in vivo gene cloning

Question 1

define in vivo

Answer 1

living organisms
(bacterial host)

Question 2

define bacterial host

Answer 2

easy to grow as replicate quickly so can get lots of DNA fragments

Question 3

define vector

Answer 3

way of transporting DNA from 1 place to another

plasmid

Question 4

what is the purpose of DNA ligase

Answer 4

forms a sugar phosphate backbone

Question 5

what is the purpose of RNA polymerase

Answer 5

makes RNA from DNA

Question 6

if a plasmid contains an area that codes for antibiotic resistance where does restriction endonucleases cut

Answer 6

restriction endonucleases have to cut at antibiotic resistance gene

Question 7

explain the process of transformation

Answer 7

when a plasmid with the DNA is inserted into a bacterial cell

need
Ca2+
higher temperature
(which makes the cell wall more permeable)

Question 8

why doesn’t it always work (3 marks)

Answer 8

• even with heat treatment and ca2+ only some bacterial cells take up the plasmid
• plasmids can close up again and they get taken up by the bacterial cell so the bacterial cell now has a plasmid with no gene
• DNA fragments can join themselves together

Question 9

how do you test the bacteria to see if a plasmid has been taken up

Answer 9

r plasmid contains 2 genes an ampicillin resistant gene and tetracycline resistant gene

Question 10

define ampicillin resistant gene

Answer 10

bacteria with that plasmid can produce an enzyme that breaks up ampicillin

Question 11

when you culture bacterial cells in ampicillin
what happens to the bacterial cell with plasmid containing ampicillin and tetracycline resistant genes
and what happens to the bacterial cell that has no plasmid

Answer 11

the bacterial cell with the plasmid containing the genes for ampicillin and tetracycline resistance will survive

the bacterial cell with no plasmid died

Question 12

what is the purpose of using marker genes

Answer 12

to see if the plasmid contains the gene/DNA fragment that we want

Question 13

what are the 3 types of marker genes

Answer 13

• antibiotic resistance
• fluorescent markers
• enzyme markers

Question 14

what are the 2 types of transcription factors

Answer 14

promotor

terminator

Question 15

how do you use antibiotic resistance gene to determine whether the plasmid contains the gene

Answer 15

then carry out replica plating

insert fragment of DNA into T gene so restriction endonuclease chops the plasmid within the T gene

put plasmid on a growth plate of ampicillin

if the DNA is in the T gene the cell will survive

if the bacterial cell has no plasmid the cell will die

if the bacterial cell has taken up a plasmid without DNA then it will survive

then grow your bacterial cell on tetracycline antibiotic

only the bacteria with the plasmid containing the T gene without any DNA will survive

Question 16

explain the process of replica plating

Answer 16

get a block of wood with velvet on the bottom that is the same size as the petri dish

stamp it down on the colony

transfer to another agar plate
(so the colonies on the first plate are the same as on the second)

Question 17

how do you use fluorescent markers to determine whether the plasmid contains the gene

Answer 17

GFP - green fluroscent protein found in jellyfish

in bacterial cell with plasmid with DNA in the GFP gene doesn’t work as the DNA is the in the middle of the gene so it doesn’t glow

in the bacterial cell with the plasmid and just the GFP gene will glow so it will fluoresce

Question 18

how do you use enzyme markers to determine whether the plasmid contains the gene

Answer 18

the lactase gene turns a particular substrate blue

by putting the DNA in the middle of the lactase gene you break the gene so they will be unable to turn blue

whereas the bacterial cells just with the plasmid have a full lactase gene and then you would discard these as they turn blue so don’t contain the DNA