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Plasmids Continued Once we insert the plasmid into the bacteria how do we know its in the bacteria and has the the right gene in it? AP Biology 2007-2008 Engineered plasmids Remember: plasmids are circular DNA from bacteria Plasmids have a antibiotic resistance genes as a selectable marker EcoRI BamHI HindIII restriction sites Selectable marker antibiotic resistance gene on plasmid ampicillin resistance selecting for successful transformation successful uptake of recombinant plasmid AP Biology plasmid ori amp resistance Selection for plasmid uptake Selection – make sure bacteria took up plasmid Antibiotic becomes a selecting agent only bacteria with the plasmid will grow on antibiotic (ampicillin) plate all bacteria grow only transformed bacteria grow a a a a a a LB plate AP Biology a a a a a a a a a a a LB/amp plate cloning Need to screen plasmids Screen – ____________________________________ _______________ Need to make sure bacteria have recombinant plasmid HOW CAN WE DO THIS: INSERT THE GENE OF INTEREST INTO A REGION OF THE PLASMID THAT CODES FOR A SEEABLE TRAIT AP Biology Example EcoRI BamHI inserted gene of interest restriction sites all in LacZ gene HindIII LacZ gene broken LacZ gene lactose blue color lactose X white color plasmid recombinant plasmid amp resistance origin of replication AP Biology amp resistance Finding your “Gene of Interest” AP Biology 2007-2008 How do we find the gene we want to insert? 1. Gene Libraries have been created 2. Use of cDNA 3. Make DNA synthetically AP Biology DNA libraries Cut up all of nuclear DNA from many cells of an organism restriction enzyme Clone all fragments into many plasmids at same time “shotgun” cloning Create a stored collection of DNA fragments AP Biology petri dish has a collection of all DNA fragments from the organism 1 Making a DNA library all DNA from many cells of an organism is cut with restriction enzymes 2 engineered plasmid with selectable marker & screening system gene of interest 3 4 clone plasmids into bacteria AP Biology all DNA fragments inserted into many plasmids But how do we find colony with our gene of interest in it? DNA library recombinant plasmids inserted into bacteria gene of interest DNA Library plate of bacterial colonies storing & copying all genes from an organism (ex. human) AP Biology ? Finding your gene of interest DNA hybridization find sequence of DNA using a labeled probe short, single stranded DNA molecule complementary to part of gene of interest labeled with radioactive P32 or fluorescent dye heat treat DNA in gel unwinds (denatures) strands wash gel with probe probe hybridizes with denatured DNA labeled probe genomic DNA AP Biology G A T C A G T A G C T A G T C A T C Find your gene in DNA library Locate Gene of Interest to find your gene you need some of gene’s sequence if you know sequence of protein… can “guess” part of DNA sequence “back translate” protein to DNA if you have sequence of similar gene from another organism… use part of this sequence AP Biology Which bacterial colony has our gene? Like a needle in a haystack! ? Colony Blots 4 Locate - expose film - locate colony on plate from film 1 Cloning - plate with bacterial colonies carrying recombinant plasmids plate plate + filter film 3 2 Replicate plate - press filter paper onto plate to take sample of cells from every colony AP Biology filter Hybridization - heat filter paper to denature DNA - wash filter paper with radioactive probe which will only attach to gene of interest Problems… Human Genome library are there only genes in there? nope! a lot of junk! human genomic library has more “junk” than genes in it Clean up the junk! AP Biology if you want to clone a human gene into bacteria, you can’t have… introns How do you clean up the junk? Don’t start with DNA… Use mRNA copy of the gene without the junk! But in the end, you need DNA to clone into plasmid… How do you go from RNA DNA? reverse transcriptase from RNA viruses retroviruses AP Biology reverse transcriptase cDNA (copy DNA) libraries Collection of only the coding sequences of expressed genes extract mRNA from cells reverse transcriptase RNA DNA from retroviruses clone into plasmid Applications need edited DNA for expression in bacteria human insulin AP Biology Make DNA synthetically Work Backwards Lets say you have a protein with the following amino acids Met, Pro, Asn, Lys, Met, Leu, Gln Find the DNA sequence that can would for it. AP Biology Problems! You also need to insert the correct promotor and control sequences so that it can be expressed AP Biology