21.2 In vivo gene cloning- the use of vectors

Question 1

cloning the DNA fragment ‘in vivo’

Answer 1

transferring the fragment to host cell using a vector

Question 2

Using the same restriction endonuclease..

Answer 2

cuts the same recognition site leaving the same bases exposed (called sticky ends)
this allows any single strands to ‘stick’ to one another as long as they have been cut by the same endonuclease.

Question 3

once sticky ends have stuck together which enzyme does what?

Answer 3

enzyme ligase joins phosphate-sugar backbone of the two sections of DNA (joining them up).

Question 4

what is a promoter

Answer 4

a region of DNA that acts as a binding site for RNA polymerase. begins process of transcription

Question 5

what is a terminator

Answer 5

a region of DNA that releases RNA polymerase and ends transcription

Question 6

what is a vector

what is a vector used for

Answer 6

a carrying unit

transports DNA into host cell

Question 7

what is a sticky end?

Answer 7

if DNA is cut in jaggedly by restriction endonuclease the single strands have exposed nucleotides which act as ‘sticky ends’

Question 8

preparation of DNA fragment for insertion (into vector) involves

Answer 8

addition of extra lengths of DNA i.e. promoter and terminator

Question 9

most commonly used vector

Answer 9

plasmid (circular lengths of DNA found in bacteria)

Question 10

Where is the plasmid ‘loop’ broken

Answer 10

plasmids almost always contain genes for antibiotic resistance.
restriction endonuclease used at one of antibiotic resistance genes to break plasmid loop

Question 11

Why use the same endonuclease to break break open the vector that you used to cut out the DNA fragment

Answer 11

ensures sticky ends of opened up vector e.g. plasmid are complimentary to sticky ends of DNA fragment and can be incorporated into vector and permanently joined by DNA ligase = recombinant DNA

Question 12

1. the process transformation happens after…
2. what is the purpose of transformation
3. what does transformation involve

Answer 12

1. the DNA has been incorporated into some of plasmids.
2. reintroducing the plasmids back into bacterial cell.
3. plasmids and bacterial cells mixed together in a medium containing calcium ions. calcium ions and change in temperature make bacterial membrane permeable- plasmids can pass through into cytoplasm

Question 13

not all bacterial cells possess DNA fragments with desired gene
3 reasons why?

Answer 13

only a few bacterial cells (as few as 1%) take us plasmids when the two are mixed together
some plasmids will have closed up again without incorporating the DNA
sometimes DNA fragment ends join together to form its own plasmid

Question 14

1. what gene can be used to identify bacterial cells that have taken up the plasmid
2. where is this gene found

Answer 14

1. the gene of antibiotic resistance which is unaffected by introduction of a new gene
2. genes for the production of the enzymes that break down antibiotics are found in the plasmids

Question 15

1. identifying the bacterial cells that have taken up the plasmid
2. drawback of this method
3. what is the next step?

Answer 15

1. all bacteria cells are grown on a medium that contains the antibiotic ampicillin
   bacteria that have taken up the plasmid will have acquired the gene for ampicillin resistance
   these bacteria are able to break ampicillin down and survive
   bacterial cells that have not taken up plasmids will not be resistant to ampicillin and therefore die
2. some cells will have taken in plasmid that have closed up without incorporating new gene and these will also have survived.
3. to identify cells without new gene and eliminate them through the use of marker genes

Question 16

1. what does using a marker gene involves

Answer 16

1. using a second separate gene on the plasmid.

Question 17

marker genes

3 reasons why is 2nd gene easily identifiable

Answer 17

may be resistant to antibiotic
may make a fluorescent protein that is easily seen
may produce an enzyme who’s action can be easily identified

Question 18

marker genes

1. using gene resistance to antibiotic
2. problems
3. how to overcome problem

Answer 18

1. uses other antibiotic resistance gene in the plasmid (the gene that was cut in order to incorporate the required gene)
   plasmids that have taken up the fragment will no longer produce the enzyme to break down antibiotic e.g. tetracycline
   can identify these bacteria by growing on a culture that contains tetracycline
2. destroys cells containing required gene
3. technique replica plaiting allows us to identify living bacteria containing required gene.

Question 19

R-plasmid carries genes for resistance to

Answer 19

ampicillin and tetracycline

Question 20

marker genes

1. fluorescent markers
2. benefits

Answer 20

1. Transfer of gene which produces a green fluorescent protein (GFP) from jelly fish into plasmid

desired gene transplanted into centre of GFP gene

any bacterial cell that has taken up the plasmid with the desired gene will not be able to produce GFP

bacterial cells that have not taken up desired gene will continue to produce GFP and to fluoresce

2. results can be obtained by viewing cells under microscope. makes process rapid

Question 21

marker genes

1. enzyme markers

Answer 21

1. Gene that produces enzyme lactase

lactase turns a particular colourless substrate blue

desired gene transplanted into gene that makes lactase

bacterial cell that has taken up the desired gene - colonies grown from it will not produce lactase

when grown on the colourless substrate they will not turn it blue

bacterial cells that have not taken up the desired protein will change the colourless substrate blue