Team:Hong Kong HKUST/Project/module3

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<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/advisors">Advisors</a></li>
<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/advisors">Advisors</a></li>
<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/instructors">Instructors</a></li>
<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/instructors">Instructors</a></li>
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<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/project">Project</a>
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<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/Project">Project</a>
<ul>
<ul>
<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/abstract">Abstract</a></li>
<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/abstract">Abstract</a></li>
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<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/modelling">Modelling</a></li>
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<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/modules">Modules Description</a></li>
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<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/characterization">Characterization</a></li>
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<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/Parts">Parts</a></li>
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<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/characterization">Characterization</a></li>
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<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/results">Results</a></li>
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<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/results">Result</a></li>
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<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/future">Future Work</a></li>
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<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/Wetlab">Wetlab</a>
<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/Wetlab">Wetlab</a>
<ul>
<ul>
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<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/notebook">notebook</a></li>
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<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/notebook">Notebook</a></li>
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<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/safety">safety</a></li>
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<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/safety">Safety</a></li>
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<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/hp">Human Practice</a>
<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/hp">Human Practice</a>
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<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/hp/Interviews">Interviews</a></li>
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<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/hp/cp">Country Profile</a></li>
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<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/hp/Presentation">Presentation</a></li>
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<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/hp/video">Videos</a></li>
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<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/hp/presentation">Presentations</a></li>
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<h6>Module Three</h6>
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<h6>Modules</h6>
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<li class="divider"></li>
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<a href="https://2013.igem.org/Team:Hong_Kong_HKUST/Project/module4">Glyoxylate Shunt</a>
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</li>
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<li>
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Protein Trafficking
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<ul><li>
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<li>
<li>
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<a href="https://2013.igem.org/Team:Hong_Kong_HKUST/Wetlab">Cell Line</a>
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<a href=#1>Overview</a>
</li>
</li>
<li>
<li>
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<a href="https://2013.igem.org/Team:Hong_Kong_HKUST/Wetlab">Cell Culture</a>
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<a href=#2>Biology of Mitochondrial Leader Sequence (MLS)</a>
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<a href=>Cell Viability</a>
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<a href=#3>Reference</a>
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                                                <li>Results</li></ul>
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Fatty Acid Quantification
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Fatty Acid Treatment
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                                <li>GC-MS</li></ul>
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<a href="https://2013.igem.org/Team:Hong_Kong_HKUST/Project/module2">FA Sensing Mechanism</a>
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<h2 class="centered">Protein Trafficking</h2>
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<h2 class="centered">Fatty Acid Quantification and Cell Viability</h2><br>
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<h3 class="centered">Targeting ACE enzyme into mitochondria by Mitochondrial Leader Sequence (MLS)</h3>
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<div class="nine columns"><p id="1"></p>
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<h3>Cell Line</h3>
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<h3>Overview</h3>
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In our project, two mammalian cell lines were used: human hepatoma cell (HepG2 cell) and human embryonic kidney 293 cell (HEK293FT). HepG2 cell was used for characterizing inducible promoters and glyoxylate systems. For higher transfection efficiency, the characterizations of mitochondria leader sequence and constitutive promoter were conducted using HEK293FT cells.  
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In our project, we introduced bacterial glyoxylate enzymes into mammalian cells to create alternative metabolic pathway. However, unlike their native environment in bacteria, the two enzymes needed to find their way through the high compartmentalized system in order to reach the citric acid cycle where they could act on.
<br>
<br>
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To guide the glyoxylate enzymes into the mitochondria where mammalian citric acid cycle resides, we produced recombinant glyoxylate enzymes by attaching the Mitochondrial Leader Sequence (MLS) to their N-termini.
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<br>
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We have also constructed the MLS in its own into standard BioBricks (<a href="http://parts.igem.org/Part:BBa_K1119000">BBa_K1119000</a> & <a href="http://parts.igem.org/Part:BBa_K1119001">BBa_K1119001</a>), and we quantitatively characterized their behavior using GFP reporter.
