Team:Washington/Alkanes/Methods

From 2011.igem.org

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<center><big><big><big><big>'''Diesel Production: Methods'''</big></big></big></big></center><br><br>
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=Basic Alkane Production Construct=
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='''Introducing the PetroBrick'''=
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[[Image:Washington_2011_PetroBrick.png|150px|frameless|border|left|link=http://partsregistry.org/wiki/index.php?title=Part:BBa_K590049]]
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In order to produce alkane, we need both acyl_acp reductase and aldehyde decarbonylase to work together in the cell. In order to achieve this goal, we use the biosynthesis method. First, we digest both acyl-ACP reductase and aldehyde decarbonylase genes on restriction sites Xbal I and Pst I. At the same time, we digest the vector on restriction sites Spe I and Pst I. Then we ligate one of the tow genes into the vector. Then we transform the complete plasmid into the cell and plate the cell. At the second day, we pick cells from the plates run a colony PCR and check the plasmid on the gel to ensure we have the right plasmid. Once we have the right plasmid. We go thorough the digestion on Spe I and Pst I on the plasmid and ligate in the other genes. Then, we repeat the same process of transformation, colony PCR, and gel checking the plasmid size. Once the gel checking shows we have the right size of plasmid, we send the plasmid out for sequencing to make sure the sequence matches.
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=Alkane Production=
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<p>In order to produce alkanes, we need both [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590032 Acyl-ACP Reductase] (AAR) and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590031 Aldehyde Decarbonylase] (ADC) to work together in the cell. To achieve this goal, we used standard cloning methods combining both to construct the [http://partsregistry.org/Part:BBa_K590025 BBa_K590025] Biobrick which contains both AAR and ADC under a high constitutive promoter, each with its own Elowitz standard RBS. This construct successfully synthesized our target product; thus, we have created a new modular alkane-producing platform:</p>
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After we have the complete assembled gene in our hand, the next step is to transform it into the cells and start the growing and alkane production process. We plate the cells after transformation and let them grow in 37 degree incubator. At the second day, we pick couple cells from the plate and inoculate them into the TB media to grow overnight. At the third day, we pallet the cells from the overnight growing TB media. Then resuspend them in sterile water and measure the OD of the cell water. Once we have the OD of the cell water, we calculate the volume of the cell water need to inoculate into each of the alkane production media at same OD.  
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[[File:Washington PetroBrick Construct.png|730px|center|frameless|border|bottom|link=http://partsregistry.org/wiki/index.php?title=Part:BBa_K590025]]
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=Alkane Extraction=
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[[File:Washington_Alkane_extraction.png|right|400px|thumb|Diagram showing the process of extraction]]
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After the cells have gone through the alkane production process, the next step is to extract alkane out of the mixture of cells and media. The method we come up here is to use acyl acetate. First, We add acyl acetate directly into the glass test tube for cell growing. Then we votex till to everything is well mixed to make sure all the alkanes go into acyl acetate. Second, we spin down the mixture by using centrifuge to form three layers (cell pallet, media, and acyl acetate), which makes it easier to take out the acyl acetate for assay.
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='''Alkane Production & Extraction'''=
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[[File:Washington_Alkane_extraction.png|right|400px|thumb|Diagram showing the process of extraction.]]<p>After we had the complete gene assembly in our hands, the next step was to transform it into cells and start them growing for alkane production. We let them grow in 37 degree shaker for 48-72 hours, in sealed glass tubes. After the cells have gone through the alkane production process, the next step is to extract the alkanes out of the cell broth. We add acyl acetate directly into the glass test tube for cell growth. Then we vortex until to everything is well mixed, to make sure all of the alkanes go directly into the ethyl acetate solvent. Next, we spin down the mixture by using a centrifuge at full speed to form three layers (cell pellet, media, and ethyl acetate supernatant). We use only the ethyl acetate layer to send for GCMS analysis. For a more extensive coverage of our media, growth, and extraction techniques, refer to our [https://2011.igem.org/Team:Washington/alkanebiosynthesis Protocols page]. </p>
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=Alkane Detection=
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Explain GCMS... again basically graphics from presentation with text
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='''Alkane Detection'''=
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==Gas Chromatography and Mass Spectrometry==
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We utilized a Gas Chromatograph / Mass Spectrometer (GCMS) to analyze alkane production concentrations. The GCMS is considered a "specific" test, because it identifies compounds specifically, not just to a category of compounds. It works by separating the individual components of a sample through a capillary column based mainly on its boiling point, similar to fractional distillation. The separated compounds generally elute from the column at different retention times, and are passed to the mass spectrometer. 
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Inside the mass spectrometer each compound is then broken down into its individual molecular components through electron beam ionization. These ions differ in mass-to-charge (m/z) ratios, creating a unique ion de-composition profile for each compound that can be used to identify it through comparison to known chemical standards. Because compounds occasionally have similar elution times or mass spec fingerprints, the combination of analyses results in reducing the chances for overlap.
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<center><gallery widths="400px" heights="300px" perrow="2">
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Image:Washington2011_Chrom.png|'''Gas chromatography:'''  A method used to separate molecules from the media extraction based on boiling point. In this image the temperature is increasing over time, and molecules with a higher boiling point are being eluted and detected by a mass spectrometer.  The ion abundance is concentration dependent and can be converted if using a standard curve
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Image:Washington2011 Spectra.png|'''Mass Spectrometry:'''  Molecules exiting gas chromatography enter an electron impact mass spectrometer.  The molecules are ionized and fragmented.  The resulting spectra is compared to a database of molecules in order to predict its chemical identity.  On the top (red) is an experimental spectra of our biologically produced C15 alkane, on the bottom (blue) is the NIST standard spectra for a C15 alkane.  The fragmentation pattern and parent ion (blue arrow, bottom right) match perfectly.
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</gallery></center>

