Team:Hong Kong HKUST/Project/module2

From 2013.igem.org

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<h3>Overview</h3>
<h3>Overview</h3>
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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. The aim of this module is to introduce an inducible system that allows tunable fatty acid uptake by sensing fatty acid concentrations. Such a system would reduce the risk of fatty acid deficiency when fatty acid concentration is below normal.
+
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.  
-
<br><br>
+
 
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Four different fatty acid induced promoters were investigated, namely: Liver Fatty Acid Binding Protein 1 (FABP1) promoter, Peroxisome Proliferator-Activated Receptor-alpha (PPAR-alpha) promoter, Glucose Regulated Protein (GRP78) promoter, Fatty Acid Metabolism Regulator Protein (FadR) and pFadBA promoter.
+
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:<br>
 +
 
 +
<br>1. Liver Fatty Acid Binding Protein 1 (FABP1) promoter;
 +
<br>2. Peroxisome Proliferator-Activated Receptor-alpha (PPAR-alpha) promoter;
 +
<br>3. Glucose Regulated Protein (GRP78) promoter; and
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<br>4. Fatty Acid Metabolism Regulator Protein (FadR) and pFadBA promoter.<br>
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<br>
<br>
</div>
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<h3>Cell Viability</h3>
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<h3>Biology behind the 4 Fatty Acid Responsive Promoters</h3>
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We worked to introduce an inducible system that allows tunable fatty acid uptake regulated by fatty acid concentrations. Fatty acid uptake was to be quantified to compare the activity of wild type cells with the activity of our engineered cells expressing inducible glyoxylate shunt.  
+
<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.
 +
<br><br>
-
High fatty acid levels are known to lead to apoptosis, so we conducted cell viability tests using MTT assay at different sodium palmitate concentrations. The objective was to determine the range of fatty acid concentrations to be introduced into our cells that would allow more than 60% viability after 24 hours incubation and/or more than 50% in 48 hours.
+
-
<br>
+
<h3>2. Peroxisome Proliferator-Activated Receptor-alpha (PPAR-alpha) Promoter</h3>
 +
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. <br><br>
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</div>
+
-
</div>
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<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 GPR78 promoter. <br><br>  
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<div class="row">
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<div class="nine columns"><p id="3"></p>
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<h3>4. Fatty Acid Metabolism Regulator Protein (FadR) and pFadBA</h3>
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<h3>Fatty Acid Quantification</h3>
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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 (promoter of operon <i>fadBA</i>) and shuts down the beta-oxidation pathway while turning on the biosynthesis pathway.
-
Two fatty acid quantification methods were investigated to measure fatty acid uptake rate of our constitutive and inducible glyoxylate systems: 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.<br><br>
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</div>
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</div>
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                            <div class="row">
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<div class="nine columns"><p id="4"></p>
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<h3>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 PPAR, are believed to transport fatty acids from the plasma membrane to intracellular receptors, and as such have a selective cooperation with the receptor. The FABP1 promoter was designed to sense fatty acid concentration and drive expression of glyoxylate genes. <br><br>
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</div>
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</div>
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+
-
<div class="row">
+
-
<div class="nine columns"><p id="3"></p>
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-
<h3>Peroxisome Proliferator-Activated Receptor-alpha (PPAR-alpha) Promoter</h3>
+
-
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 is constitutive, white elevated extracellular palmitate amount to 150uM increases expression of genes by over 4 times in 48 hours. In our project, PPAR-alpha promoter was used to sense fatty acid and perform inducible glyoxylate shunt.  
+
</div>
</div>
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<div class="row">
<div class="row">
<div class="nine columns"><p id="3"></p>
<div class="nine columns"><p id="3"></p>
-
<h3>Glucose Regulated Protein (GRP78) Promoter</h3>
+
<h3>Cell Viability</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, causing endoplasmic reticulum stress (ERS). This in turn, activates the unfolded protein response (UPR) that consists of three transmembrane proteins: IRE1, PERK and ATF6. Three signals constitutively activate the GRP78 promoter with the help of other factors, such as NF-Y, ERSF, YY1 and cleaved ATF6, acquired from the normal stress response followed by UPR. The activated GRP78 promoter by high fatty acid concentration is used to drive increased expression of glyoxylate genes.  
+
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.
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<div class="nine columns"><p id="5"></p>
<div class="nine columns"><p id="5"></p>
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<h3>Fatty Acid Metabolism Regulator Protein (FadR) and pFadBA</h3>
+
<h3>Fatty Acid Quantification</h3>
-
FadR is a bacterial transcription factor that regulates lipid metabolism of fatty acid biosynthesis and beta-oxidation. The binding of FadR is inhibited by fatty acyl-CoA compounds, which are intermediates of fatty acid degradation. In the absence of fatty acid, a constitutively expressed fatty acid metabolism regulator protein FadR binds to Pfad promoter (pFadBA) and inhibits the expression of glyoxylate genes.  
+
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.
<br>
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Revision as of 19:57, 27 September 2013


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 pFadBA 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 GPR78 promoter.

4. Fatty Acid Metabolism Regulator Protein (FadR) and pFadBA

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 (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 Ines Pineda Torra et al. “Characterization of the human PPARalpha promoter: Identification of a functional Nuclear Receptor Response Element.”