Lecture 4: Plasmids Flashcards
what is genetic transformation
the insertion of a gene into an organism
what are some uses for genetic transformation, and examples
- agriculture genes coding for resistance against frost, pest or spoilage being introduced to plants
- bioremediation bacteria that can be genetically trannsformed w genes enabling them to digest oil spills
- medicine diseases caused by defective genes can betrated by gene therapy
true/false all vectors are plasmids, but not all plasmids are vectors
true
what is a vector
a plasmid that has been manipulated to make it a useful tool
what is a plasmid
- small, circular, double stranded, extrachromosomal elements found in some strains of bacteria
- range from 1 to 200 kb
what is the copy number of plasmids in a cell
from 1-1000
how are plasmids normally drawn
as a double circle (to represent the 2 DNA strands)
what kind of tension are plasmids under
super helical tension (negative supercoiling)
why are plasmids under so much tension
cause they’re closed circular double-stranded DNA molecules
true/false because all plasmids have the same molecular weight, they migrate at the same rate on an agarose gel
- false
- different topological forms of plasmid DNA migrate at diff rates
rank which different topological forms of plasmid DNA migrate the quickest vs the slowest
- slowest
- concatamers
- nicked circular form
- linear form
- supercoiled form
- quickest
what are the genes encoded on plasmid DNA
- the genes required for its own replication and propagation
- Origin of replication (ori)
- Gene for partitioning of plasmids into each daughter cell during cell division
- Plasmids also often contain genes that confer advantages to their bacterial hosts
true/false Genes required for plasmid replication and propagation are stored on plasmid DNA
true
what is put in plasmids to create vectors
- Selectable markers that add the thing you want
- Multiple cloning sites
- Regulatory signals
what do selectable markers do when developing cloning vectors
- antibiotic resistant gene
- allows only bacteria containing plasmid to grow in presence of antibiotic
what do multiple cloning sites do when developing cloning vectors
requires removal and addition of various restriction enzyme sites so a region on the plasmid would contain a number of unique rstriction enzyme sites
what do regulatory signals do when developing cloning vectors
- regulate expression of cloned genes
- promotor sequences to allow expression (transcription) of gene cloned into plasmid
what induces the promotor sequence in the pGLO plasmid
sugar arabinose
what resistance gene is in the pGLO plasmid
ampicillin
what happens in DNA cloning
- the fragment of DNA contianing the gene of interest is isolated
- then the fragment of DNA containing the gene is inserted into a plasmid to produce a recombinant DNA molecule
- introduce the recombinant DNA molecule into a host cell
- after that, plate the transformed bacteria under selection so that cells containing the plasmid will grow and form a colony
what are the diff ways bacteria can receive DNA
- transformation free DNA enters cell
- transduction DNA containing virus introduces DNA
- conjugation plasmid in donor cell introduces DNA
describe conjugation/ bacterial mating
- a natural plasmid transfer
- responsible for the spread of antibiotic rsistance in bacteria for viruses
what is the most simple way to introduce foreign DNA in the lab
CaCl2 treatment and heat shock method
describe how genes can be cloned using bacteria
- we start w a circular, double-stranded plasmid DNA (cloning vector)
- it gets cleaved with a restriction nuclease
- DNA ligase covalently links a DNA fragment that we want cloned into the vector
- this plasmid DNA vector is introduced into a bacterial cell
- the cell culture produced hundreds of Dmillions of new bacteria
