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Team:Hong Kong HKUST/experiment/exp1
From 2013.igem.org
Cell Viability & Fatty Acid Quantification
Introduction
For one of our human practice projects, our team investigated the current development of synthetic biology in some of the Asian countries. While doing research, we found out that synthetic biology is really a new field in science and that is not well-known in Asia. In fact, when some of our members tried to research information about synthetic biology in Cantonese, we could not find any webpages dedicated to share information about synthetic biology for the public in Hong Kong. To address this observation, we have decided to use an easily accessible platform called Google Blogger. This blog will be used as mean to introduce synthetic biology, share our experience and our project, and report current events related to synthetic biology. It will be written in Chinese and English so that most people in Hong Kong can read it easily and pay more attention to this new field in science. By providing holistic and informational blog posts, we aim at raising the public awareness to synthetic biology.
A Sneak Peek of Our Blog
CLICK HERE TO VISIT OUR BLOG!
Cell Culture and Transfection
We cultured HEK 293FT cells following ATCC’s standard procedure, except that we have used DMEM with 10% FBS and 1% penicillin/streptomycin for culture medium. For transfection, we have followed the manufacturer’s protocol of LipofectamineTM 2000 (Invitrogen). We have used serum free and antibiotics free DMEM to form DNA-lipofectamine complex.
Cell Line
In our project, two mammalian cell lines were used: human hepatoma cell (HepG2 cell) and human embryonic kidney 293 cell (HEK293FT). HepG2 cell was used for characterizing inducible promoters and glyoxylate systems. For higher transfection efficiency, the characterizations of mitochondria leader sequence and constitutive promoter were conducted using HEK293FT cells.
Cell Culture
HepG2 and HEK293FT cells were maintained in DMEM supplemented with 10% heat-inactivated FBS and 50ug/mL penicillin, 50ug/mL streptomycin at 37℃in a humidified atmosphere containing 5% CO2. Cells were transfected in petri dishes and multi-well plates with different construct Lipofectamine 2000 (Invitrogen; Carlsbard, CA) according to manufacturer’s protocols. Any GFP signals were observed under fluorescent microscope or under confocal microscope if necessary.
Cell Viability
We designed to introduce an inducible system that allows tunable fatty acid uptake by sensing fatty acid concentrations. Fatty acids uptake was to be quantified to compare the activities of wild type cells and cells expressing inducible glyoxylate shunt.
To facilitate expression of inducible glyoxylate shunt in human hepatoma cell line (HepG2 cell), cell viability at different sodium palmitate concentration was measured. While a high fatty acid level is known to lead apoptosis, the cell viability test ensured maintenance of a stable cell line for transfection.
We used MTT assay to test cell viabilities in different fatty acid concentrations. The objective was to determine a range of optimal concentrations of fatty acids to be introduced into HepG2 cell and achieve more than 60% viability after 24 hours incubation and/or more than 50% in 48 hours.
MTT Assay Description
MTT assay measures the enzymatic activity of oxidoreductase enzymes that only show activity when the cells are alive. MTT, a tetrazolum dye, is reduced into an insoluble formazan, giving a purple color. Organic solvent such as DMSO can be used to dissolve the formazan. Absorbance at 570 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 incubation gradient concentration of sodium palmitate from 0 mM to 1.0mM, and 2.0mM were added into each row. After adding the sodium palmitate, we have incubated the cells for 24 hours and 48 hours respectively. MTT reagent was added and formazan formation was observed and measured using spectrophotometer.
