in vivo gene cloning Flashcards
define in vivo
living organisms
(bacterial host)
define bacterial host
easy to grow as replicate quickly so can get lots of DNA fragments
define vector
way of transporting DNA from 1 place to another
plasmid
what is the purpose of DNA ligase
forms a sugar phosphate backbone
what is the purpose of RNA polymerase
makes RNA from DNA
if a plasmid contains an area that codes for antibiotic resistance where does restriction endonucleases cut
restriction endonucleases have to cut at antibiotic resistance gene
explain the process of transformation
when a plasmid with the DNA is inserted into a bacterial cell
need
Ca2+
higher temperature
(which makes the cell wall more permeable)
why doesn’t it always work (3 marks)
- 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
how do you test the bacteria to see if a plasmid has been taken up
r plasmid contains 2 genes an ampicillin resistant gene and tetracycline resistant gene
define ampicillin resistant gene
bacteria with that plasmid can produce an enzyme that breaks up ampicillin
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
the bacterial cell with the plasmid containing the genes for ampicillin and tetracycline resistance will survive
the bacterial cell with no plasmid died
what is the purpose of using marker genes
to see if the plasmid contains the gene/DNA fragment that we want
what are the 3 types of marker genes
- antibiotic resistance
- fluorescent markers
- enzyme markers
what are the 2 types of transcription factors
promotor
terminator
how do you use antibiotic resistance gene to determine whether the plasmid contains the gene
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
explain the process of replica plating
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)
how do you use fluorescent markers to determine whether the plasmid contains the gene
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
how do you use enzyme markers to determine whether the plasmid contains the gene
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
