Team:Washington/alkanebiosynthesis cloning


Alkane Biosynthesis Cloning

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.

After we have the complete assembled gene in our hands, 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.