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CBG Bacteria HELEP > CBG Lecture 17: Bacterial Transformation > Flashcards

CBG Lecture 17: Bacterial Transformation Flashcards

1
Q

in what bacteria was transformation first discovered

A

Streptococcus pneumoniae

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2
Q

what happens in transformation

A

bacteria take up extrogenous DNA from enviro - either linear DNA or plasmids

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3
Q

what is a use of transformation

A

transfer genes between bacteria

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4
Q

what can cotransformation be used for

A

to map genes on the bacterial chromosomes

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5
Q

what is the “transforming principle”

A

ssDNA

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6
Q

how is exogenous DNA incorporated into the bacterial chromosome

A

by breakage andinsertion process - via recombinaiton (2 crossovers) the after replication and division ONE of the resulting cell is transformed

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7
Q

after bacterial transformation (replication and division) how many of the bacterial cells are transformed

A

1

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8
Q

what is the bacterial transformation process analogous to

A

HFr and F- crosses

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9
Q

what is competence

A

the cells in popn of bacteria that are able to be transformed

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10
Q

how did Avery know that DNA was the transforming principle

A

because DNAse broke it down

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11
Q

how many crossovers are needed for integration of ss exogenous DNA into bacterial chrom

A

2

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12
Q

is the transfer of material permanent

A

yes

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13
Q

what happens if transfer is interrupted

A

transferred DNA is broken down and F- cell remains F- cell

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14
Q

why does natural transformation occur

A

nutrition: source of nucleotides
repair: uptake of homologous DNA allows damage repair by recombination
Diversity: inc gen diversity ny HGT

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15
Q

how does natural transformation increase diversity

A

by HGT

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16
Q

how does natural transformation help dna repair

A

uptake of homologous DNA allows famage repair by recombination

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17
Q

how is natural transformation a source of nutrition

A

source of nucleotides

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18
Q

what are the ways of artificial transformation

A

chemical: CaCl2 @ 4deg
electroporation: electric field (10-20kV/cm) change permeability of membrane then membrane repair resores it

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19
Q

what condns for artificial chemical transformation

A

CaCl2 @ 4deg

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20
Q

what condns for artificial electroporation transformation

A

electric field 10-20kV/cm change permeability of membrane

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21
Q

what is the basic structure of cloning vector

A

origin of replication - essential for propagation daughter cells
antibiotic resistance - essential for selection
polylinker - unique area for insrting foreign DNA with unique restriction sites

22
Q

what is a polylinker

A

unique area for inserting foreign DNA with unique restriction sites

23
Q

what is the ori essential for

A

propagation of daughter cells

24
Q

why is antibiotic resistance good for a cloning vector

A

essential for selection

25
Q

what is the rate of cotransformation of two genes inversely proportional to

A

the distance between them

26
Q

what is the distance between two genes inversely proportional to

A

the rate of cotransformation of two genes

27
Q

what % of transformants of cotransformants

A

40%

28
Q

what are 40% of transformants

A

cotransformant

29
Q

what gives streptococci its smooth appearance

A

polysacchs

30
Q

why are no streptoccoi pneumoniae virulent

A

because rough (no polysacchs) and no capsule therefore can be killed by immune system

31
Q

what xperiment did Griffith do

A

extract dead bac, transform living cells

mix IIR and dead heat killed IIS - killed mice

32
Q

who did experiment where extract dead bac, transform living cells

A

Griffith

33
Q

what is IIIR/IIS

A

IIR - rough strain of streptococci as no smooth polysach on surface
IIS = smooth

34
Q

outline process of transformation in depth

A

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

35
Q

when inducing bacterial transformation eg. mix Arg- with arg+, what types of cells are produced

A

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

36
Q

what does bacterial transformation competence require

A

stressful condns

37
Q

how many membranes does gram + have

A

1

38
Q

how many membranes does gram - have

A

2

39
Q

GIVE AN example of a gram positive bacteria that is naturally competentt

A

streptococcus pneumoniae

40
Q

give an example of a gram negative naturally competent bacteria

A

neisseria gonorrhoea

41
Q

what does the com system involve

A

can induce competence
comA - protein responsible for the reception of dsDNA on surface cell
dsDNA nicked by nucleases and ssDNA transported through COMEC channel

42
Q

what is comA involved in

A

protein responsible for the receptio n of dsDNA on the surface

43
Q

what is the COMEC channel

A

channel involved in competence where ssDNA transported through to get into recipient cell

44
Q

where is the com system on a gram - bacterium

A

on inner membrane

45
Q

what is difference between gram + and gram - com system

A

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

46
Q

what is PilQ

A

a secretin involved in the com system of a gram - bacteria whihc allows ssDNA to reach import system

47
Q

give an example of a bacteria used for artificial transformaiton

A

e coli (gram -ve)

48
Q

what colour does XGal go

A

blue colour

49
Q

what do white clones mean

A

theyve received resistant plasmids and also the gene

50
Q

what fragments are donor cell wildtypes cut into

A

cut it in 20kB fragments

CBG Bacteria HELEP (3 decks)

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