Wisconsin/Project
From 2007.igem.org
Contents |
Project Overview
Reprogramming gene expression is one of the main goals of synthetic biology. In the past, most groups have focused on cis-regulatory elements such as promoter to control transcription. We decided to explore artificial transcription factor (ATF), a trans-regulatory element, for controlling genes. Our project consists of characterizing ATF and testing ATF in mammalian cells.
Artificial Transcription Factor
Background
Artificial transcription factor (ATF) has two domains: DNA-binding domain and regulatory domain. The DNA-binding domain provides sequence specific targeting and the regulatory domain can up- or down-regulate gene expression. ATF design is modular, meaning one can mix and match different DNA-binding motifs with various transcription factors.
We decided to use zinc fingers for DNA-binding. In general, a zinc finger is 30 amino acids long, is stabilized by a single zinc ion to form a certain protein structure (beta-beta-alpha), and can interact with a specific 3 base-pair motif in DNA (Segal & Barbas, 2001). Regulation of gene activity is achieved by a ZFP recognizing a specific DNA sequences, binding to it, and recruiting other molecular machinery that will activate or repress the gene being targeted. It is this ability to target specific sites on DNA and then induce cellular activity in a controlled and predictable manner that is causing such anticipation. Researchers are learning to manipulate zinc-fingers artificially, so that they can be used to control gene expression in a meaningful way (Ansari, 2003, Segal et al., 1999). Our iGEM project has centered on further developing these ideas with ZFP's, and experimenting with real world applications, in particular to use ZFP’s as an artificial transcription factor (ATF). This would, we hope, create a powerful, new class of Bio-brick.
Design
We designed an ATF that can recognize 9 base pairs and characterized it with CSI array.
Cognate Site Identity (CSI) Microarray
We used CSI microarrays to quantify how well zinc fingers bind to DNA sequences. The CSI array contains every permutation of 9bp DNA sequence. This allows us to test the entire sequence space in an unbiased manner.
BCL-2 and Cell Fate Regulation
Background
Doxrubicin is a widely used drug to treat cancer. However one main limitation of its use is cardiotoxicity. Accumulated dose of doxrubicin can cause oxidative stress to cardiomyocytes and trigger apoptosis. Our goal is to up-regulate BCL-2 (a pro-survival protein) and prevent cells from dying under mild stress.
Design
Using zinc-finger concepts, we designed an in vivo gene regulation system. We chose to target the Bcl-2 gene, because it makes an empirically important protein, and offers a good system for conducting experiments in vivo by its involvement in cellular disease states. We prepared a highly developed ATF, one that is programmed to bind the promoter region of Bcl-2 and is subject to control by tetracycline itself.
Experiment
We tested our construct for function in mammalian cells. The experiments were conducted by introducing our ATF into mouse fibroblast cells, adding tetracycline in certain conditions, and then measuring protein levels made by the cells. Our project design predicts that cells that have the ATF and receive a dose of tetracycline should have increased Bcl-2 gene expression. We would then be able to detect this up-regulated gene activity by measuring the subsequent increase production of Bcl-2 protein by western blot.
