Team:Hong Kong HKUST/Project/module2
From 2013.igem.org
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To construct this inducible system, we searched for candidates that could regulate gene expression on the level of transcription while responding to fatty acid levels. Four different fatty acid induced promoters were then investigated, namely:<br> | To construct this inducible system, we searched for candidates that could regulate gene expression on the level of transcription while responding to fatty acid levels. Four different fatty acid induced promoters were then investigated, namely:<br> | ||
- | <br>1. Liver Fatty Acid Binding Protein 1 (FABP1) | + | <br>1. Liver Fatty Acid Binding Protein 1 (FABP1) Promoter; |
- | <br>2. Peroxisome Proliferator-Activated Receptor-alpha (PPAR-alpha) | + | <br>2. Peroxisome Proliferator-Activated Receptor-alpha (PPAR-alpha) Promoter; |
- | <br>3. Glucose Regulated Protein (GRP78) | + | <br>3. Glucose Regulated Protein (GRP78) Promoter; and |
- | <br>4. Fatty Acid Metabolism Regulator Protein (FadR) and | + | <br>4. Fatty Acid Metabolism Regulator Protein (FadR) and FadBA Promoter.<br> |
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<h3>Biology behind the 4 Fatty Acid Responsive Promoters</h3> | <h3>Biology behind the 4 Fatty Acid Responsive Promoters</h3> | ||
- | <h3>1. Liver Fatty Acid Binding Protein 1 (FABP1) | + | <h3>1. Liver Fatty Acid Binding Protein 1 (FABP1) Promoter</h3> |
Fatty acid binding proteins (FABPs) are lipid-binding proteins that regulate fatty acid uptake and transfer between extra-and intracellular membranes. There are 9 different FABPs identified with tissue-specific distribution, including FABP1 in liver. Some, such as Peroxisome Proliferator-Activated Receptor (PPAR), are believed to transport fatty acids from the plasma membrane to intracellular receptors, and as such have a selective cooperation with the receptor to activate gene transcription. | Fatty acid binding proteins (FABPs) are lipid-binding proteins that regulate fatty acid uptake and transfer between extra-and intracellular membranes. There are 9 different FABPs identified with tissue-specific distribution, including FABP1 in liver. Some, such as Peroxisome Proliferator-Activated Receptor (PPAR), are believed to transport fatty acids from the plasma membrane to intracellular receptors, and as such have a selective cooperation with the receptor to activate gene transcription. | ||
<br><br> | <br><br> | ||
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<h3>3. Glucose Regulated Protein (GRP78) Promoter</h3> | <h3>3. Glucose Regulated Protein (GRP78) Promoter</h3> | ||
- | GRP78 (HSPA5) is involved in the folding and assembly of proteins in the endoplasmic reticulum (ER). High concentration of fatty acids disrupts cell homeostasis, leading to endoplasmic reticulum stress (ERS). This in turn will activate the unfolded protein response (UPR) that consists of three trans-membrane proteins: IRE1, PERK and ATF6. The signals from these three proteins, when integrated together, will activate the GRP78 promoter. Other factors such as NF-Y, ERSF, YY1, which are normally acquired from the normal stress response followed by UPR, also play a role in activating the | + | GRP78 (HSPA5) is involved in the folding and assembly of proteins in the endoplasmic reticulum (ER). High concentration of fatty acids disrupts cell homeostasis, leading to endoplasmic reticulum stress (ERS). This in turn will activate the unfolded protein response (UPR) that consists of three trans-membrane proteins: IRE1, PERK and ATF6. The signals from these three proteins, when integrated together, will activate the GRP78 promoter. Other factors such as NF-Y, ERSF, YY1, which are normally acquired from the normal stress response followed by UPR, also play a role in activating the GRP78 promoter. <br><br> |
- | <h3>4. Fatty Acid Metabolism Regulator Protein (FadR) and | + | <h3>4. Fatty Acid Metabolism Regulator Protein (FadR) and FadBA Promoter</h3> |
