CBG Lecture 17: Bacterial Transformation

Question 1

in what bacteria was transformation first discovered

Answer 1

Streptococcus pneumoniae

Question 2

what happens in transformation

Answer 2

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

Question 3

what is a use of transformation

Answer 3

transfer genes between bacteria

Question 4

what can cotransformation be used for

Answer 4

to map genes on the bacterial chromosomes

Question 5

what is the “transforming principle”

Answer 5

ssDNA

Question 6

how is exogenous DNA incorporated into the bacterial chromosome

Answer 6

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

Question 7

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

Answer 7

1

Question 8

what is the bacterial transformation process analogous to

Answer 8

HFr and F- crosses

Question 9

what is competence

Answer 9

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

Question 10

how did Avery know that DNA was the transforming principle

Answer 10

because DNAse broke it down

Question 11

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

Answer 11

2

Question 12

is the transfer of material permanent

Answer 12

yes

Question 13

what happens if transfer is interrupted

Answer 13

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

Question 14

why does natural transformation occur

Answer 14

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

Question 15

how does natural transformation increase diversity

Answer 15

by HGT

Question 16

how does natural transformation help dna repair

Answer 16

uptake of homologous DNA allows famage repair by recombination

Question 17

how is natural transformation a source of nutrition

Answer 17

source of nucleotides

Question 18

what are the ways of artificial transformation

Answer 18

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

Question 19

what condns for artificial chemical transformation

Answer 19

CaCl2 @ 4deg

Question 20

what condns for artificial electroporation transformation

Answer 20

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

Question 21

what is the basic structure of cloning vector

Answer 21

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

Question 22

what is a polylinker

Answer 22

unique area for inserting foreign DNA with unique restriction sites

Question 23

what is the ori essential for

Answer 23

propagation of daughter cells

Question 24

why is antibiotic resistance good for a cloning vector

Answer 24

essential for selection

Question 25

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

Answer 25

the distance between them

Question 26

what is the distance between two genes inversely proportional to

Answer 26

the rate of cotransformation of two genes

Question 27

what % of transformants of cotransformants

Answer 27

40%

Question 28

what are 40% of transformants

Answer 28

cotransformant

Question 29

what gives streptococci its smooth appearance

Answer 29

polysacchs

Question 30

why are no streptoccoi pneumoniae virulent

Answer 30

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

Question 31

what xperiment did Griffith do

Answer 31

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

Question 32

who did experiment where extract dead bac, transform living cells

Answer 32

Griffith

Question 33

what is IIIR/IIS

Answer 33

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

Question 34

outline process of transformation in depth

Answer 34

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

Question 35

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

Answer 35

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

Question 36

what does bacterial transformation competence require

Answer 36

stressful condns

Question 37

how many membranes does gram + have

Answer 37

1

Question 38

how many membranes does gram - have

Answer 38

2

Question 39

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

Answer 39

streptococcus pneumoniae

Question 40

give an example of a gram negative naturally competent bacteria

Answer 40

neisseria gonorrhoea

Question 41

what does the com system involve

Answer 41

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

Question 42

what is comA involved in

Answer 42

protein responsible for the receptio n of dsDNA on the surface

Question 43

what is the COMEC channel

Answer 43

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

Question 44

where is the com system on a gram - bacterium

Answer 44

on inner membrane

Question 45

what is difference between gram + and gram - com system

Answer 45

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

Question 46

what is PilQ

Answer 46

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

Question 47

give an example of a bacteria used for artificial transformaiton

Answer 47

e coli (gram -ve)

Question 48

what colour does XGal go

Answer 48

blue colour

Question 49

what do white clones mean

Answer 49

theyve received resistant plasmids and also the gene

Question 50

what fragments are donor cell wildtypes cut into

Answer 50

cut it in 20kB fragments