Dave Gray's Build-A-Gene Class Notes

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The goal of the project is to add a gene to E. coli bacteria that will cause it to generate a protein that is phosphorescent under UV light. It is an end-to-end introduction to one of the routes used to introduce foreign genetic material to a living organism.

The process we will use will not actually splice the DNA into the genome of the E. coli. It turns out that the DNA of bacteria naturally occurs in a crumpled up ring (like a rubber band stuffed into a bottle cap.) It is possible that a small section of the DNA can separate into its own little ring (a “plasmid”) and function alongside the larger DNA molecule just as effectively as if it was attached. It acts as a sort of separate, tiny, ring-shaped chromosome. (I would think this would sometimes be disruptive by separating segments of DNA needed to assemble a protein – have to ask about that.) A good visualization can be seen here. In effect, we will be building a plasmid and adding it to the E. coli.

What we are building
This shows the plan of the plasmid we will be assembling. First we amplify the vector + terminator and the promoter + RBS segments. Next, we assemble the emGFP coding sequence from oligonucleotides. Then we splice these together.

Session Notes

BUGSS:Build-a-Gene Main Page | Glossary