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Chapter 20

MCB 3020L Chapter Notes - Chapter 20: Calcium Chloride, Electroporation, Plasmid


Department
Microbiology
Course Code
MCB 3020L
Professor
Scheurle Daniela
Chapter
20

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Chapter 20
Chapter 20: Bacterial Transformation- Part B
Methods that have been developed to produce competency in naturally
incompetent bacteria and to transform these bacteria artificially
One method for this is the treatment of bacterial cells with an ice-cold calcium
chloride (CaCl2)
o Exact mechanism by which this makes the bacteria more permeable is not
well known or understood
o What IS known is that this treatment allows foreign DNA to pass through the
membrane and into the cell
o A concentration of 100 mM makes the bacterial cell membranes more
permeable
Another method: electroporation: the bacterial cells are subjected to high voltage
electrical impulses that destabilize the cell membrane, resulting in increased
permeability and enabling DNA to pass into the cells
Plasmids: small circular pieces of extrachromosomal, double-stranded DNA that can
be found in a variety of bacterial species
o Replicate independently
o Range in size from a few thousand base pairs (bp) to more than 100 kilo base
pairs (kb)
Plasmid cloning vectors: plasmids that are used for transformation expiriements and
therefore have been genetically engineered
This lab: making E. coli cells articificially competent by using the CaCl2 method
o We are using the pGLO plasmid
Contains several reporting genes, most notably for the Green
Flourenscent Protein (GFP) and the beta-lactamase antibiotic
resistance gene (bla)
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