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pGLO Bacterial Transformation Green Fluorescent Protein or GFP What is transformation? Uptake of foreign DNA, often a circular DNA called a plasmid GFP Bacterial chromosomal DNA Amp Resistance pGLO plasmids What is a plasmid? • A plasmid is a small circular piece of DNA (about 2,000 to 10,000 base pairs) that contains important genetic information for the growth of bacteria. A plasmid containing resistance to an antibiotic is used as a vector. • The gene of interest is inserted into the vector plasmid and this newly constructed plasmid is then put into E. coli that are sensitive to ampicillin. • Vector - Something that is used to transfer something else (a mosquito is a vector for the organism that causes malaria) The bacteria are then spread over an agar plate that contains ampicillin. • The ampicillin provides a selective pressure because only bacteria that have acquired the plasmid can grow on the plate. • Therefore, as long as you grow the bacteria in ampicillin, it will need the plasmid to survive and it will continually replicate it, along with your gene of interest that has been inserted to the plasmid. Mouse under blue light (left) Same mouse under normal light (right) Mouse blood vessels (green-GFP) in tumor (red-DsRed). Mouse with brain tumor expressing DsRed. Three 60 day old kittens. Two have been genetically modified to make red fluorescent protein. All three look similar under normal light, but when irradiated with blue light only the two genetically modified kittens glow red. What is the GFP gene? • GFP is a green fluorescent protein that normally is found in jellyfish. Engineering Green Fluorescent Protein (GFP) • GFP has been added to : – rabbits – rats – mice – frogs – flies – worms – and countless other living things S. Stevens 6/14/2006 GFP requires the sugar Arabinose in order to turn on the GFP Gene. • Called the arabinose operon of E.coli: • Arabinose, a 5 carbon sugar, serves as energy and carbon source for the bacteria. • There are 3 genes (araB, araA, & araD) that need to be expressed for the transport and breakdown of arabinose. • These genes are only expressed when the sugar arabinose is present in the environment. These three ara genes are clustered together and make up the arabinose operon. The ara regulatory sequences precede the 5’ end of the araB gene. • They are “upstream” from the coding sequences. • The promoter region is the site that binds the RNA polymerase enzyme (required for transcription). • Just upstream from the promoter lies the binding site for the arabinose gene activator, araC. • This activator protein is bound to the regulatory sequence and changes shape when the sugar, arabinose, binds to it. • This shape change allows RNA polymerase to bind at the promoter sequence & transcription is initiated. • The three genes, araB, araA, and araD are transcribed and translated to produce the three enzymes required to breakdown arabinose. • As the arabinose is used up and broken down, AraC has no arabinose to bind to it, AraC returns to its original shape, RNA polymerase binding does not occur and transcription is then turned off. Overview: • A. AraC is blocking the binding of RNA polymerase for transcription of the gene responsible for degradation of arabinose. • B. Arabinose binds to araC causing a change that reveals the promoter region. • C. RNA polymerase binds to promoter. • D. Transcription of the arabinose operon is achieved. pGLO Plasmid Cloning of Plasmid TRANSFORMATION • THE PROCESS BY WHICH BACTERIA CELLS PICK UP AND INCORPORATE DNA FROM DEAD BACTERIA CELLS. • THEY TAKE UP DNA FROM DEAD CELLS OF THE SAME OR A CLOSELY RELATED SPECIES Genomic Bacterial DNA S. Stevens 6/14/2006 Plasmid S. Stevens 6/14/2006 Antibiotic Resistance Amp resistant E. Coli on Left E. Coli on Right S. Stevens 6/14/2006 Competent Cells • Cells must be made competent before they can absorb the plasmids that carry the new genes. • In this lab, we will make our cells competent by soaking them in CaCl2 solution and heat shocking them.