment

Fatty acid solution was mixed with ethanol and chloroform. After acidifying by hydrochloric acid and refluxing in a water bath for 30 min, the organic layer containing fatty acids was collected and extracted by diethyl ether and petroleum ether solution. Again the organic layer was extracted out to be dried before sodium hydroxide was added. Then after derivatisation by boron trifluoride and bromotetradecane, the organic layer was collected into GC-MS vials for analysis.

GC-MS

GC-MS is a very useful tool to quantify volatile compounds effectively. For our experiment, we conducted calibration tests using known concentrations of fatty acids. However, it was difficult to reach a conclusion due to lack of internal standards and an uncertain amount of sample loss.