Accelerated Biology Transformation Lab

Transformation Process by which a bacterium “takes up” and expresses “outside” DNA resulting in a new genetic trait that is stable and can be passed on to other generations. Can be Recombinant DNA or natural DNA

Recombinant DNA Animation DNA in which one or more genes have been inserted from a different organism (Basically, combining the DNA of two different organisms) Recombinant DNA Animation

Natural DNA Oswald Avery – Transforming Principle

How Does Transformation Work? Bacterial cells must be in a particular physiological state competency Basically, it means that the bacterial cell wall is permeable to macromolecules (like DNA) We will be transforming the bacteria E. Coli, so the E. Coli will need to “take up” the GFP gene.

How Can We Make the E.Coli Competent? We will be treating it with CaCl2 and abruptly alternating it between hot and cold temperatures. This will allow the cell wall and cell membrane of the E.Coli to become permeable and allow DNA molecules to pass through it.

Plasmids To carry foreign DNA into cells, plasmids are often used A plasmid is an extra piece of circular DNA found in most bacteria Many plasmids also can carry genes that are resistant to certain antibiotics

What Will Our Plasmid Look Like? Our plasmid will contain the gene for bioluminescence and a gene that is resistant to the antibiotic ampicillin (amp)

So, What’s the Goal? The goal is to express fluorescent proteins (gfp) in transformed bacterial cells. Gene Expression – a gene is expressed or “turned on” when transcription occurs, but cells do not always need to produce all of the proteins for which their genes code

Gene Expression in Prokaryotes Example: Lac Operon Earliest studies were done with how genes control the metabolism of the sugar lactose in E. Coli Lactose is found naturally in cow’s milk When you drink cow’s milk, the presence of lactose stimulates E. Coli to produce 3 enzymes.

Gene Expression in Prokaryotes Example: Lac Operon

What is an Operon? 1. Structural Genes – Code for a protein ie. Code for the enzyme that allows E. Coli to digest and use lactose 2. Promoter – DNA segment that recognizes the enzyme RNA polymerase & thus promotes transcription 3. Operator – DNA segment that serves as a binding site for an inhibitory protein that blocks transcription & prevents protein synthesis from occurring

What’s the Operon? Those 3 together (structural genes, promoter, operator) for an operon Lac operon – named that because the structual genes coded for the enzyme that digests lactose Found that these genes for utilizing lactose were only “turned on” when lactose was present

Lac Operon Animation Lac Operon Animation

So, How Can Genes Be Turned “Off” and “On” Repression In the absence of lactose, a protein called a repressor attaches to the operator; and it prohibits the RNA polymerase from binding to the structural genes, and transcription can’t occur

So, How Can Genes Be Turned “Off” and “On” Activation When lactose is present, it temporarily binds to the repressor protein on the operator and removes it. Now, RNA polymerase can transcribe the structural genes and the enzyme (protein) is produced to break down the lactose

To begin, we need to be able to “turn on “ the cloned gfp gene in the recombinant plasmid. This can be turned on by the presence of a sugar molecule called arabinose Arabinose will bind to and inactivate the lac repressor RNA polymerase can recognize the lac promoter, and begin transcription and the proteins will be produced that contain bioluminescence and ampicillin resistance