23 BIO 326R Plasmids - BIO 326R Plasmids Plasmids Small...

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BIO 326R Plasmids
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Plasmids Small (usually) autonomously replicating extrachromosomal DNA Conjugative or non-conjugative Usually circular (we will only consider circular plasmids) B form DNA Usually possess negative supercoils (i.e. they are underwound), probably due to DNA gyrase activity. This might help maintain the DNA in a more “open” configuration to facilitate transcription and translation. Must have means of replication Origin of replication – specific sequence on plasmid ori, oriV rep gene(s) coded on plasmid allow initiation at plasmids origin or replication Origin of replication helps determine copy number, host range and compatibility with other plasmids Commercial plasmids usually use the ColE1 origin. Confer some selective advantage to host cell, but only under certain conditions e.g. Metabolic activities, antibiotic resistance With lack of selective pressure, they can and will be lost, (despite mechanisms in place to prevent loss). Loss of a plasmid is called curing. Curing can be accomplished by lack of selection or intercalating agents (Acridine orange, ethidium bromide).
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Plasmid maps Protein encoding ORFs designated by block arrows (arrow indicates direction of transcription) Unique restriction sites noted and given numerical position “Unique” means that there is only one site for that enzyme in this plasmid A large number of unique restriction sites are found within the MCS (Multiple Cloning Site) Commercial plasmids will have Antibiotic resistance bla, AmpR, tet are most common. called a selectable marker Origin of replication Cloning site
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Copy number: relaxed vs. stringent Too many copies = too much of a burden to cell, Less than two just prior to division = plasmid is gradually lost i.e. if a cell with only one plasmid divides, only one daughter cell can inherit the plasmid Copy number is a function of the origin of replication Relaxed plasmids will have higher copy number (30 or more/ cell) Too many copies will present undue burden on cell and slow growth To confuse things further, relaxed plasmids can be high or low copy number ColE1 ori plasmids produce ~30 / cell if the plasmid also contains an rop gene. They produce 100-200 / cell if the plasmid lacks the rop gene. Obvious point: many commercially available plasmids lack the rop gene. Stringent plasmids will have a low copy number (1-2 / cell) Very low copy number requires regulation of partitioning – random distribution to daughter cells would result in rapid loss of plasmid
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Partitioning Stringent plasmids (1-2 copies / cell) must regulate distribution of DNA to daughter cells Plasmid P1 partitioning: parS : cis acting sequence on plasmid parB : site specific DNA binding protein encoded on plasmid (parB protein binds parS site on plasmid DNA) parA : protein with ATPase activity, also encoded on plasmid. Binds parB:DNA complex and uses ATP to move plasmid – much of movement process not well understood
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23 BIO 326R Plasmids - BIO 326R Plasmids Plasmids Small...

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