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Molecular Genetics and Microbiology
Johanna Rommens

MGY376Monday November 21 Transformationgetting DNA into a host One of the three ways in which foreign DNA gets into bacteria Other two are conjugation and transductiono Conjugation mediated by plasmidsMating system involving conjugative pillus joining two bacteria o Transduction via phageDNA in phage headsTransformation just naked DNA going into bacteria In this lab using calcium chlorideartificialo Hijacking methods involving natural transformationo Some bacteria like hemophilus and nesseria are competent all the time Bacteria can take up DNA Have system where transport DNA into cell and its selective for own DNAspecific sequences recognized if these sequences present DNA uptake and incorporation into genomes by recombination Otherwise degradation Even E coli has similar systemcalled kai sequencesrecognized and shuts down degradative mechanismo Gram positive bacteria have competencea developmental process induced Alternative to sporulationturned on by quorum sensing and nutritional state of celly Turns on system that takes up DNAy Preferentially taking up own DNAUsing plasmid in lab to transformo To carry genes to be expressed in E colio Plasmid replicates autonomously so do not need recombination for it to get into cello Only need one plasmid to get in and then replicate and get more of ito pNBV1plasmidPUC originhigh copy number so many copies of plasmid in cell Pal originallows replication in broad host range of different microbacteriay So functional in both E coli and mycobacteriaHydromycinselectiono pNBV1 is negative controlo other two plasmids PSLR2 and PHNLScontain HNS gene which is basic protein of E coli that binds to DNA and has several roles binds to shut down genes binds ATrich sequences so tends to shut down foreign DNA foreign DNA when in any species of DNA seems to get more AT rich than the ATrichness of endogenous sequencesnot sure why ex Pathogenicity islands HNS binds to sequences and then shuts down transcription of these region silences foreign genes Once foreign DNA is established slowly over time the organism adapts to it incorporates these functions turning them on conditionally depending on the function Prevents harmful functions by Using HNS
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