Latest revision as of 01:56, 29 September 2011


Diesel Production: Methods


Introducing the PetroBrick

In order to produce alkanes, we need both [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590032 Acyl-ACP Reductase] (AAR) and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590031 Aldehyde Decarbonylase] (ADC) to work together in the cell. To achieve this goal, we used standard cloning methods combining both to construct the [http://partsregistry.org/Part:BBa_K590025 BBa_K590025] Biobrick which contains both AAR and ADC under a high constitutive promoter, each with its own Elowitz standard RBS. This construct successfully synthesized our target product; thus, we have created a new modular alkane-producing platform:

Washington PetroBrick Construct.png



Alkane Production & Extraction

Diagram showing the process of extraction.

After we had the complete gene assembly in our hands, the next step was to transform it into cells and start them growing for alkane production. We let them grow in 37 degree shaker for 48-72 hours, in sealed glass tubes. After the cells have gone through the alkane production process, the next step is to extract the alkanes out of the cell broth. We add acyl acetate directly into the glass test tube for cell growth. Then we vortex until to everything is well mixed, to make sure all of the alkanes go directly into the ethyl acetate solvent. Next, we spin down the mixture by using a centrifuge at full speed to form three layers (cell pellet, media, and ethyl acetate supernatant). We use only the ethyl acetate layer to send for GCMS analysis. For a more extensive coverage of our media, growth, and extraction techniques, refer to our Protocols page.


Alkane Detection

Gas Chromatography and Mass Spectrometry

We utilized a Gas Chromatograph / Mass Spectrometer (GCMS) to analyze alkane production concentrations. The GCMS is considered a "specific" test, because it identifies compounds specifically, not just to a category of compounds. It works by separating the individual components of a sample through a capillary column based mainly on its boiling point, similar to fractional distillation. The separated compounds generally elute from the column at different retention times, and are passed to the mass spectrometer.

Inside the mass spectrometer each compound is then broken down into its individual molecular components through electron beam ionization. These ions differ in mass-to-charge (m/z) ratios, creating a unique ion de-composition profile for each compound that can be used to identify it through comparison to known chemical standards. Because compounds occasionally have similar elution times or mass spec fingerprints, the combination of analyses results in reducing the chances for overlap.


Gas chromatography: A method used to separate molecules from the media extraction based on boiling point. In this image the temperature is increasing over time, and molecules with a higher boiling point are being eluted and detected by a mass spectrometer. The ion abundance is concentration dependent and can be converted if using a standard curve

Mass Spectrometry: Molecules exiting gas chromatography enter an electron impact mass spectrometer. The molecules are ionized and fragmented. The resulting spectra is compared to a database of molecules in order to predict its chemical identity. On the top (red) is an experimental spectra of our biologically produced C15 alkane, on the bottom (blue) is the NIST standard spectra for a C15 alkane. The fragmentation pattern and parent ion (blue arrow, bottom right) match perfectly.

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