Difference between revisions of "Part:BBa K203106"

Revision as of 15:42, 20 October 2009

HMG-CoA synthase promoter (sterol regulated)

This part is a natural HMG CoA synthase promoter responsive to SREBP and when placed upstream of other genes could induce their transcription in response to lipid addition to the medium.

Usage and Biology

SREBP induction. Upon Sterol depletion, the interaction of SCAP and Insig in the ER membrane is inhibited, resulting in cleavage of SREBP, and migration to the nucleus. [http://en.wikipedia.org/wiki/File:WikF1.png Image is public domain.]

Sterol regulatory element-binding protein (SREBP) is a transcription factor involved in the regulation of sterol metabolism. In cells with high concentration of cholestrol SREBP is present in an inactive form anchored to the endoplasmatic reticulum or the nuclear envelop. If the cholesterol concentration decreases SREBP is cleaved by the proteases site-1 protease and site-2 protease resulting in a release of the aminoterminal domain of SREBP. Two additional proteins (Scap and Insig) are needed to regulate this process in a way that the cleavage occurs exclusively during lack of sterol. The aminoterminal domain of SREBP is translocated into the nucleus and binds to the DNA consensus sequence TCACNCCAC. The binding causes an up regulation of the genes needed for cholesterol synthesis. See reference at [http://2009.igem.org/Team:Heidelberg/Eucaryopedia#SREBP Eucaryopedia].

Sequence and Features

Functional Parameters

The natural HMG CoA synthase promoter was integrated into Part:BBa_K203100 and was induced by Lipoprotein Deficient Serum in [http://2009.igem.org/Team:Heidelberg/Eucaryopedia#HeLa HeLa cells]. The 0,5% LDS DMEM (1% Glucosamin, 1% Pen/Strep, 1% non-essential aminoacids) medium was put on cells after transfection for two days. After that, 1% hydroxypropyl-β-cyclodextrin (HPCD) medium was put on for 3 hours and then, 0,5% LDS DMEM (1% Glucosamin, 1% Pen/Strep, 1% non-essential aminoacids) medium was used for incubation for 3 hours. Promoter activtiy was then roughly characterized analogous to [http://2009.igem.org/Team:Heidelberg/Project_Measurement#Two_units_for_promoter_activity_in_mammalian_cells REU] by TECAN (automated plate fluorescence reader).

BBa_K203106 is a natural HMG CoA synthase promoter induced approximately 35% by Lipoprotein Deficient Serum. The HMG CoA synthase promoter is coupled to GFP and is integrated into Part:BBa_K203100. The GFP fluorescence was measured by TECAN once (à three replicates). Background fluorescence was substracted, and fluorescence levels are plotted relative to Part:BBa_K203112. The standard deviation is represented by the error bars. Inactive condition corresponds to Full medium plus Cholsterol and LDL