Dave Gray's Build-A-Gene Class Notes

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.

Terms Used

• 5' - The "upstream" end of DNA. The end of the DNA or RNA strand that has the fifth carbon in the sugar-ring of the deoxyribose or ribose at its end. If the 5' end is missing, the DNA cannot be extended by joining additional neucleotides beyond that point.
• 3' - The downstream end of DNA. Can attach to a 5' end.
• Anneal - Bind two strands of DNA.
• Amplify - Make copies of a DNA template.
• Denature - Separate the two strands of DNA. This is done by heating the DNA to near the boiling point of water.
• GFP – Green Florescent Protein - GFP variants contain about 750 nucleotides. We order 60 NT sets (about 20 pieces)
• Nucleobases - These are the coding components of DNA and only come in four flavors – A C T and G (adenine, thymine, cytosine and guanine). A always binds with T and C with G. A fifth nucleobase U (uracil) replaces T (thymine) in RNA and lacks the 5' methyl group. A and G are called purines. C T and U are pyrimidines.
• Nucleotides – A building block of DNA consisting of a nucleobase, a five-carbon sugar (either ribose or 2-deoxyribose), and one or more phosphate groups. The 5-carbon sugar and phosphate group(s) make up the “backbone” of the DNA.
• Vector - A DNA molecule used as a vehicle to artificially carry foreign genetic material into another cell.
• PCR – Polymerase Chain Reaction – A process for replicating (“amplifying”) DNA or a portion of a DNA molecule.
• Primer - A strand of nucleic acid that serves as a starting point for DNA synthesis. It is required for DNA replication because the enzymes that catalyze this process, DNA polymerases, can only add new nucleotides to an existing strand of DNA. The polymerase starts replication at the 3'-end of the primer, and copies the opposite strand.
• Promoter – A region of DNA that initiates transcription of a particular gene. Can be strong, weak or function only in the presence of a certain chemical.
• RBS - Ribosome Binding Site - Point at which protein synthesis starts
• Terminator - Stops MRNA synthesis.

Session Notes

• Session 1
• Session 2
• Session 3

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