- | FadR is a bacterial transcription repressor that regulates lipid metabolism and determines the bi-stable switch between fatty acid biosynthesis and beta-oxidation. The binding of FadR to the operator is inhibited by fatty acyl-CoA compounds, which are intermediates of fatty acid degradation. When the cellular environment is deficient in fatty acids, FadR binds to | + | FadR is a bacterial transcription repressor that regulates lipid metabolism and determines the bi-stable switch between fatty acid biosynthesis and beta-oxidation. The binding of FadR to the operator is inhibited by fatty acyl-CoA compounds, which are intermediates of fatty acid degradation. When the cellular environment is deficient in fatty acids, FadR binds to P<sub><i>fad</sub></i> (promoter of operon <i>fadBA</i>) and shuts down the beta-oxidation pathway while turning on the biosynthesis pathway. |
</div> | </div> |
Revision as of 23:27, 27 September 2013
![](https://static.igem.org/mediawiki/igem.org/c/c7/BANNER1_%281%29.png)
Fatty Acid Sensing Mechanism
Overview
In 2009, Prof. James Liao's research group at UCLA published their findings that mice expressing synthetic glyoxylate shunt had increased resistance to diet-induced obesity. To engineer this behavior in mice, they introduced glyoxylate shunt genes to mouse liver cells, employing a constitutive promoter for expression of the said genes. Though not demonstrated in mice, we worry that this glyoxylate shunt, when constantly turned on in human cells, could incur a fitness cost by continuing to burn off energy when the environment is not so energy rich. Thus, we are working to put this glyoxylate shunt under regulation by an inducible system, which would allow tunable fatty acid uptake by sensing fatty acid concentrations. Such a system should reduce the risk of energy or fatty acid deficiency when the surrounding fatty acid concentration is not too high. To construct this inducible system, we searched for candidates that could regulate gene expression on the level of transcription while responding to fatty acid levels. Four different fatty acid induced promoters were then investigated, namely:1. Liver Fatty Acid Binding Protein 1 (FABP1) Promoter;
2. Peroxisome Proliferator-Activated Receptor-alpha (PPAR-alpha) Promoter;
3. Glucose Regulated Protein (GRP78) Promoter; and
4. Fatty Acid Metabolism Regulator Protein (FadR) and FadBA Promoter.
Biology behind the 4 Fatty Acid Responsive Promoters
1. Liver Fatty Acid Binding Protein 1 (FABP1) Promoter
Fatty acid binding proteins (FABPs) are lipid-binding proteins that regulate fatty acid uptake and transfer between extra-and intracellular membranes. There are 9 different FABPs identified with tissue-specific distribution, including FABP1 in liver. Some, such as Peroxisome Proliferator-Activated Receptor (PPAR), are believed to transport fatty acids from the plasma membrane to intracellular receptors, and as such have a selective cooperation with the receptor to activate gene transcription.2. Peroxisome Proliferator-Activated Receptor-alpha (PPAR-alpha) Promoter
The peroxisome proliferator – activated receptors (PPARs) function s transcription factors to regulate expression of genes. The expression of PPAR-alpha can be up-regulated by increased fatty acid concentration in mammalian liver cells. The promoter of PPAR-alpha has a basal expression level. However, when it is stimulated with an extracellular palmitate concentration of 150uM, the activity of the promoter will increase by over 4 folds within 48 hours.3. Glucose Regulated Protein (GRP78) Promoter
GRP78 (HSPA5) is involved in the folding and assembly of proteins in the endoplasmic reticulum (ER). High concentration of fatty acids disrupts cell homeostasis, leading to endoplasmic reticulum stress (ERS). This in turn will activate the unfolded protein response (UPR) that consists of three trans-membrane proteins: IRE1, PERK and ATF6. The signals from these three proteins, when integrated together, will activate the GRP78 promoter. Other factors such as NF-Y, ERSF, YY1, which are normally acquired from the normal stress response followed by UPR, also play a role in activating the GRP78 promoter.4. Fatty Acid Metabolism Regulator Protein (FadR) and FadBA Promoter
