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Team:Washington/Alkanes/Methods
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(→Basic Alkane Production Construct) |
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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. | 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. | ||
Revision as of 03:35, 16 September 2011
Basic Alkane Production Construct
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.
Alkane Production
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.
Alkane Extraction
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.
Alkane Detection
Explain GCMS... again basically graphics from presentation with text
