CBG Lecture 17: Bacterial Transformation Flashcards
in what bacteria was transformation first discovered
Streptococcus pneumoniae
what happens in transformation
bacteria take up extrogenous DNA from enviro - either linear DNA or plasmids
what is a use of transformation
transfer genes between bacteria
what can cotransformation be used for
to map genes on the bacterial chromosomes
what is the “transforming principle”
ssDNA
how is exogenous DNA incorporated into the bacterial chromosome
by breakage andinsertion process - via recombinaiton (2 crossovers) the after replication and division ONE of the resulting cell is transformed
after bacterial transformation (replication and division) how many of the bacterial cells are transformed
1
what is the bacterial transformation process analogous to
HFr and F- crosses
what is competence
the cells in popn of bacteria that are able to be transformed
how did Avery know that DNA was the transforming principle
because DNAse broke it down
how many crossovers are needed for integration of ss exogenous DNA into bacterial chrom
2
is the transfer of material permanent
yes
what happens if transfer is interrupted
transferred DNA is broken down and F- cell remains F- cell
why does natural transformation occur
nutrition: source of nucleotides
repair: uptake of homologous DNA allows damage repair by recombination
Diversity: inc gen diversity ny HGT
how does natural transformation increase diversity
by HGT
how does natural transformation help dna repair
uptake of homologous DNA allows famage repair by recombination
how is natural transformation a source of nutrition
source of nucleotides
what are the ways of artificial transformation
chemical: CaCl2 @ 4deg
electroporation: electric field (10-20kV/cm) change permeability of membrane then membrane repair resores it
what condns for artificial chemical transformation
CaCl2 @ 4deg
what condns for artificial electroporation transformation
electric field 10-20kV/cm change permeability of membrane
what is the basic structure of cloning vector
origin of replication - essential for propagation daughter cells
antibiotic resistance - essential for selection
polylinker - unique area for insrting foreign DNA with unique restriction sites
what is a polylinker
unique area for inserting foreign DNA with unique restriction sites
what is the ori essential for
propagation of daughter cells
why is antibiotic resistance good for a cloning vector
essential for selection
what is the rate of cotransformation of two genes inversely proportional to
the distance between them
what is the distance between two genes inversely proportional to
the rate of cotransformation of two genes
what % of transformants of cotransformants
40%
what are 40% of transformants
cotransformant
what gives streptococci its smooth appearance
polysacchs
why are no streptoccoi pneumoniae virulent
because rough (no polysacchs) and no capsule therefore can be killed by immune system
what xperiment did Griffith do
extract dead bac, transform living cells
mix IIR and dead heat killed IIS - killed mice
who did experiment where extract dead bac, transform living cells
Griffith
what is IIIR/IIS
IIR - rough strain of streptococci as no smooth polysach on surface
IIS = smooth
outline process of transformation in depth
bac take up exogenous ssDNA if competent
dsDNA pieces specifically bind onto the bacterial envelope+once bound on surface, exonuclease nich dsDNA and therefore only a ssDNA moves into bacterial cell
to ecome integrated, ssDNA inserted into chrom of recipient via 2 crossover (recombination)
after replication&division, on of resulting cells transformed
when inducing bacterial transformation eg. mix Arg- with arg+, what types of cells are produced
Arg+ transformed
unchanged Arg- and dsDNA
unintregrated ssDNA not taken up, and Arg-
untransformed Arg- but ssSNA not taken up because only dsDNA binds on surface
what does bacterial transformation competence require
stressful condns
how many membranes does gram + have
1
how many membranes does gram - have
2
GIVE AN example of a gram positive bacteria that is naturally competentt
streptococcus pneumoniae
give an example of a gram negative naturally competent bacteria
neisseria gonorrhoea
what does the com system involve
can induce competence
comA - protein responsible for the reception of dsDNA on surface cell
dsDNA nicked by nucleases and ssDNA transported through COMEC channel
what is comA involved in
protein responsible for the receptio n of dsDNA on the surface
what is the COMEC channel
channel involved in competence where ssDNA transported through to get into recipient cell
where is the com system on a gram - bacterium
on inner membrane
what is difference between gram + and gram - com system
gram + has com system on outside as it only has one membrane
gram - has com system on inner membrane, but has PilQ secretin on outer membrane allowing ssDNA to reach import system
what is PilQ
a secretin involved in the com system of a gram - bacteria whihc allows ssDNA to reach import system
give an example of a bacteria used for artificial transformaiton
e coli (gram -ve)
what colour does XGal go
blue colour
what do white clones mean
theyve received resistant plasmids and also the gene
what fragments are donor cell wildtypes cut into
cut it in 20kB fragments
