Paris/July 11
From 2007.igem.org
Preparation of LB agar plate for E.coli transformed by pKS::DGAT
We decided to test the synthesis of triglyceride by E.coli transformed with pKS::DGAT on LB culture. Knowing that when bacteria are in presence of long chain fatty acid (oleate for example), fatty acid are transported into the cytoplasm by fadL (fatty acid degradation), are added acetyl-CoA by fadD and then the fatty acyl-coA is able to bind fadR and repress it. FadR is a repressor of the expression of fadL (the transporter) and the degradation machinery. Culture in presence of long chain fatty acid enhances the transport of fatty acid into the cytoplasm to be dregraded by beta-oxidation. We would like to use this enhanced transport to synthesize triglyceride by DGAT (substrate: long chain fatty acil-CoA and diacylglycerol).
We prepared different medium:
- LB agar + ampicillin (100µg/mL) +/- IPTG (0.4mM) +/- sodium oleate (0.5mM and 2mM) + Nile red (0.5µg/mL of medium). - Minimum medium + ampicillin (100µg/mL) +/- IPTG (0.4mM) +/- sodium oleate (0.5mM and 2mM) + Nile red (0.5µg/mL of medium).
We spread DH5alpha pKS::DGAT (liquid culture from 07/04/2007).
