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22 Cards in this Set
- Front
- Back
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What do rep genes allow?
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Initiation at plasmid's origin of replication
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What does the origin of replication help determine?
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Copy number
Host range (?) Compatibility with other plasmids |
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What is a common origin of replication that commercial plasmids use?
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ColE1 origin
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What will prevent plasmids from being lost through evolution?
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Selective pressure prevents curing (loss of plasmids)
Limitation of intercalating agents (Acridine orange, ethidium bromide) |
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Where are a large number of unique restriction site found in a plasmid?
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the MCS (Multiple Cloning Site)
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What controls the copy number?
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The origin of replication
rop genes |
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What is the function of rop genes?
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Reduce the copy number per cell
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In relation to copy number, what are the different kinds of plasmids?
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Relaxed -- 30 or more per cell
Stringent -- 1-2 per cell |
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What do Stringent plasmids require in order to prevent rapid loss of plasmids?
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Partitioning -- regulated distribution to daughter cells
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Describe Plasmid P1 partitioning.
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parS -- cis acting sequence on plasmid
parB -- site specific DNA binding protein encoded on plasmid (parB binds parS site on plasmid DNA) parA -- motor protein with ATPase activity, also encoded on plasmid. Binds parB:DNA complex and uses ATP to move plasmid |
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What determines whether the host range of a plasmid in wide or narrow?
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Origin of replication
ColE1 plasmids only replicate in E. coli Broad host range plasmid will often encode all genes necessary for initiating replication |
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Which proteins made by the E. col F plasmid cause a cell harboring the plasmid to be addicted to the plasmid?
Describe their function. |
CcdB -- can bind and inactivate DNA gyrase (lethal)
CcdA -- binds and inactives CcdB, but is less stable than CcdB due to rapid degradation by a host protease |
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What are requirements for cloning vectors?
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High copy number
Ease of cloning (unique restriction sites) Minimum size Selectable marker (e.g. beta lactamase for ampicillin resistance) |
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How do the types of restriction endonucleases act?
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Type I -- recognize specific site but cut DNA at random location far from recognition site
Type II -- recognize specific site and cut within or adjacent to that site. ***Widely used. Very reliable Type III -- cleave DNA about 25 bp from recognition site |
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How can endonucleases distinguish host and page DNA?
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The endonucleases cannot cleave METHYLATED host DNA
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Describe the behavior of Type II restriction enzymes.
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Form dimer which specifically recognizes and cleaves a palindromic DNA sequence
Both phosphodiester backbones are cut such that the cleavage is symmetrical -- blunt or sticky ends |
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What is the process to construct a library?
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Digest both the plasmid and foreign DNA with EcoR1
Mix and ligate If foreign DNA inserts into plasmid, the plasmid will now have 2 EcoR1 sites the the foreign sequence in between |
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If you insert foreign DNA to plasmid, what happens to the LacZ protein?
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You won't translate a working LacZ protein because the betalactamase is disrupted, rendering it non-functional
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What does IPTG do?
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It is chemical which induces transcription of the LacZ gene
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What is X-gal?
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Commerical reagent, undergoes colorimetric change when treated with beta galactosidase -- changes from colorless to dark blue
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What qualities make for a good host cell for plasmids?
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No endogenous restriction enzyme
Ability to take up DNA (plasmid) at an acceptable rate (competence) Ability to support replication of plasmid (ori of the plasmid must be functional in host cell) No endogenous beta galactosidase activity (would cause all colonies to be blue -- no screening possible) Susceptible to antibiotic selection (for screening purposes) Must not recombine DNA insert with host genome (recA- == deficient in DNA recombination) |
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What does uncut plasmid have 3 bands on a agarose gel?
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Different apparent weights of supercoiled (fastest), linear, and open circle (slowest) plasmids
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