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
true/false CaCl2 and heat shock methods are easy and have a high transfer efficiency
- false
- they are easy but have a low efficiency
describe the CaCl2 treatment and heat shock method
- the bacterial cells are incubated with cold solution of CaCl2
- heat shock the cells to open pores in the cell membrane
- this allows the foreign DNA to enter
- then the cells recover and pores close
true/false in the CaCl2 treatment and heat shock method, less than one cell will take up the plasmid/vector intended
true
Why is a good selectable marker important in the CaCl2 treatment and heat shock method?
This is because if only 1% of cells take the vector and there is 1000, how do you know which one has the marker, you kill the rest of them (ex: antibiotic resistence)
true/false electroporation has high transformation efficiencies
true
describe the process of electroporation
- Short pulses of current create temporary pores or holes in the cell membrane
- the DNA can then enter the cell
- then the cells are allowed to recover
what methods can be used for prokaryotic AND eukaryotic transformation
- electroporation
- lipofection
- ballistic/ gene gun
what is the efficiency of electroporation
10^8 to 10^9
what happens during lipofection
- DNA is encapsulated in an artificial liposome (micelle)
- DNA/ liposome complex is added to cells
- this complex becomes closely associated w the negatively charged cell membrane and eventually fuses w it
describe how genes are cloned using bacteria
- start w a circular, double-stranded plasmid DNA cloning vector
- it is cleaved by a restriction nuclease
- DNA ligase covalently links a DNA fragment we want to clone into the vector
- this forms recombinant DNA
- the recombinant DNA is introduced to a bacterial cell
- the cell culture produces hundreds of millions of new bacteria
what is the deterrent of electroporation
slightly more dangerous and more expensive
Why is lipofection not commonly used if it is one of the best methods for transformation?
Because it is extremely expensive. One liposome can cost anywhere from 10-20$
What is Microinjection for DNA transformation?
Using a very refined glass micropipette, plasmids are injected into a cell
What happens in the Ballistic/gene gun method?
- Gold particles coated with DNA and plasmids is shot at the cell at high velocity
- This allows stable transformation or transient expression of new genes