FadR is a bacterial transcription repressor that regulates lipid metabolism and determines the bi-stable switch between fatty acid biosynthesis and beta-oxidation. The binding of FadR to the operator is inhibited by fatty acyl-CoA compounds, which are intermediates of fatty acid degradation. When the cellular environment is deficient in fatty acids, FadR binds to Pfad (promoter of operon fadBA) and shuts down the beta-oxidation pathway while turning on the biosynthesis pathway.Cell Viability
We are working towards to introducing an inducible system that allows tunable fatty acid uptake regulated by fatty acid concentrations. To test our promoters, fatty acid has to be added in the cell culture medium. It is however known that high fatty acid levels could lead to apoptosis by inducing stress responses. So in order to determine the range of fatty acid concentration suitable for testing, we conducted cell viability tests using MTT assay under different sodium palmitate concentrations. Our desired concentration range should keep at least 60% of cells alive after 24 hours incubation and/or at least 50% alive in 48 hours.Fatty Acid Quantification
To differentiate between the fatty acid amount added to the medium versus the actual fatty acid amount inside the medium, we investigated two fatty acid quantification methods: 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 use the fatty acid quantification kit due to time limitations.References
1 Guzman, Carla et al. "The human liver fatty acid binding protein (FABP1) gene is activated by FOXA1 and PPARα; and repressed by C/EBPα: Implications in FABP1 down-regulation in nonalcoholic fatty liver disease." Biochemica et Biophysica Acta (BBA) - Molecular and Cell Biology. 1831.4 (April 2013): 803-818. Web. 23 Sep. 2013.2 Ann Vogel Hertzel, et al (2000) “the Mammalian Fatty Acid-binding Protein Multigene Family: Molecular and Genetic Insights into Function” Elsevier Science
3 Guor Mour Her, et al. (2003) “In vivo studies of liver-type fatty acid binding protein (L-FABP) gene expression in liver of transgenic zebrafish (Danio rerio)”
4 Ines Pineda Torra et al. “Characterization of the human PPARalpha promoter: Identification of a functional Nuclear Receptor Response Element.”
5 Christopher D. Swagell et al. (2004) “Expression analysis of a human hepatic cell line in response to palmitate." Biochemical and Biophysical Research Communications.
6 Yibin Xu, et al. (2001) “The FadR-DNA Complex. Transcriptional control of fatty acid metabolism in Escherichia Coli.” JBC Papers in Press
7 Yuren Wei, et al. (2006) “Saturated fatty acids induce endoplasmic reticulum stress and apoptosis independently of ceramide in liver cells.” Am J Physiol
8 Do-Sung Kim, et al. (2007) “Effects of triglyceride on ER stress and insulin resistance” Biochemical and Biophysical Research Communications.
9 Mingqing LI, et al. “ATF6 as a Transcription Activator of the Endoplasmic Reticulum Stress Element: Thapsigargin Stress-Induced Changes and Synergistic Interactions with NF-Yand YY1.” Department of Biochemistry and Molecular Biology, and the USC/Norris Comprehensive Cancer Center, Keck School of Medicine of the University of Southern California, Los Angeles.
10 https://2012.igem.org/Team:NTU-Taida
11Life Technologies. (n.d.). Vybrant® mtt cell proliferation assay kit. Retrieved from http://www.lifetechnologies.com/hk/en/home/references/protocols/cell-culture/mtt-assay-protocol/vybrant-mtt-cell-proliferation-assay-kit.html"
12Sigma-Adrich. (2012). Free fatty acid quantification kit. Retrieved from http://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/Bulletin/1/mak044bul.pdf