transformation Flashcards
what is this the definition for? a virus infects a bacterium and some bacterial DNA is entrapped in the viral capside; this virus infects another bacterium and transfers the DNA of the previous bacterium
transduction
what is transformation?
the bacteria gains a trait by taking in DNA from its environment
what is conjugation?
Transfer of DNA from one bacterium (F+) to other (F-) through sex pilus and with the help of F factor plasmid is
are bacteria diploid?
no
what is the impact of bacteria being haploid? are they easy to grow?
yes easy to grow and test traits, also show no dominance or recessivity
how is there bacterial variation?
from errors e.g DNAP
what is an auxotroph?
a mutant organism, especially a microorganism, that has a nutritional requirement not shared by the parent organism. (it cannot synthesize compound needed for growth)
what is a prototroph?
an organism able to grow and survive on minimum conditions (it provides its own compounds needed for growth)
when did Griffith perform his experiments?
1928
what were griffiths observations?
virulen strain=smooth non virulent=rough then in mice experiments mix of heat killed smooth and non virulent still caused mice to die –> discovered transformation
what did Frederick Griffith discover in 1928
transformation
what did Avery,Macleod and McCarty demonstrate?
they showed the ‘transforming principle’ was DNA
how did Avery,Macleod and McCArty demonstrate DNA was the transforming principle?
added RNAse,protease and DNAase to the same mix(heat killed virulent and non virulent strain) and only the one without DNA stayed non virulent
in transformation what type of DNA attached onto the cell and what type is taken up?
dsDNA binds to cell,endonucleases mean only ssDNA is actually moved into the cell and this ssDNA is inserted into the bacterial chromosome by recombination
what 3 problems can occur in transformation?
1)dsDNA may not attach to cell 2)ssDNA may not be taken up into cell 3)ssDNA may not be incorporated into the chromosome by recombination
which process allows ssDNA to get into the bacterial chromosome in transformation?
recombination
in transformation is the cell the info is being picked up from dead?
yes
why undergo transformation? (3)
nutrition,repair and variety (adaptation)
if two donor genes are located closely on the chromosome what is there a greater chance of?
they will be carried on the same piece of transforming dna and therefore greater chance of double transformation
what can transformation be used for?
to assess linkage (test for a departure from the product rule)
in transformation does the donor DNA replace a large percent of the recipients chromosome?
no, only a small percent (cotransformation is because thE genes are in close proximity to one another)
What are PLQ?
pores that allow DNA to reach import system
what is ComEA protein responsible for?
reception of dsDNA on cell surface which is then nucked by endonucleases to become ssDNA
what do endonucleases in a competent bacterial cell do?
nick the dsDNA to make it ssDNA
what is the ssDANA transported through?
ComE
what can lead to the COM system developing?
stress and high density
why do bacteria undergo transformation?
nutrition,repair,variety
what is the pillus function?
no known function,perhaps involved in conjugation or pulling up DNA as it contracts and retracts
what are two methods of artificial transformation?
cacl2 transformation and electroporation
what is competence?
competence refers to the state of being able to take up exogenous DNA from the environment
what system does natural transformation involve?
COM system
what does electroporation do?
artificial transformation-electric field 10-20kV/cm changes permeability of bacterial membranes
what voltage is used in artificial electroporation?
10-20kV/cm
is artificial more or less efficient than natural?
less
explain Cacl2 transformation briefly?
add CaCl2 which promotes binding of plasmid to LPS and calcium ions attract negative DNA. The plasmid DNA can then enter cells after Heat shock (changes membrane permeability) e.g from 4 degrees to 42 for e.coli
what is required in CaCL2 transformation to allow plasmid DNA to enter? at what temps?
heat shock from 4 to 42 for e.coli
what does the Calcium ion in calcium chloride do in artificial transformation?
promotes plasmid binding to Lippopolysacharide and the negative DNA is attracted to it
what is a polylinker?
area for inserting foreign DNA (unique restriction sites are found in this region)
where are unique restriction sites found?
polylinker region
T or F: the rate of cotransformation of 2 genes is inversely proportional to the distance between them?
True. The quicker the transformation the closer they are.
what is b galactosidase encoded by?
lacZ
in blue white screening do both e.coli and the plasmid have a mutant lacZ?
no, e.coli has mutant lacZ(deletion mutant) and the plasmid has a functional lacZ
what does the lacZ in the plasmid have? (blue/white screening)
A MCS (multi cloning site)
what can MCS in plasmid be cut by? (blue/white screening)
restriction enzymes
will blue colonies have an inserted vector?
no, blue colonies have vector with uninterrupted lacZalpha therefore have no insert
in blue/white screening what are the cells grown in the presence of?
Xgal