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<h3>Cell Culture</h3>
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<h3>Biology of Mitochondrial Leader Sequence (MLS)</h3>
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HepG2 and HEK293FT cells were maintained in DMEM supplemented with 10% heat-inactivated FBS and 50ug/mL penicillin, 50ug/mL streptomycin at 37℃in a humidified atmosphere containing 5% CO2. Cells were transfected in petri dishes and multi-well plates with different construct Lipofectamine 2000 (Invitrogen; Carlsbard, CA) according to manufacturer’s protocols. Any GFP signals were observed under fluorescent microscope or under confocal microscope if necessary.
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<p>In eukaryotes, the signal sequence guides the translocation of the newly synthesized peptide.</p>
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<p>The story starts with the MLS attached to the N-terminus of the protein of interest. Once the protein of interest is synthesized, this preprotein would remain unfolded by associating with chaperons. The preprotein will stay in the cytosol until the MLS gets recognized by receptor of the TOM complex on the outer mitochondrial membrane. Binding of the MLS to the receptor will trigger the feeding of the peptide through the translocation channel. Afterwards, the MLS will then be handed over to a TIM complex which sits on the inner membrane, which will then open up the channels on the inner membrane and allow the peptide to pass through. Once the peptide is through the double membranes, mitochondrial chaperone will be involved in pulling the peptide into the mitochondria and refold the protein. Lastly, the MLS will be cleaved by signal peptidase and dissociate from the transported peptide. (Alberts, 2002) <br>For the MLS we used, four additional amino acid residues (Ile-His-Ser-Leu) will be left at the N-terminus of the protein after the cleavage.(Invitrogen, 2012)</p>
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<img src="https://static.igem.org/mediawiki/2013/8/87/MLS_mechanism.png" >
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<h3>Cell Viability</h3>
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We designed to introduce an inducible system that allows tunable fatty acid uptake by sensing fatty acid concentrations. Fatty acids uptake was to be quantified to compare the activities of wild type cells and cells expressing inducible glyoxylate shunt.
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<h3>Reference</h3>
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To facilitate expression of inducible glyoxylate shunt in human hepatoma cell line (HepG2 cell), cell viability at different sodium palmitate concentration was measured. While a high fatty acid level is known to lead apoptosis, the cell viability test ensured maintenance of a stable cell line for transfection.<br><br>
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Alberts, B., Bray, D., Hopkin, K., Johnson, A., Lewis, J., Raff, M., Roberts, K., & Walter, P. (2010). <i>Essential cell biology.</i> (3rd ed., p. 505). UK: Garland Science.<br><br>
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We used MTT assay to test cell viabilities in different fatty acid concentrations. The objective was to determine a range of optimal concentrations of fatty acids to be introduced into HepG2 cell and achieve more than 60% viability after 24 hours incubation and/or more than 50% in 48 hours.<br><br>
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pCMV/<i>myc</i>/mito Invitrogen.(2012).pShooter™ Vector(pCMV/<i>myc</i> vectors).Retrieved from http://tools.lifetechnologies.com/content/sfs/manuals/pshooter_pcmv_man.pdf <br><br>
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<h5><b>MTT assay description</b></h5>
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Alberts, B. (2002). <i>Molecular biology of the cell.</i> (4th ed., pp. 1050-1061). New York:Garland Science.
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CMTT assay measures the enzymatic activity of oxidoreductase enzymes that only show activity when the cells are alive. MTT, a tetrazolum dye, is reduced into an insoluble formazan, giving a purple color. Organic solvent such as DMSO can be used to dissolve the formazan. Absorbance at 570 is measured using a spectrophotometer to quantitatively determine the amount of formazan formation.<br><br>
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In our experiment, HepG2 cells were seeded into a 96-well plate. After one day incubation gradient concentration of sodium palmitate from 0 mM to 1.0mM, and 2.0mM were added into each row. After adding the sodium palmitate, we have incubated the cells for 24 hours and 48 hours respectively. MTT reagent was added and formazan formation was observed and measured using spectrophotometer.
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<br><br>
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<h5><b>Results</h5></b>
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From the MTT assay, we observed that after 24 hours of incubation with palmitic acid, around 45% of cell viability can be maintained even at 2.0mM. For 48 hours incubation, cell viability varied for different concentrations. To maintain 50% cell viability after 48 hours incubation with palmitic acid, we concluded to experiment within range from 0mM to 0.32 mM of palmitic acid. Click on the pictures beside for a detailed result on cell viability.
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<h3>Fatty Acid Quantification</h3>
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Two fatty acid quantification methods were investigated to measure fatty acid uptake rate of constitutive and inducible glyoxylate system: 1) Gas Chromatography-Mass Spectrophotometry (GC-MS), and 2) Fatty acid quantification kit (Sigma Aldrich). While we managed to measure fatty acid amount in cell culture medium using GC-MS, fatty acid quantification kit could not be tested due to limitation of time.<br><br>
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<h5><b>Fatty Acid Treatment</b></h5>
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In measuring fatty acid, fatty acid solution was mixed with ethanol and chloroform. After acidifying by HCl and refluxing in water bath for 30 min, the organic layer containing fatty acid was collected and extracted by diethyl ether and petroleum ether solution. Again the organic layer was sucked out to be dried before NaOH was added. Then after derivatisation by BF3 and bromotetradecane, the organic layer was collected into GC-MS vial for analysis.
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<br><br>
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<h5><b>GC-MS</b></h5>
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GC-MS is a very useful tool to quantify volatile compounds effectively. For our experiment, we conducted calibration test using known concentrations of fatty acids. However, it was difficult to reach a conclusion due to lack of internal standards and uncertain amount of sample loss for<br>
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Latest revision as of 12:42, 28 October 2013

