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.

Cell Viability

We worked to introduce an inducible system that allows tunable fatty acid uptake regulated by fatty acid concentrations. Fatty acid uptake was to be quantified to compare the activity of wild type cells with the activity of our engineered cells expressing inducible glyoxylate shunt.

High fatty acid levels are known to lead to apoptosis, so we conducted cell viability tests using MTT assay at different sodium palmitate concentrations.

The objective was to determine the range of fatty acid concentrations to be introduced into our cells that would allow more than 60% viability after 24 hours incubation and/or more than 50% in 48 hours.

MTT assay description

The MTT assay measures the enzymatic activity of oxidoreductase enzymes that only show activity when the cells are alive. When the aforementioned enzymes are active, MTT, a tetrazolium dye, is reduced into an insoluble formazan, giving a purple color. Organic solvents such as DMSO can be used to dissolve the formazan. Absorbance at 570nm is measured using a spectrophotometer to quantitatively determine the amount of formazan formation.

In our experiment, HepG2 cells were seeded into a 96-well plate. After one day of incubation, a gradient concentration of sodium palmitate from 0 mM to 1.0mM, and 2.0mM was added into each row of wells. After adding the sodium palmitate, we incubated the cells for 24 hours or 48 hours. MTT reagent was added and formazan formation was observed and measured using a spectrophotometer.

Results

From the MTT assay, we observed that after 24 hours of incubation with palmitic acid, around 45% viability can be maintained even at 2.0mM. For 48 hours incubation, cell viability varied for different concentrations. To maintain 50% viability after 48 hours incubation with palmitic acid, we decided to conduct subsequent experimentation within the range of 0mM to 0.32 mM palmitic acid. Click on the pictures to the side for our specific findings on cell viability.

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.