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Lecture 35: "Expressing YFG & DNA Fingerprinting"

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Western University
Biochemistry 2280A
Mel Usselman

Biochemistry Lecture No. 35: Expressing YFG & DNA Fingerprinting th Thursday November 29 , 2012 Expression Of YFG: Q) What sorts of things must be considered when inserting your human protein for expression in E. Coli? Prokaryotic organisms differ in that their genes do not contain introns, and share different promoters, translational sequences as well as post-translational modifications. The issue with introns is largely solved through the use of cDNA and the different promoters and translational mechanisms can be easily accommodated by the design of the plasmid you choose in cloning YFG. In expression plasmids, it is necessary that they have a bacterial promoter as well as a ribosome-binding site in front of the integrated cDNA of YFG to be expressed. The promoter that is used is usually an inducible promoter whereby the human protein is not expressed until the bacterial cell has reached maturation, so as not to negatively impact the development of the E. Coli. Once you’ve induced the protein, the protein is isolated from E. Coli, purified (protein homogenization and chromatography) and checked for activity. This result is the expression of YFG. Site-Directed Mutagenesis: -Site-directed mutagenesis is process used when you want to alter a gene to create a new gene that has lost its function or gained a new function. In the example that YFP contains an aspartate (GAC) which is leading to insolubility, you can convert it to an alanine codon (GCC) to overcome this problem. The key to the procedure is the use of synthetic oligonucleotides. The first step is to separate the plasmid DNA strands (containing YFG) by heating and then a oligonucleotide primer (containing the GCC codon change) will bind to the aspartate site in the plasmid, creating a mismatch that can be hybridized. The subsequent addition of DNA polymerase and DNA ligase will synthesize the new DNA strand and seal up the nick at the end respectively. The result is a double-stranded plasmid that contains a mismatch at the favoured position. This DNA is then transformed into E. Coli, in which some individuals will have the mutant sequence (GCC), while others will retain the wild-type sequence (GAC). By way of DNA sequencing, the mutant colonies are checked for the right genetic code. Theoretical yields are about a 50:50 mutant-to-wild type ratio, while actual yields are somewhere closer to 10:1. Transgenic Organisms: -Transgenic organisms (or genetically-modified organisms) are organisms in which their genomes have been permanently altered by genetic engineering. There are three main kinds of genetic changes possible in transgenic organisms: gene knockout, gene replacement and gene addition. Through the process of homologous recombination, you can create an organism with unique features for use in research or to produce a unique product. In gene knockout, you simply remove the coding region of the gene of interest from the organism (no active gene is present). Gene knockout is very easy to perform on model organisms and it serves as a definitive test for determining the function of a gene/protein (e.g. non-spoiling tomato). Gene addition requires the insertion of another active gene into the organism. Gene addition is the basis of biotechnology as it can create an organism that expresses novel proteins (e.g. golden rice). Gene replacement is the slight tinkering of an active gene through site-directed mutagenesis in order for its protein to function differently. Gene replacement allows for the examination of an altered gene’s function or the creation of a gene with altered features (e.g. non- allergenic
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