Transcript Vectors for Even Larger Genomic DNA Inserts

Microbial Genetics (Micr340)
Lecture 7
Plasmids
Plasmids
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DNA molecules other than chromosomes
Widely present in most bacteria
Roles: adaptation, evolution, pathogenesis
Usually encode genes not essential
Mostly circular
Sizes: a few kb to >100 kb
Naming Plasmids
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Many name after genes they carry or
functions of the plasmids
In the beginning:
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ColE1, producing Bacteriocin to kill E. coli
Tol, degradation of toluene
Ti, Tumor initiation in plants
RK2, resistance to amp, tet, kanamycin
Now: pXXNNNN. eg, pHX200
Plasmid
structure
Plasmid purification
Plasmid purification
Plasmid replication
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Replicons: DNA molecules that can replicate
autonomously in cell (chromosome, plasmid)
All replicons have at least one origin of
replication (ori)
For plasmid ori, it is called oriV (vegetative)
Two mechanisms of replication:
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Theta replication
Rolling-circle replication
Theta replication
Rolling-circle replication
Function of ori region
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Replication related genes close to ori.
Host range determination
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Narrow host range
Broad host range
Regulation of copy-number
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Relaxed plasmids – high copy-number
Stringent plasmids – low copy-number
Regulation
of replication:
ColE1 plasmids
Random plasmid curing
Plasmid Partition
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Plasmid avoid being lost from dividing cells by
carrying partitioning systems
The functions involved in these systems are
called par functions
cis-acting site: parS
trans-acting site: parA and parB
Incompatibility
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Many bacteria contain multiple types of
plasmids and can coexist for many
generations
Not all types of plasmids can stably coexist in
same cell; some interfere with each other’s
replication or partition and one of them will
be lost
This is called plasmid incompatibility
Incompatibility
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Two plasmids that cannot stably coexist are
members of the same incompatibility (Inc)
group
If two plasmids can stably coexist, they
belong to different Inc group
Coexistence of different Inc groups
Coexistence of
same Inc groups
Measuring curing of a plasmid
Finding the
ori of a plasmid