CBG Lecture 17: Bacterial Transformation Flashcards Preview

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Flashcards in CBG Lecture 17: Bacterial Transformation Deck (50):
1

in what bacteria was transformation first discovered

Streptococcus pneumoniae

2

what happens in transformation

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

3

what is a use of transformation

transfer genes between bacteria

4

what can cotransformation be used for

to map genes on the bacterial chromosomes

5

what is the "transforming principle"

ssDNA

6

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

7

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

1

8

what is the bacterial transformation process analogous to

HFr and F- crosses

9

what is competence

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

10

how did Avery know that DNA was the transforming principle

because DNAse broke it down

11

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

2

12

is the transfer of material permanent

yes

13

what happens if transfer is interrupted

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

14

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

15

how does natural transformation increase diversity

by HGT

16

how does natural transformation help dna repair

uptake of homologous DNA allows famage repair by recombination

17

how is natural transformation a source of nutrition

source of nucleotides

18

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

19

what condns for artificial chemical transformation

CaCl2 @ 4deg

20

what condns for artificial electroporation transformation

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

21

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

22

what is a polylinker

unique area for inserting foreign DNA with unique restriction sites

23

what is the ori essential for

propagation of daughter cells

24

why is antibiotic resistance good for a cloning vector

essential for selection

25

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

the distance between them

26

what is the distance between two genes inversely proportional to

the rate of cotransformation of two genes

27

what % of transformants of cotransformants

40%

28

what are 40% of transformants

cotransformant

29

what gives streptococci its smooth appearance

polysacchs

30

why are no streptoccoi pneumoniae virulent

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

31

what xperiment did Griffith do

extract dead bac, transform living cells
mix IIR and dead heat killed IIS - killed mice

32

who did experiment where extract dead bac, transform living cells

Griffith

33

what is IIIR/IIS

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

34

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

35

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

36

what does bacterial transformation competence require

stressful condns

37

how many membranes does gram + have

1

38

how many membranes does gram - have

2

39

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

streptococcus pneumoniae

40

give an example of a gram negative naturally competent bacteria

neisseria gonorrhoea

41

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

42

what is comA involved in

protein responsible for the receptio n of dsDNA on the surface

43

what is the COMEC channel

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

44

where is the com system on a gram - bacterium

on inner membrane

45

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

46

what is PilQ

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

47

give an example of a bacteria used for artificial transformaiton

e coli (gram -ve)

48

what colour does XGal go

blue colour

49

what do white clones mean

theyve received resistant plasmids and also the gene

50

what fragments are donor cell wildtypes cut into

cut it in 20kB fragments