21.2 in vivo cloning - the use of vectors Flashcards
what is the promotor region?
binding to transcription factors & RNA polymerase to start transcription
what is the terminator region?
RNA polymerase is released & stops transcription
how are the gene inserted into a vector? (2)
1- the plasmid & gene are cut with the same restriction endonuclease - both have complementary sticky ends
2- the cut plasmids & genes are joined & mixed by DNA ligase to form the recombinant plasmid
why are plasmids used as a vector?
plasmids almost carry genes for antibiotic resistance
how is DNA transformed into plasmids? (2)
1- recombinant plasmids & bacteria (host cells) are mixed in Ca2+ ion medium
2- Ca2+ ions & changing temperature causes membrane to be permeable & plasmid moves into cytoplasm
what are the 3 reasons for unsuccessful transfomation of recombinant plasmids?
1- only ~1% takes up plasmid
2- some plasmids would close up before gene was inserted
3- DNA fragments / genes joined together to form their own plasmid
how is the cell that has taken up a plasmid identified?
bacteria is grown on antibiotic & only bacteria that has taken up the plasmid will survive & be visible
following up, in which 3 ways the cell that has taken up a plasmid & has the desired gene identified?
1- anti-biotic resistance marker genes
2- fluorescent marker genes
3- enzyme markers
give 5 advantages of in vivo cloning.
1) useful for introducing gene into other organisms (eg. gene therapy)
2) no risk of contamination
3) very accurate
4) cuts out specific gene
5)transformed bacteria could produce large quantities of gene products
why is there a much smaller chance of contamination compared to in vivo cloning?
on,y contamination of dna fragments - no chance of inserting contaminant dna due to genes & plasmids cut with the same restriction endonuclease
