Cheat sheet:Ecoli: small, needs few nutrients, reproduces fast, aerobic/anaerobic; grows rapidly with a doubling time of around 22 minutes. Also, it can be easily transformed with foreign DNA making it useful in recombinant DNA experiments. S. cervisiae: yeast, eukaryotic, similar cell cycle to humans, post translational modification/ alternative splicing. Stanford University (Cohen) and UCSF (Boyer) $255 million. The eukaryotecell will splice out the introns during transcription while a prokaryotecell will not, leading to a much larger protein with altered structure and function. Euks have nucleus and compartmentalization. Bacteria have promotersin front of operons. Operator region before coding dna. Repressorsbinds to operator region. Effectorcan bind a repressor to change conformation. Effector can be same as end product: negative feedback loop. Repressor can be inactive until compressor binds it. activatorcan bind operator region. Effector can activate inactive activator protein. Translation 5’-3’. HOW TO CLONE GENE.First, cut p1 and p2 with the restriction enzymes, EcoR1 and NotI. These sites flank gene A and are present in p2 allowing for transfer of a minimal amount of desired DNA. Next, perform a ligation reaction with the small fragment of p1 containing gene A and the large fragment from p2 containing the AmpRgene (isolate fragments through separation on a gel). Then transform the ligation reaction product into E. coli competent cells. Select the transformed cells for ampicillin resistance. Isolate plasmid DNA from the selected cells and perform a check digest to check for the insertion of gene A into p2. Finally, sequence the cloned plasmid from positive check digests to confirm insertion of gene A==First, the gene for the protein is PCRed from the bacterial genome using two primers designed to incorporate restriction endonuclease sites for cloning. Next, the strand of PCRed DNA and the expression plasmid are cut with the proper restriction endonucleases. The desired fragments are isolated by gel separation and attached using a ligation reaction. The ligation reaction products are then transformed into E.coli. The transformed cells are selected by growing the cells on the antibiotic corresponding to the selective marker on the plasmid. Finally, the plasmids in the selected cells are checked using restriction digests and sequencing. What else could the gene be important for that would inhibit antibiotic synthesis? (1 pt) The mutation could be located in a global regulator gene that is involved in other processes or in a gene responsible for the production of a precursor to erythromycin. Having a genome sequence helped identify this protein since the 24 amino acid sequence determined experimentally could be compared to protein sequences identified from the genome sequence. Putative protein sequences can be identified from a genome sequence by searching for open reading frames. Mutagenesis
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