Amplifying In Vivo pt2 - Identifying transformed cells

general method

using marker genes

why are marker genes used

show gene has been taken up/ only cells that show marker will contain gene

3 ways of identifying

antibiotic resistant markers

fluorescent markers

coding for an enzyme

how do antibiotic resistant markers work

antibiotic resistance gene inserted into plasmid containing desired gene

host cells grown on agar plate

colonies transferred to another agar plate containing antibiotic- replica plating

host cells containing plasmid will survive and reproduce- form colonies

host cells not containing plasmid will die

how do fluorescent markers work

GFP gene inserted into plasmid

grow bacteria on agar

expose colonies to UV light

the ones containing the desired gene will fluoresce

how does enzyme coding work

gene that codes for marker eg lactase inserted into plasmid

grow bacteria on agar containing colourless substance

those containing desired gene will contain gene for producing lactase and turn solution blue

what needs to be done to the host cell when identifying it

reproduce so produces many copies of gene

produce protein coded for by gene that has been inserted into plasmid

advantage of in Vivo rather than PCR

lower risk of contamination

very accurate and specific

can be then used to produce large quantities of protein coded for by gene