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Lecture

Cloning (Continued)

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Department
Biochemistry
Course
Biochemistry 2280A
Professor
Derek Mc Lachlin
Semester
Fall

Description
Brandl Lecture 9 Notes 11/28/2012  E. coli are the most commonly used organism for expressing YFP  Q: Which of the following need to be considered when expressing your favorite human protein in E. Coli? A) Human and E. coli promoter structures are different B) Introns are found in human genes C) Different mechanisms of translational initiation D) Different protein modifications occur in prokaryotic and eucaryotic cells E) All of the above Expressing YFP in E. Coli  If plasmid is chosen correctly, it will have a promoter and ribosome binding site just upstream from the restriction site  Gene will be inserted into cloning site which will put the gene in a position where it will be expressed  See figure 10-24 Producing YFP  The promoter used is usually inducible because some human proteins are toxic to bacteria  Purify protein from E. coli  Check protein for purity and activity (whether it is functional or not)  YFP is now ready for use Site-Directed Mutagenesis  Custom designing YFG  How do we create a gene with altered sequence and in turn altered function?  You believe that an aspartate within your favorite protein may be leading to insolubility  To overcome this, we must convert a codon for Asp to a codon for Ala o Site directed mutagenesis of a protein coding gene o Asp → Ala, GAC → GCC  Start with double-stranded plasmid and denature it, obtaining single-stranded plasmid o Synthetic oligonucleotide primer containing desired mutated sequence  Anneal/hybridize oligonucleotide to the single-stranded plasmid  Strand completion by DNA polymerase and DNA ligase, making it a double-stranded molecule  Transform recombinant molecule into cells – replication and segregation into daughter cells o Some colonies will have the wild type gene o Some colonies will have the mutant gene Transgenic Organisms  Genetically modified organisms  An organism that has had its genome permanently altered by genetic engineering  GMOs are valuable in research and biotechnology Genetic Changes  Three different types of genetic changes are possible o Gene replacement o Gene knockout o Gene addition  These are all done using a procedure called homologous recombination (same process used to repair DNA)  Gene knockout: o Taking a gene and removing it, no active gene is present
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