plasmid-08 - THE GENETICS AND USEFULNESS OF PLASMIDS Paul...

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1 THE GENETICS AND USEFULNESS OF PLASMIDS Paul A. Gulig I. Plasmids - General A. Extrachromosomal DNA, usually circular B. Usually encode ancillary functions for in vitro growth C. Can be essential for specific environments: virulence, antibiotics resistance, use of unusual nutrients, production of bacteriocins (colicins) D. Must be a replicon - self-replicating genetic unit II. Replication A. Plasmid DNA must replicate every time host cell divides or it will be lost B. Host cells do not “spit out” plasmid DNA C. Two functions of replication 1. DNA replication 2. partitioning (making sure each progeny cells receives a plasmid) D. high copy (>20) and low copy (<5) plasmids E. high copy plasmids are usually small; low copy plasmids can be large F. partitioning is strictly controlled for low copy, but loose for high copy G. plasmid replication requires host cell functions (DNA polymerase, etc.) H. copy number is regulated by initiation of plasmid replication I. plasmids are incompatible when they cannot be stably maintained in the same cell because they interfere with each other’s replication. J. Models of replication 1. F plasmid a. large (100 kb) b. low copy (1-2 copies/cell) c. self transmissible d. requires protein synthesis (chloramphenicol-sensitive) e. repE gene encodes RepE protein f. RepE protein binds to origin of replication ( oriS ) and initiates DNA replication g. RepE binds to the repE promoter and activates transcription h. RepE binds to the copA/incC locus and causes plasmids to bind together via RepE thereby inhibiting replication (coupling) 2 . ColEI plasmid a. small (6.6 kb) b. high copy (20 copies/cell) c. non-transmissible d. does not require de novo protein synthesis for replication (chloramphenicol amplifiable) e. RNA-II is transcribed through the origin of replication, gets cut by RNaseH and serves as the primer for DNA replication f. RNA-I is transcribed in the opposite orientation and is complementary to RNA-II. g. binding of RNA-II and RNA-I prevents initiation of replication (RNA-I is a negative regulator) h. the Rom/Rop protein made by the rom/rop gene stabilize the binding of RNA-I and RNA-II (also negative regulator)
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3 III. PLASMIDS FOR FUN AND PROFIT Choosing a plasmid vector is like shopping for a new car. You have to take into account what you are going to use it for, how much power you want under the hood, whether it is for the road or all-terrain, how big, the color, and various other options that might make life easier (or more difficult). A. Use.
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This note was uploaded on 12/13/2011 for the course GMS 6038 taught by Professor Gulig during the Fall '11 term at University of Florida.

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plasmid-08 - THE GENETICS AND USEFULNESS OF PLASMIDS Paul...

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