Protein Trafficking

Overview

In our project, we introduced bacterial glyoxylate enzymes into mammalian cells to create alternative metabolic pathway. However, unlike their native environment in bacteria, the two enzymes needed to find their way through the high compartmentalized system in order to reach the citric acid cycle where they could act on.
To guide the glyoxylate enzymes into the mitochondria where mammalian citric acid cycle resides, we produced recombinant glyoxylate enzymes by attaching the Mitochondrial Leader Sequence (MLS) to their N-termini.
We have also constructed the MLS in its own into standard BioBricks (BBa_K1119000 & BBa_K1119001), and we quantitatively characterized their behavior using GFP reporter.

Biology of Mitochondrial Leader Sequence (MLS)

In eukaryotes, the signal sequence guides the translocation of the newly synthesized peptide.

The story starts with the MLS attached to the N-terminus of the protein of interest. Once the protein of interest is synthesized, this preprotein would remain unfolded by associating with chaperons. The preprotein will stay in the cytosol until the MLS gets recognized by receptor of the TOM complex on the outer mitochondrial membrane. Binding of the MLS to the receptor will trigger the feeding of the peptide through the translocation channel. Afterwards, the MLS will then be handed over to a TIM complex which sits on the inner membrane, which will then open up the channels on the inner membrane and allow the peptide to pass through. Once the peptide is through the double membranes, mitochondrial chaperone will be involved in pulling the peptide into the mitochondria and refold the protein. Lastly, the MLS will be cleaved by signal peptidase and dissociate from the transported peptide. (Alberts, 2002)
For the MLS we used, four additional amino acid residues (Ile-His-Ser-Leu) will be left at the N-terminus of the protein after the cleavage.(Invitrogen, 2012)

Reference

Alberts, B., Bray, D., Hopkin, K., Johnson, A., Lewis, J., Raff, M., Roberts, K., & Walter, P. (2010). Essential cell biology. (3rd ed., p. 505). UK: Garland Science.

pCMV/myc/mito Invitrogen.(2012).pShooter™ Vector(pCMV/myc vectors).Retrieved from http://tools.lifetechnologies.com/content/sfs/manuals/pshooter_pcmv_man.pdf

Alberts, B. (2002). Molecular biology of the cell. (4th ed., pp. 1050-1061). New York:Garland Science.

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